POSTERS - European Journal of Anatomy

POSTERS

Anatomy Teaching

P-1 THE TEACHING OFANATOMYIN FOURIBEROAMERICAN DENTALSCHOOLS.

Machado-Martínez, M.1, Martínez-Amaya,C .2, Va l d i v i a - G a n d u r, I.3, Manzanares-Céspe-des, M.C. 4

1 Instituto Superior de Ciencias Médicas de Vi l l aClara, Cuba. 2 Universidad de Los Andes, Mérida,Venezuela. 3 Universidad de Antofagasta, Chile. 4U n i -versidad de Barcelona, España.

The teaching of Human Anatomy has experienced anenormous evolution during the last years. The new tea-ching methods and technologies, the implementation ofdifferent Health professions requiring a specific forma-tion, and the new international educational context haveinfluenced very noticeably the way the Anatomy is taughtand studied in our Faculties. Our Universities belong tofour different geographic areas and differ in many aspects,both philosophical and organizational.

This first report offers a general view of the teachingof the Anatomy in four Iberoamerican dental schools,developed around some main points: a general view ofthe anatomy courses, both obligatory and compulsory.This description deals with the comparison of the gene-ral contents of the programs, the calendar, the coursesstructure, the evaluation methods, the number of the pro-fessors, and the material recourses disponible for the stu-dents. This document has been prepared using the samemethodology that permitted the redaction of the Firstand the Second “Report on the Teaching of the A n a t o m yin the Spanish Dental Schools” presented respectively in1996 and 1998 by the Board nominated by the SpanishAnatomical Society (Sociedad Anatómica Española,S A E ) .

Tha main objective of our group is to discuss the exis-ting problems in teaching Human Anatomy in Dentistry,to suggest some general recommendations regarding theteaching of Anatomy in the dentistry degree, and finally,to develop a common basis for the preparation and deve-lopment of the different Anatomy courses for the studentsof the Iberoamerican dental faculties. The Iberoamericandimension, in which the spanish language and numerouscultural aspects are shared by a community of more than400 milion people, mades necessary an effort for stregt-hening the collaboration between professionals, both tea-chers and clinicians, dedicated to the Odontology.

We conclude that it is important to establish and main-tain a strong relationship between the teachers of Ana-tomy in Dentistry in order to discuss programs and tea-ching or evaluating methods and especially to obtainupdated information about all the different factorsinfluencing the Teaching of both the Basic and the Clini-cal Sciences in Dentistry.

P-2 ADAPTING THE TEACHING OF H U M A NA N ATO M Y TO THE EUROPEAN HIGHERE D U C ATION A R E A( E H E A ) .

Jové, M., Arredondo, Z., Torrente, M., Sempe-re, T., Cobos, P., Piera, V.

Universitat Rovira i Virgili. Facultat de Medicina iCiències de la Salut (Reus). Unitat d’AnatomiaHumana i Embriologia.

The process of integration into the European HigherEducation Area should be initiated with the intention toimprove the teaching-learning process and teachingo rganization. In this paper we present one of the modelsfor coping with this process in the teaching of humananatomy in the medical, physiotherapy and humannutrition and dietetic studies of the Universitat Rovira iVi rg i l i .

Having defined the profile of education and compe-tencies, and within the framework of the current curri-culum, we used the following methodology: first, weselected the competencies in human anatomy requiredby the medical students; then we defined the learningobjectives, related these objectives to the competenciesto be evaluated and proposed the learning methodology;f i n a l l y, we calculated the number of hours of workrequired by the students. This methodology was used inthe three subjects of the medicine curriculum (HumanA n a t o m y, Anatomy of the Locomotor Apparatus andAnatomical Techniques); two subject on the physiothe-rapy curriculum, and in one subject on the human nutri-tion and dietetic curriculum.

P-3 PROBLEM-BASED LEARNING APPLIEDTO LARGE GROUPS.

Maranillo, E., Mirapeix, R., Reig, J.

Universitat Autònoma de Barcelona (UAB).

As a result of the Bologna Declaration and the intro-duction of the European Credit-Transfer System, there isa pressing need to redesign the objectives of university-student education as well as the methodology applied inobtaining such objectives.

Problem-based learning (PBL), on the basis of articu-lating real-life problems, aims to assist student acquisitionor development, in an on-going form, of a series of abili-ties: the integration of knowledge and skills; self-assess-ment; teamwork, oral discourse, argumentation and criti-cal reasoning; the ability to relate and synthesiseinformation, etc. That is, this system aims at teaching stu-dents to be more self-sufficient in the processes both ofwork and of learning.

Nevertheless, the main difficulty that PBL faces wit-hin Spanish universities is student overcrowding in theclassrooms and lecture halls.

Lecturers teaching N e u roanatomy and the SenseOrgans (in the Anatomy and Embryology Unit, UAB) putPBL into practice for one week with two student groups,each comprising 100-150 students. Each session lasted1h, corresponding to the hour of theory usually given atthis time. At the start of the class, the lecturer presentedstudents with a clinical case to solve within 30 minutes.Students worked on this in groups of three, each groupbeing asked to provide two copies of their solution: onefor the lecturer and the other for subsequent correctionand self-assessment of the clinical case. Each group wasresponsible for bringing to class the necessary biblio-graphy with which to solve the problem. Once the versionfor the lecturer had been handed in, the whole class thenassessed the various group solutions, with the lecturerthen finally going on comment on and discuss these, com-plementing his or her explanation with images or additio-nal information.

XXII Congress of the Spanish Society of Anatomy. ABSTRACTS

31European Journal of Anatomy, 2 0 0 5 , vo l. 9 (Suppl. 1)

P-4 OPINION OF ANATOMY STUDENTS ONTHE NEWTECHNOLOGIES. OUR EXPE-RIENCE WITH THE ANILLO DIGITA LDOCENTE (DIGITALTEACHING RING).

Escolar, J.D.1, Escolar, M.A.1, Blasco, J.1, Mar-tínez, C., Ros, L.H.2

1 Department of Human Anatomy and Histology.2 Department of Radiology. University of Zaragoza.

INTRODUCTION: Over four centuries have passed sinceFray Luis de León delivered his classes from the pulpit.Over all this time, methodology and technology havebeen incorporated into the teaching process. Six years agothe University of Zaragoza put the Anillo Digital Docen-te (ADD) on its website for use in the educational process.The ADD enables teachers and students to access andexchange different kinds of information.

MATERIALANDMETHODS : By means of the ADD we aredeveloping a kind of semi-attendance based teaching forthe study of Anatomy, by placing the following docu-ments on the website:

1. Schedule indicating each day’s activity.2. Sheet of concepts for each topic, with practical

objectives and bibliography.3. Subject syllabus.4. Multiple-choice questions for self assessment.5. Interactive atlas.At the end of first and second year Anatomy in the

studies for a Degree in Medicine, the students’ level ofsatisfaction was assessed.

RESULTS: From the results, we would point out: thatthe ADD is a tool that the students were not to keen onusing. They do not consider the ADD essential for thestudy of Anatomy. Regarding the material placed in theADD, the multiple-choice questions followed by the inte-ractive atlas were the most commonly used and the sche-dule was not used at all. A considerable number of stu-dents find the access complicated. Most of them considerthat the ADD has helped them to improve their studyhabits.

CONCLUSION: We propose that the use of the ADD forteaching Anatomy can help the teaching objectives of thesubject to be achieved.

P-5 I M PA C T O F ELECTRONIC MAILINGON T U TO R I Z ATION OF A N ATO M I C A LP R A C T I C U M .

Solá, R., Mias, C.

Anatomia Humana, Ciencias Médicas Básicas.Facultad de Medicina. Universidad de Lleida.

The authors present their experience in the use of theelectronic mailing to meliorate the answer of the studentsduring the realisation of the clinical anatomical practices,in a subject named Clinical Anatomy in the third year ofMedicine in our Faculty.

The students matriculated in this subject must madetwo clinical practices. The first one is in the operationroom and the other one in the Trauma consultation in ourHospital in order to make anatomical observations. Thestudents give a script that must be completed by themduring the practice and later they could send it by mailingto the responsible teacher before the next fifteen days. The

teacher correct the practice and send another time it to thestudent for new corrections. So the students could reachthe wished knowledge.

The authors have revised the total name of electronicmailing related with the two practices, and have measuredthe degree of participation of the students. They haveobserved that in a total of 106 students matriculated in thesubject, only a 32% have send no mailing. The rest, 72students, the 9,7% have send only one mailing, the 16%two mailings, the 22,2% five mailings and the 41,6%more than five mailings.

The first conclusion is that the use of the electronicmailing is a good instrument to control the work of thestudents during the practices, specially when there are ageographic difference between the Faculty and the Hospi-tal. The second conclusion is about the time that repre-sents to the teacher this control, but it has the advantagethat could be made out of the Faculty. The third conclu-sion is related with the objectives of the practices thatcould been reached more satisfactorily after severalcorrections by the teacher. The last conclusion is to consi-der this control as a new valuation of the student’s work,that must be considered in this moment of change in theanatomical learning.

P-6 SCIENTIFIC ILLUSTRATION AND MEDI-C A LT E A C H I N G .

Plaza Moreno, P., González Hernández, T.,Pérez Delgado, M.M.

Department of Anatomy, Faculty of Medicine, Uni -versity of La Laguna, Tenerife, Spain.

Most advances in Medicine have depended on the abi-lity to visualize the structure and science of the body.Being able to clearly visualize the basic science has oftenmade the difference between medical success and failure.It is not simply a matter of academic and scientific exerci-se. Data needs to be put into a form our senses understand.

Art and Medicine have been intertwined as far back asthe Stone Age. Scientific illustration has been widelyused, from ancient times, as a media to show informationfrom one person to other, to show the anatomy and phy-siology of the human body, and also as a media to showdifferent medical pathologies and congenital malforma-tions to the observer, that can be either the physician, thepatient, the student and/or the teacher.

The first known anatomical illustration is a Paleolithicdrawing in the El Pindal cave in Spain, showing a leaf-shaped area locating the heart. Egyptian doctors, by 4000BC utilized the first illustrations to accompany scientifictext. In the Medieval Ages, artists and physicians oftenworked together, and this collaboration led to great advan-ces in anatomical education during the Renaissance.

New technologies, the use of computers, havebrought new possibilities and improvements in the use ofscientific illustration as a media of communication bet-ween the teacher and the student and /or the physsicianand the patient.

Our study presents scientific representations of diffe-rent stages of the human embryos development, and alsoscientific illustrations of the changes in the woman perineduring the baby delivery. Both types of illustrations havebeen realized using the Photoshop Program in a computer,based on a previous obtained image. We describe the dif-ferent steps in the process to get the final image.


32 European Journal of Anatomy, 2 0 0 5 , vo l. 9 (Supp l. 1)

We can appreciate in our results that this technique,the application of the Photoshop program on the initialdrawing realized by the scientific illustrator, or on theimage obtained from the scan of a previous photography,offers us a big improvement in the final illustration, resul-ting in a great media for education and for communicationbetween the medical and the patient.

P-7 THE INFLUENCE OF HISTOLOGY ANDEMBRYOLOGY COURSES ON STUDENTA C H I E V E M E N T IN GROSS HUMANANATOMY COURSES.

Elizondo-Omaña, R.E., García Rodríguez,M.A., Morales-Gómez, J.A., Guzmán López, S.

Department of Anatomy. School of Medicine, Univer -sidad Autónoma de Nuevo León .

In the School of Medicine of the Universidad A u t ó n o-ma de Nuevo León, there is a clear division between pre-clinical and clinical subjects. Gross human anatomy istaught for one semester along with other subjects thatinclude an introduction to medicine, art appreciation andcreative thought, and the subjects of histology and embr-yology are taught during another semester, which alsoincludes oral and written communication. Half of first yearstudents are found registered in the gross human anatomycourse, while the other students study histology and embr-y o l o g y. This study’s objective is to establish the existenceof a difference in achievement in the anatomy course afterhaving taken other morphological science courses.

For the study, regular students were included whowere registered in the anatomy course during the March-August 2004 semester. They agreed to participate in thesame and they answered a survey, a pre-test and a post-test. Students who dropped out of the course and thosewho did not take the tests and/or answer the survey wereeliminated from the study.

There is no relationship (r=0.19) between the type ofhigher middle education (public, private or technical pre-paratory school) and achievement in the human anatomycourse.

For second semester students who took histology andembryology in the previous semester, it was observed that89.64% of them credited at least one of the two subjects,and 67.24 % credited both of them, while those who didnot credit either of them made up 10.34 %. A weak posi-tive correlation (r=0.43) was found between the academicsituation (passing-failing) in the subjects of histology andembryology and their achievement (grade) in the humangross anatomy course. Students who passed histology andembryology did not necessarily have higher grades in theanatomy course, while those who did not pass either ofthem tended to have lower achievement. For that reason,the academic situation in histology and embryology cour-ses is not a factor for predicting achievement in the ana-tomy course.

P-8 E M B RY O - F E TA L D E V E L O P M E N T B YECHOGRAPHY.

Prados, J.1, Sabatel, R.M.1, Melguizo, C.2,Carrillo, E.1, Velez, C.2, Marchal, J.A.3, Bou-laiz, H.3, Rodriguez, F.3, Martinez, A.3, Fernán-dez, J.E.1, Caba, O.1, Alvarez, L.1, Aranega, A.1

1 Universidad de Granada. 2 Universidad de Almería.3 Universidad de Jaén.

The embryology is an important subject not only inthe student formation of Medicine and Surgery but in thatof any student in relation to Health Sciences. However,the embryology is sometimes hard to understand for thepupil causing a formative lack which makes the compre-hension difficult. The student feels unable to understandthe main happenings which take place during the embryodevelopment and also the base of the appearing of somecongenital defects. In the last two decades have appearedconsiderable advances in obstetric ultrasonography.Nowadays, besides the bi-dimensional transvaginal echo-graphy it is possible to obtain three-dimensional imagesand even what it has been call 4D images or 3D images inmovement. The clinical applications of ultrasound inclu-de confirmation of pregnancy and multiple gestation, esti-mation of gestational age, localization of placenta, moni-toring of fetal wellbeing and is also useful in prenataldiagnosis and fetal therapy. The first-trimester fetal echo-graphy is accurate in detecting major structural abnorma-lities. Prenatal surgical therapy can also be offered to fetu-ses with simple anatomic defects that have predictablydevastating developmental consequences. However,appropriate use of these new tools will not only provideinformation for better clinical assessment of fetal diseasebut also allows the student in an easier way to understandthe more important anatomic modifications that take placein this development period and it is also useful for theknowledge of the abnormal changes which may cause pat-hologic alterations.

P-9 A N ATO M I C A L DISSECTION FOR PRE-GRADE STUDENTS: EXPERIENCE A F -TER 12 YEARS OF P R O G R A M M E DC O U R S E S .

Barbosa, M., Pastor, J.F., De Paz, F.J., Gil,J.A., Barbosa E.

University of Valladolid.

In our university, in general, not every student canpractice anatomical dissection due to the low number ofcorpse donations. Thus, 12 years ago we began organizingstandardized dissection and anatomical techniques cour-ses during the month of July. This course is 50 hours long,distributed in 3 weeks, with classes during the morning,from 10:00 to 14:00. The number of students has variedfrom 20 to 32, depending on the number of the teacherscollaborating each year. On the years on in which thenumber of applications exceeded the number of vacanciesthe selection has been based on the marks obtained duringthe year on the subjects related to Anatomy and Embryo-logy. This courses were for free but nowadays it costs¤180 (this prize includes a dissection kit, rubber glovesfor every day and a diploma).

During this course the students either dissect an ana-tomical region or practice on different dissection techni-ques, as dyeing cerebral cuts, dyeing and transparentationon embryos or synthetic resin inclusion of anatomical pre-parations. The student is tutored all through the processesby a teacher who shows him or her how to use the medi-cal instruments and all the steps he or she has to follow inorder to correctly fulfil the work.



The purpose of these courses is multiple: 1) To intro-duce the interested students on the dissection techniques.2) To provide them with dissection material for the annualpractices. 3) To promote handling aptitudes, and to getused to the material and surgical techniques. 4) The com-pilation of photographic material for teaching and investi-gation purposes.

On the last day of the course the students answer a sur-vey about the development, the contents and the expe-rience during the course. The three most valued thingsare: 1) the relationship teacher-student, 2) the relationshipamong the students and 3) The increasing in knowledge.

The fact that the course did not have any other valuethat the personal experience and enrichment, on the firstyears, and the latter change to serve as official class hourson the last five years, has constituted a turning point, foron the first period the quality and interest of the studentswas obvious, while when the course implied the obtainingof recognizable hours of lessons, the attitude and qualityof the projects has gone down.

A very difficult factor to value is how the experienceon dissection can influence the latter tendency to choosesurgical specialities or, on the contrary, to make the stu-dent realize that those are not the most suitable techniquesfor him or her.

P-10 G R O S S - A N ATO M Y DISSECTIONS: ATOOL TOWARDS TO SELF-LEARNING.

Arroyo-Jiménez, M.M., Marcos, P., Martínez-Marcos, A., Artacho-Pérula, E., Insausti, R.

Universidad de Castilla-La Mancha, Albacete, Spain.

Tr a d i t i o n a l l y, anatomical teaching is based on lecturesand laboratory sessions, which include prossections, plasticmodels and radiological images. The students cope with thelearning of Human Anatomy mostly by means of memoris-tic techniques. There are different opinions about the vali-dity of dissections and/or prossections as good techniquesfor the learning of A n a t o m y. We think that the direct contactwith the cadaver provides and excellent resource for bothlearning and teaching of Human A n a t o m y. For this reason,in both first and second years of the Medical curriculum ofthe UCLM we carry out a student based learning methodo-l o g y, trying to increase time efficiency and the student’se ffort to learn. Students of Human Anatomy and Embrio-logy disciplines have the possibility to perform two dissec-tions in different anatomical regions without the teacher’shelp once finished their theoretical training. They must iden-tify by themselves the anatomical structures pointed in aguide at the beginning of this activity, helped by appropria-te books and atlases. When dissection is finished, the teacherevaluates it and answers the possible questions. We showhere the results of a test answered by the students after thedissection procedure, in order to demonstrate the importan-ce of such activities for Anatomy learning. In this work, weattempt to validate the efficiency of the student self-learningby means of these dissections, by comparing the resultsobtained for each dissection as well as the results obtainedin first and second years.

P-11 GENERAL ANATOMY TEACHING PRO-CESS FOR ODONTO L O G Y S T U D E N T SAT THE UNIVERSITY OF SEVILLE.

Arteta, D., Jiménez-Castellanos, J., Jiménez,A., Catalina, C., Carmona, A.

Departamento de Anatomía y Embriología Humana.Universidad de Sevilla.

Spanish Anatomical Society (SAE) has establishedgeneral standards for the teaching of General Anatomy inOdontology. Here, we report our experience in the Uni-versity of Seville where we made a study based on threeanonymous surveys answered by a group of students ofGeneral Anatomy along the academic year 2003-04. Wewanted to investigate several questions as: previous requi-rements with which the students arrive to our subject,their expectations before the beginning of the course, andthe degree of satisfaction reached upon ending it, amongothers. The surveys were accomplished at the beginning,in the middle and, finally, at the end of the course andbefore the final exam. In good accordance with the ans-wers given educational strategies were implemented toimprove the assimilation of General Anatomy by the stu-dents. To conclude, we made a topic review taking intoaccount the situation in other Spanish and European Uni-versities in order to suggest some proposals to achievethat the content of our present academic program could beadapted to the new deals that implies the European creditsystem, and to reach the most complete training and aca-demic formation of the future odontologists.

P-12 TEACHING CLINICAL A N ATO M Y TOSURGICAL INTERNS.

Llusá, M., Pacha, D., Carrera, A., Morro, M.R.,Leon, M., Forcada, P.

Departamento de Anatomía y Embriología Humana.Facultad de Medicina. Universidad de Barcelona.

We present our experience in the Department ofHuman Anatomy and Embriology of the University ofBarcelona in teaching clinical anatomy for surg i c a linterns.

Our teaching method consists in an introduction inclu-ding a theoretical revision and a demonstration of prosec-tion about the lesson to be given in each session. Most ofthe time of the course is dedicated to the anatomical dis-section performed by the interns, and at the same timespecialist doctors show them the relationship with the sur-gical approaches of the region. The interns are suppliedwith bibliography to induce them to look for informationabout surgical anatomy.

We also use complementary methods such as videos(Acland’s video atlas of human anatomy) and videos thatwe have made ourselves.

The course lasts 250 hours: 100 theory and 150 practice.

P-13 THIEL’S METHOD AS SELECTED TECH-NIQUE FOR TEACHING HUMAN SURGI-CALANATOMY.

Garcia-Martinez, V., Lopez-Sanchez, C.

Human Anatomy and Embryology, Faculty of Medi -cine, University of Extremadura, Badajoz. Spain.

In the last few years, many selective surgical approa-ches, mainly supported by minimally invasive surgical



techniques, have been developed under the knowledge ofa precise and detailed anatomy. Several studies on cada-veric specimens have shown to be very helpfully for sur-gery, and many others specialities (interventional radio-logy, endoscopy, oftalmology...); however, the traditionalfixation of cadavers by means of formaline solutions doesnot allow the realization of different experimental surgicaltechniques, i.e. the gas injection into the peritoneal cavityduring laparoscopic surgery. Attempting to get a betterpreservation of the anatomical structures in human cada-vers, we have developed a technique based in a modifica-tion of Thiel´s method. This technique showed somedisadvantages, as defficient central nervous system fixa-tion. The combination of intrarterial and intravenous fixa-tion, as well as the combination with solutions of higherformaline concentrations, allow a better preservation ofthe human cadavers, mainly foccused for teaching inhuman surgical anatomy.

P-14 THE PROFESSIONAL PROFILE OF THEPROFESSOR OF HUMAN A N ATO M YAND EMBRYOLOGY.

Lafuente, J.V.

Universidad del País Vasco/ Euskal Herriko Unibert -sitatea. Dpto. de Neurociencias.

The professional profiles of the teachers of HumanAnatomy in the different degrees and diploma courses inwhich it appears as a syllabus subject vary widely.

The “Bolonia 2010” objective has made the restructu-ring of further education a subject of media debate. Someaspects of the changes are so far-reaching that they havenot been allowed to transcend the walls of university.

An important part of the running of any project is theselection of the personnel who are going to start it up andimplement it. This makes the criteria used in the selectionof these personnel a key issue.

In this communication, we are going to present someideas which we hope will lead to a debate within the SAE.We would particularly like to highlight these two ideas:

1.- Scientific societies, like the SAE, are the rightforums in which to produce, through the process the mem-bers think fit, documents that set out the recommendedprofessional profile for the different types of Universityteacher of Human Anatomy and Embryology envisaged inlaw. Any kind of guide to the career would be of great useto young people who are thinking about working in ourfield. It would provide those who are beginning work inthe profession with, to coin a fashionable phrase, a “routemap” of their future career. The Society could provide thecriteria to define the “desirable profile” or the “minimumrequirements” of a University teacher of Human Anatomyand Embryology.

2.- The configuration of Human Anatomy and Embr-yology as a professional speciality, a medical/non-clini-cal speciality like others in the Health field. To achievethis, the first thing would be to agree on a “specialitysyllabus” which should contain the “list” of the things the“apprentice” would have to do, courses, training periodsin laboratories and even in hospitals (“if they learn toget-h e r, they will work together”). Access to an official trai-ning course could be made through an agreed series ofpreliminary studies. This training itinerary must be accre-dited and certified.

Systemic, Topographic and Clinical Anatomy

P-15 C O M PA R ATIVE A N ATO M Y O F T H EANTERIOR PORTION (PARS NERVINA)O F THE JUGULAR FORAMEN INHUMANS AND IN GREAT APES.

Potau, J.M.1, Bardina, X.2, Ciurana, N.1, Villal-ba, N.1

1 Universidad de Barcelona. 2 Escuela Universitariade Fisioterapia “Gimbernat”.

In the present study the relation between the area ofthe anterior portion of the jugular foramina and the esti-mated size of the pharynx was compared in humans andin three species of great apes (orang-outang, gorilla andchimpanzee). It is our aim to find anatomic differencesthat may be related with the different phonatory capabili-ties in the groups studied. The jugular foramen, that islocated between the occipital bone and the petrous portionof the temporal bone, is divided in an anterior and a pos-terior portion by a fibrous tract that runs between theintrajugular processes. The internal jugular vein runsthrough the posterior part as the continuation of the sig-moidal sinus. The glossopharyngeal, vagus and accessorynerves run through the anterior portion (pars nervina)together with the inferior petrosal sinus. As it is wellknown, the three mentioned nerves play a significant rolein the innervation of the phonatory organs like the larynx,the pharynx and the soft palate. Our results show thathumans have a larger area of the anterior portion of thejugular foramina in relation to the size of the pharynxcompared to great apes. No significant differences wereobserved among the three species of great apes studied.This finding could imply a greater relative size of theglossopharyngeal, vagus and accessory nerves in humans,that could be related to the higher speed and precision ofthe muscles of the larynx, the pharynx and the soft palatethat are needed for human speech. In addition, the relationbetween the area of the anterior portion of the jugularforamina and the size of the pharynx was studied in diffe-rent species of hominids, showing that the members of thegenus Australopithecus have values similar to great apes,while the members of the genus Homo have values simi-lar to modern humans.

P-17 SECTIONAL ANATOMY OF THE EAR.I N T R AT E M P O R A L S PACES. A P P L I C A-TION FOR THE UNDERSTANDING OFCAT AND NMR IMAGES.

Carretero, J., Blanco, E., Rubio, M., Sierra, E.,Riesco, J.M., Benito, J., Cambero, J., González,R., Rubio, L., Vázquez, R.

Department of Human Anatomy and Histology andLaboratory of Neuroendocrinology, Institute of Neu -rosciences of Castilla y León, University of Salaman -ca, Spain.

Understanding the elements forming the parts and thelimits of the three portions of the ear within the temporalbone is difficult owing to their complexity, size, and orien-tation, and because they are deep structures surrounded bybone, are hard to dissect, and are not always approachable.

New technologies for the clinical exploration of theear –nuclear magnetic resonance and computerised axial



tomography– allow these portions to be explored. Theresults obtained demand that clinicians must recycle theirknowledge about structures that may have been forgotten,poorly understood or even overlooked. This implies con-siderable difficulties in the interpretation of imagesbecause some physicians may not have clear concepts asregards the spatial organisation of the ear.

With the objective of fostering a better understandingof the structures and spaces of the intratemporal ear inundergraduate students and offering a review of this issuein life-long training programs, we have performed anexhaustive study on the identification of the intratemporalstructures and spaces of the ear, collecting multiple sagit-tal, horizontal and axial sections on the major axis of thepetrous part. We complete these with oblique axial andhorizontal sections, in many cases correlating them withCAT or NMR images similar to those used in clinicalpractice.

Part of this work is reflected in the poster, which iscompleted with a generous number of images summari-sing the study.

P-18 INTERNAL EAR BY RM (3D FIESTA).

Hernández-González, L.C.1, Montoya, J.2, Ló-pez-Muñiz, A .1, Martínez deVega, V2, Viaño, J.2

1 Universidad Oviedo. 2 Clínica Nª Sª del Rosario.Madrid.

The development of the new sequences in MagneticResonance (MR) allows establishing new protocols forthe study of the internal ear and the internal auditory canal(IAC). All the studies were made in patients who had cli-nical syntomatology: tinitus, hipoacusis... It was made inan equipment of MR Signa EXCITES 1.5TGE. The hi-resimages strongly harnessed of the internal ear in T2 provi-de an optimal resistance between the nerves of the faceacoustic package and the F.C.E. and between the bony andmembranous labyrinth that contains perilympha. T h eimportance of the sequence was shown 3D fiesta in thecribate and valuation of congenital and inflammatory pat-hology. The obtaining of images 3D with high involutionin T2 of the internal ear supposes a great advance in thestudy and evaluation of the pathology of the ear in thisregion. It allows a suitable valuation of the anatomicalstructures that compose it. It has the high sensitivity todiscard and to diagnose pathology. It is of short durationand it does not need the administration of intravenousresistance. High-resolution MR imaging of the inner earwith a heavily T2-weighted 3D sequence: 3D FIESTA hasbeen performed successfully at 1.5 T. Normally, the highsignal from the perilymphatic and endolymphatic spacesis homogeneous and images of sufficient technical qualitywere obtained to enable the recognition of the and one-half turns of the cochlea, similarly, we could visualize theposterior labyrinth with the vestibule and the semicircularcanals (lateral, superior, and posterior) joined by the cruscommune. This approach allowed examine the fluid chan-nels within the cochlea and separate demonstration ofscala vestibuli and scala tympani, this is of particular rele-vance in the planning of cochlear implantation, in whichdetailed preoperative assessment of cochlear morphologyis important. MR imaging also provides the ability todiagnose aplasia or hypoplasia of the vestibulocochlearand facial nerves. The cochlear nerve may be definedseparately from the superior and inferior vestibular and

from the facial nerves within the IAC. 3D FIESTA is alsouseful in the correct diagnosis of congenital pathologysuch as malformation of the semicircular canals (as thelateral semicircular canal is the last to form embryologi-cally, its malformation is one of the most common) isola-ted or in association with other inner ear anomalies: ves-tibular and utriculosaccular anomalies and cochleardysplasias.

P-19 NASAL HOLE (Foramina nasalia) VARIA-BILITY IN A POPULATION FROM CAS-TILLA Y LEÓN.

Pastor, J.F., De la Iglesia, M., De Paz, F.J., Gil,J.A., Barbosa, M., Delgado, J.M.

University of Valladolid.

The nasal holes (Foramina nasalia) can allow vesselsand/or nerves to go through. The holes of a vascular ori-gin allow small veins or arteries to go through them.These are branches of the angular vessels, which connectthe nasal cavities to the outside. If the holes have a ner-vous origin, they let terminal braches of the external nasalnerves, branches of the anterior ethmoidal nerves to gothrough, these nerves are going to innervate the skin onthe back and sides of the nose up to the very tip. There canbe one or more nasal nerves, and thus one or more nasalholes. When the holes are absent there is no vascular com-munication and the nerves have to pass between the infe-rior edge of the nasal bone and the lateral nose cartilage.The lack of references on osteology in the Spanish popu-lation makes it necessary for systematic studies to becarried out on these populations.

The nasal holes of 86 men and 55 women were stu-died. All of them with a known age (over 20 years) andcoming from Palencia and Valladolid. They all belong tothe osteological collection (665 crania) of the Museum ofAnatomy of the University of Valladolid. From the 141crania, a total of 268 nasal bones were studied. The dete-riorated and pathological ones were rejected.

The mean was 2 holes present, the same for men andfor women, being the range between 0 and 8 holes. Thenumber of holes found more frequently is 1 for bothsexes. The presence of only one hole is more frequentlyfound in left bones, being especially significant inwomen. The presence of more than one hole predomina-tes on the right side. The absence of holes has a similarfrequency for both sexes (8.9%), having the bilateralabsence a frequency of 2.1% (3 crania). No significantdifferences related to age have been found.

The number, arrangement and size of the nasal holesare intimately related to the degree of pre and postnataldevelopment of certain soft structures, as the vessels andthe nerves. The development of the vascular and nervouspatterns is going to be influenced by external factors,mainly during the postnatal period, factors which are verydifficult to control. Due to it, the variability of these nasalholes is considered to be a discontinuous, discrete, nonmetric, or epigenetic character.

There are not conspicuous sexual differences in thenasal region on humans; moreover, the nose has lost struc-tural and functional importance over the millennia, thesmell sense not being essential for us, the humans. On theother hand, the plastic and repairing surgery has beengiven a high profile during the last few years, being the



nose operations the most common ones. In these kinds ofsurgeries the surgeon has to know exactly the places werethe vascular-nervous pedicles are located.

The obtained results are consistent with the onesPublisher for Danish and Japanese populations.

P-20 A N ATOMIC DIFFERENCES IN CEN-T R A L C O R N E A L THICKNESS, A N T E-RIOR CHAMBER DEPTH, LENS THICK-NESS, VITREOUS CHAMBER DEPTH,AND AXIAL LENGTH BETWEEN NOR-MALAND DRY EYES.

Sanchis-Gimeno, J.A.1, Erades, D.2, Herrera,M.2, Alonso, L.3, Rahhal, M.S.3, Sánchez DelCampo, F.4, Martínez-Soriano, F.1

1 University of Valencia. 2 University of Alicante. 3 Rahhal Ophthalmology Clinic. 4University MiguelHernández, Elche.

OBJECTIVE: To quantify the anatomic differences incentral corneal thickness, anterior ocular chamber depth,lens thickness, vitreous chamber depth, and ocular axiallength between normal and dry eyes.

METHODS: The central corneal thickness, the ocularanterior chamber depth, the lens thickness, the vitreouschamber depth and the ocular axial length were measuredin 70 normal subjects and in 58 subjects with dry eye. Thecentral corneal thickness was measured with scanning-slitcorneal topography while the ocular anterior chamberdepth, the lens thickness, the vitreous chamber depth andthe ocular axial length were measured with applanationultrasound biometry.

RE S U LT S: The central corneal thickness was 0.558±0.30mm and 0.532±0.34 mm in normal and dry eyes respecti-vely (p<0.001). The mean ocular anterior chamber depthwas 3.17±0.23 mm and 2.93±0.35 mm in normal and dryeyes respectively (p=0.002). The lens thickness was4.49±0.42 mm in the dry eye patients and 4.71±0.32 mm inthe normal subjects (p=0.022). The vitreous chamber depthwas 16.75±1.75 mm and 15.54±1.34 mm in normal and dryeyes respectively (p=0.001). The ocular axial length was24.58±1.73 in normal subjects and 23.07±1.48 in dry eyesubjects (p<0.001).

CONCLUSIONS: The quantitative ocular anatomy valuesare lower in dry eye subjects.

P-21 C O R N E A L A N ATO M I C A L S T U D Y I NPATIENTS WITH BULLOUS KERATO-PAT H Y SUBJECTED TO STRUMALMICROPUNCTURE.

Zabaleta-Meri, M.1, Francés-Muñoz, E.2

1 Dept. Anatomía y Embriología Humana. Universi -dad de Valencia. 2 Servicio de Oftalmología. Hospi -tal Universitario “La Fe”.

The Bullous Keratopathy (BK) is one of the main indi-cations for penetrating keratoplasty. However, a long wai-ting time before receiving this treatment beside that thereis a group of patients in which this treatment is contrain-dicated has raised the development of alternative palliati-ve therapies that treat the secondary effects of the epithe-lial damage observed in BK, like corneal persistent

erosions. One of these therapies is the anterior strumalmicropuncture. The aim of our work was to study the ana-tomical changes induced by the anterior strumal micro-puncture on the cornea of patients with BK and to rese-arch its mechanism of action.

We performed the anterior strumal micropuncturetechnique in 8 patients with BK: six of them were pre-viously given a diagmosis of pseudophakic BK and 2 ofthem were phakic patients with secondary BK and endot-helial dystrophy of Fuchs. Patients answer a questionarywhere they evaluated the severity of their ocular pain andthey were subjected to penetrating keratoplasty in an ave-rage interval of eight months. In three of the cases we pro-ceeded with a fixation of the receptor button with 10%gluteraldehide, fixed in paraffin and dye with Hematoxili-ne-Eosine, then we studied subepithelial reaction andstrumal neurovascularization in each of the epithelia.

Patients experienced a significant and stable improve-ment at least during the eight-months of pursuit: 7 of thempresented a almost total reapplication of the corneal epit-helium and one patient presented a reduction in the num-ber of blisters. In this anatomopathological study of theserial slides of the corneal buttons we found differentdegrees of subepithelial fibrosis. In the places weremicroinjection was performed, we found ruptureson onthe Bowman’s membrane and strumal fibrosis that affec-ted the external third in each place of the puncture.

Strumal micropunctures have the advantage of decre-asing the risk of bacteria keratitis and the appearance ofcorneal neurovascularization, which is a known risk fac-tor of reject of penetrating keratoplasty.

The exact mechanism by which the epithelium sticksto the struma are still unknown.

P-22 A N A LYTIC FUNCTION OF T R I S O M I CCHILDREN’S PA L ATA L VA U LT S U R FA C E .

Panchón, E.1, Jornet, A.1, Panchón, A.1, San-chez, F.2

1 Universidad de Alicante. 2 Universidad Miguel Her -nández de Elche.

Metric studies have shown that lineal dimensions ofpalatal vaults are different in Down’s syndrome (DS) indi-viduals than in normal (N) controls. But, it is difficult tocompare these numerical results because different pointsof reference were used in order to define their longitudi-nal parameters and their measuring techniques were diffe-rent as well. So these conclusions were partially contro-versial with respect to palatal length and width. Also thereare papers that show specific shapes for Down Syndromepatients, as the one called gothic palate and “u” or “w”palatal shapes in children.

In a former work adult’s palate vault morphometry ofpatients with Down’s syndrome was studied and compa-red to the normal population. The purpose of this study isto determine how the craniofacial morphology of indivi-duals affected by the Down’s syndrome diverges fromhealthy Spanish people in a child’s population. In order toreach this purpose, the lineal dimensions (depth, widthand maximum height) were obtained from dental castsand the morphometric shapes of the palatal vaults weredetermined. We used a moiré topography method whichallows to found the lineal dimensions and the analyticalfunction, z=f(x,y), which is representative of palatal shapefor Spanish patients of trisomy 21.


37European Journal of Anatomy, 2 0 0 5 , vol. 9 (Suppl. 1)

Moire topography is used for quantitative determi-nation of palatal vaults shape of in Down’s syndrome(DS) patients and control normal (N) groups from Spa-nish people. 40 patients with trisomy 21 (20 males and20 females), aged from 9 to 11 years old, and 40 healthycontrols (20 males and 20 females), aged from 9 to 11years old, were studied. We found a characteristic pala-tal morphology associated to Down’s syndrome, withd i fferent ratios for the three dimensions in relation tonormal population.

P-23 I N F O R M ATIC A P P L I C ATION FOR PRO-CESSING AND GEOMETRIC V I S U A L I S A-TION OF A N ATO M I C A L - R A D I O L O G I-C A L IMAGES OBTAINED USINGD I F F E R E N T DIAGNOSTIC T E C H N I Q U E S .

Juanes, J.A.1, Prats, A .2, Riesco, J.M.1, Carre-ra, A .2, Potau, J.M.2, Velasco, M.J.3, V á z q u e z ,R .1

1 Universidad de Salamanca. Instituto de Neurocien -cias de Castilla y León. 2 Universidad de Barcelona.3 Hospital “Río Hortega”, Valladolid.

Amira is a powerful informatic tool for advancedvisualisation, data analysis and the geometric reconstruc-tion of anatomical structures, based on radiological ima-ges obtained using the customary diagnostic techniques,supporting DICOM (Digital Imaging and Communica -tions in Medicine) formats.

Here we offer a three-dimensional reconstruction ofthe skull from Computed Tomography sections.

The processing of 3D images is supported by power-ful automatic and interactive segmenting tools. T h ereconstruction of algorithms permits easy creation ofpolygonal models from segmented objects. It is possibleto generate volumetric meshes that are appropriate foradvanced simulations of finite elements.

The informatic application discussed here is able toexecute, almost in real time, volumetric representationswith large amounts of data: higher than 10 megabytes.The volumetric images thus generated can be combinedwith any type of polygonal representation.

Furthermore, Amira is able to compute anatomical-radiological sections of random orientation in 3D data,and it is possible to combine multiple sections.

Polygonal separation is another interesting feature ofAmira and can be used to adaptively reduce the number oftriangles in an exterior model.

Once the geometric image has been generated, it ispossible to practice different user-specified sections onthe plane and rotate the structure in any spatial plane. Thismeans that it is possible to visualise the whole anatomicalimage in an integrated fashion.

The tool is available for the PC and UNIX environ-ments and for PC stations based on Linux, permittingrapid visualisation and treatment of radiological images.

To conclude, Amira allows easy three-dimensionalreconstruction, providing innovative and potent algorithmsfrom the image processing and from the computational geo-m e t r y. There is no doubt that this potent informatic applica-tion will allow the productivity of radiological images to bee n h a n c e d .

P-24 CORRELATION BETWEEN THE RADIO-LOGICALANATOMY FINDS AND CLINI-CAL EXPLORATION IN THE LUMBARPAIN.

Marchal, J.A.1, Jiménez, I.2, Delgado-Marti-nez, A.D.1,3, Carrillo, E.4, Boulaiz, H.1, Rodrí-guez-Serrano, F.1, Martínez-Amat, A.1, Vélez,C.5, Prados, J.C.4, Melguizo, C.5, Álvarez, L.3,Aránega, A.4

1 Dept. Ciencias de la Salud, Universidad de Jaén. 2 Hospital Comarcal de Úbeda (Jaén). 3 Serv. DeOrtopedia, Complejo Hospitalario de Jaén. 4 Dept.Anatomía y Embriología Humana, Universidad deGranada. 5 Dept. Neurociencias y Ciencias de laSalud, Universidad de Almería.

The low back pain is a problem of health today thatoriginates a great number of consultations in primary care–is the second motive after the cold– and in urgencies.The family doctor has two methods to approach this pro-blem. The first one is the correct physical exploration thateast of a clear way to the reason that originates them,being able to discard of this form serious processes. Thesecond one, as complementary exploration, is the study ofanatomic parameters by X-ray photography, whichthough in the majority of the cases it is normal, in occa-sions there are detected alterations that orientate towardsthe reason of the clinic. These two explorations mustallow the doctor to handle the backache and to value deri-vation to the specialized attention for the accomplishmentof other tests.

We lead to end a patients’ random systematical sam-pling that they came to the Service of Urgencies with lum-bar pain, for four months. The number of quiet casesbelonged to 30. We gathered the following variables: theage, the sex, the personal precedents, the time of evolu-tion, the form of appearance and characteristics of thepain, the location and irradiation, and the activities thatwere aggravating it. We realize the physical explorationaccording to the guides of performance for primary careand, for the radiological exploration of the lumbosacralcolumn; two projections were realized with the measure-ment of ten parameters.

The results of the present study showed than the 65-y e a r-old major patients presented: osteoporosis(p=0,002), osteophytes (p=0,018; c2 test), and alterationsin the articular process (p=0,012 test c2 test). Possiblymore cases would present with lumbar spinal stenosis(p=0,073) and pedicle breaks (p=0,059). In them theradiological quality was the worst (p=0,046). The wides-pread pain might have minor presence of osteophytes(p=0,096). The low back pain of more than twelve weeksare associated with: osteoporosis (p=0,016), osteophytes(p=0,06) and process elements upset (p=0,008).

We conclude that though the accomplishment of X-rayphotography’s is not indispensable, we have found impor-tant relations between the clinical and the radiologicalinformation, which can be of great usefulness in the clini-cal daily practice.

P-25 C L I N I C A L A N ATO M Y APPLIED TOR E G I O N A L ANESTHETIC T E C H N I Q U E S .

Carrera, A., Sala-Blanch, X., Prats, A., Morro,R., Pacha, D., Forcada, P., Llusá, M.


38 European Journal of Anatomy, 2 0 0 5 , vol . 9 (Supp l. 1)

Departamento de Anatomía y Embriología Humana.Facultad de Medicina. Universidad de Barcelona .

Regional anaesthesia and plexus blockade are veryuseful in surgery on the extremities and have a lot ofadvantages for the patients. However, their use is not wideextended due to the high rate of failures because of thedifficulty of the percutaneous approach of the nerves.Despite the use of the neurostimulation, which help tolocalize the nerves, these techniques require a good know-ledge of the anatomical regions where the nerves passthrough.

In our department we have made some specific pre-parations in order to organize courses of techniques onregional anaesthesia of plexus and peripheral nerves ofthe extremities. These preparations are focused in theareas where the anesthetic approaches are performed, sothat, they permit to assay the different techniques andposteriorly verify where the tip of the needle is locali-zed. They are specially designed to be able to remove,from superficial to profound, the different anatomicalstructures which the needle must pass through. The ner-ves and vessels keep their anatomical disposition andr e l a t i o n s .

We have also made sections in special anatomical pla-nes which help understanding the direction of the approa-ches of the nerves. They are also useful in the correlationwith the ultrasound images, which have recently beenintroduced for the localization of the nerves.

During the course and to integrate the anatomical con-cepts with the functional and clinical aspects, there areanaesthesiologist and professors of anatomy who combi-ne their knowledge.

P-26 D I S TA L FOREARM TO U R N I Q U E TW I T HFOREARM BLOCK: A U S E F U L T E C H -NIQUE FOR LONG-LASTING HAND S U R G E RY.

M a rchal, J.A.1, Delgado-Martinez, A . D .1 , 2,Marchal, J.M.3, Carrillo, E.4, Martínez-Amat,A1, Rodríguez-Serrano, F.1, Boulaiz, H.1, Ará-nega, A.4

1Dept. Ciencias de la Salud, Universidad de Jaén.2Departamento de Cirugía Ortopédica, ComplejoHospitalario de Jaén. 3Dept. de Anestesia. ComplejoHospitalario de Jaén. 4Dept. de Anatomía y Embrio -logía, Universidad de Granada.

INTRODUCTION AND OBJECTIVES: Tourniquet has beenrecently placed at the forearm for hand surgery, but whenusing local anesthesia or peripheral blocks, patientsusually do not tolerate the tourniquet more than 20 minu-tes. To avoid this, and get a longer tourniquet tolerance, atechnique which combines nerve blocks at the distal fore-arm with the tourniquet placed at the same level has beendeveloped. The objective of this work is to test the use-fulness of this technique in Dupuytren fasciectomies, anusual long-lasting hand surgery.

MATERIAL AND METHODS: A complete distal forearmblock at 5 cms (3 finger breadths) proximal to the palmarwrist crease is performed with mepivacaine 2%. A 8-cmwide cuff is later set at two finger breadths proximal to thepalmar wrist crease, and inflated 75 mmHg above systo-lic blood pressure. 13 patients scheduled to hand surgerylasting between 20 and 60 minutes underwent this techni-

que (Dupuytren fasciectomy in all cases). Pain during sur-gery, quality of ischemia and complications during sur-gery and 6 months after surgery were registered.

RESULTS: VAS pain (0-10) was rated as 0.89 at the sur-gical site and 0.61 at the tourniquet. A bloodless field wasachieved in all cases. 2 cases required supplemental anest-hesia at the beginning of surgery. No nervous or vascularcomplications were found at follow-up.

CO N C L U S I O N: This is a simple, painless, safe anduseful technique for hand surg e r y. It is mainly indicatedfor surgeries of the hand lasting between 20 and 60minutes, when regional or general anesthesia are nots u i t a b l e .

P-27 THE ANTEROLATERAL THIGH FLAP:A N ATO M I C A L VA R I ATIONS OF T H EVASTUS LATERALIS NERVE.

Santana Majan, M.J.1, Felici, N.2, Sanchezdel Campo, F.1, Herrera, M.3, Terol, F. F.1,A l m e n a r, V.1

1 Dpto. de Histología y Anatomía Humana, Universi -dad Miguel Hernandez. 2 U.O. Chirurgia Plastica eRicostruttiva Azienda ospedaliera “S.Camillo – For -lanini” Roma, Italia. 3 Dept. Interuniversitario deOptica, Universidad de Alicante.

During the past 20 years, the neural anatomy of manyflaps has been investigated, although no extensive studieshave been reported yet on the anterolateral thigh (ALT )flap. The goal of this study was to describe the anatomi-cal relation between the vastus lateralis nerve and thedescending branch of lateral circumflex femoral arterysupplying the anterolateral thigh flap. One hundred andfourteen anterolateral thigh flaps were elevated in 23bilateral cadavers and 65 patients (three of them werebilateral) and the vastus lateralis nerve, descendingbranch of lateral circunflex femoral artery and the mainperforator of the flap were dissected. For this porpoisewe proposed a classification of motor branches into threetypes. Type 1 : The nerve is medial to the descendingbranch of lateral circumflex femoral artery and cross itfrom medial to lateral distally to the connection of mainp e r f o r a t o r. Type 2 : The nerve is medial to the descendingbranch of lateral circumflex femoral artery (LCFA) andcross it from medial to lateral proximal and superficial tothe connection of main perforator. Type 3 : The nerve ismedial to the descending branch of LCFA and cross itfrom medial to lateral proximal and deep to the connec-tion of main perforator.

MATERIAL AND METHODS: The flap dissections weremade with a skin incision at 1 cm medial to the mark ofintermuscular septum. The intermuscular septum was theline between the anterior superior iliac spine and the supe-rolateral border of the patella. The perforating arterieswere preoperative identified with a Doppler probe. Theidentification and dissection of perforating arteries wasperformed subfascially. The dissection of arteries wasmade under surgical loupe.

RESULTS: Our study shows that the type 1 was presentin a 55%, the type 2 was present in a 35% and the type 3was present in a 14% of the cases.

CONCLUSION: Variations between the localisation ofthe nerve to the perforating artery of the flap is high. Type2 pedicular flap is necessary to sectioned the nerve inorder to rotate the flap adequately.


39European Journal of Anatomy, 2 0 0 5 , vol. 9 (Suppl . 1)

P-28 ANATOMIC VARIATIONS OF CALCANE-AN ARTICULAR FACETS.

Úbeda García, F1, Fernández-Villacañas, M.2,Salmerón Martínez, E.1, Moreno Cascales, M.2,Doménech, G.2

1 Servicio de Cirugía Ortopédica y Traumatología.Hospital Universitario Morales Meseguer (Murcia).2 Departamento de Anatomía Humana y Psicobiolo -gía. Facultad de Medicina. Universidad de Murcia .

Knowledge of the anatomo-functional features of thedifferent articulations forming the ankle joint is necessaryto understand the fisiopathology, biomechanics and possi-ble surgical procedures on the ankle and hindfoot. Theexistence of multiple morphological variations of the lit-tle articular facets which participate in these joints struc-ture is particularly interesting in the composition of thetalocalcaneonavicular articulation.

We have studied 1056 calcaneus belongling to theH a m a n n - Todd osteology collection at the ClevelandMuseum of Natural History and 56 calcaneus belongingto the Anatomy Department at the Medical School ofMurcia University, in order to find significant differencesbetween different existing types of calcaneus, attending tothe articular facets variations. “Bunning and Barnett clas-sification of subtalar facet configuration” was used, dis-tinguishing four types of joints, depending on the facetsdisposition: joined or separated (types A, Transitional, Band C).

We found significant differences between types B andTransitional of both collections. No significant differen-ces were observed between he other types.

Significant differences found between Transitionaland B may be explained by measurement subjective fac-tors due to the erosion suffered by the bone along thetime. Does the existence of these morphological varia-tions question the enlargement surgery procedures of cal-caneus by osteotomy?. In feet: 1.-Flat Foot in children. 2.-valgus and abducted in adults, secondary to an insuffi-ciency of the posterior tibial-tendon.

REFERENCES:RAGAB, AA. et al. “Implications of subtalar joint anatomic

variation in calcaneal lengthening osteotomy” J Pediatric Orthop23:79-83, 2003.

GOLANO, Pau.; FARIÑAS, Oscar.; SÁENZ, Ivan. “The Anatomyof the Navicular and periarticular structures” Foot Ankle Clin NAm 9:1-23, 2004.

DE PALMA, L.; y Cols. “Anatomy and Embryology of thetalocalcaneal joint” Foot Ankle Surg 9-1:7-18, 2003.

P-29 S TATIC-DYNAMIC BEARING SYSTEMFOR LEG AND FOOT DISSECTION. ANEW DESIGN.

Úbeda García, F1, Moreno Cascales, M. 2, Sal-merón Martínez, E.1, Fernández-Vi l l a c a ñ a s ,M.2, Doménech, G.2

1 Servicio de Cirugía Ortopédica y Traumatología.Hospital Universitario Morales Meseguer (Murcia).2 Departamento de Anatomía Humana y Psicobiolo -gía. Facultad de Medicina. Universidad de Murcia .

At the Dissection Rooms of Human Anatomy Univer-sitary Departments there are a wide spread kind of bearingsystems designed for lower limbs from cadaver. The pur-pose of such designs is fix, as well as possible, the sample

to be dissected in order to get the best accuracy and com-fort during the dissection.

METHOD:We show the design and manufacturing pro-cess of a bearing-system for lower limb that was develo-ped for the “I Curso Teórico-Práctico de Anatomía y Ciru-gía de Pie y Tobillo”, hold in the “Universidad de Murcia”in January, 2005. A) The technical survey and the assem-ble of parts are showed. B) The practical performancesare, also, showed.

CONCLUSIONS: The bearing system designed has beena great helpful tool to do the dissection and to get accessto the surgical pathways of foot.

b) The bearing system has strength, ergonomical cha-racteristics and versatility.

P-30 COMPARING FOUR DIFFERENTPROXI-M A L S C I ATIC APPROACHES FORANESTHESIA IN PEDIATRIC PATIENTS.A PRELIMINARY STUDY

Doménech, P.1, Roqués, V.1, Moreno Cascales,M.2, Hernández, F.1, Hernando, J.1, Fernández-Villacañas, M.2, Doménech, G.2, Castaño, I.1

1 Servicio de Anestesia y Reanimación. Hospital Uni -versitario Virgen de la Arrixaca (Murcia). 2 Departa -mento de Anatomía de la Universidad de Murcia.

The sciatic nerve block provides optimal surgicalanesthesia for foot and ankle surgery offering significantadvantages in terms of postoperative analgesia. The pro-ximal sciatic nerve approaches for anesthesia in pediatricpatients are at the moment a strict impose of the approa-ches previously described in the adult patient. No speci-fics sciatic approaches has been reported in the pediatricpopulation. The aim of our study is to compare four diffe-rent adult approaches in children undergoing foot andankle surgery and over lower-extremity of three differentnew born cadavers.

METHODS: The data from 44 sciatic punctures of chil-dren between 6 months and 11 years, 6-52 kg of weightand 65-155 cm of height, scheduled for ankle or foot sur-gery are reported. All sciatic nerve blocks were performedusing a nerve stimulator. After induction of general anest-hesia the children was positioned in lateral modified sim’sposition and direction of the needle was inserted perpen-dicular to cutaneal plane in all cases. 12 Labat’s classicalapproaches (L) were performed, based on the bony rela-tionship of the posterior superior iliac spine and the grea-ter trochanter; 12 Winnie modification of Labat’s classicalapproaches (W), adding in an additional landmark, thesacral hiatus; 10 Casal’s posterior sciatic approaches,based on the relationship of posterior iliac spine and thesacral hiatus; and 10 Rucci’s sciatic approaches, adding toall of the foremencioned references the ischial tuberosity.Presence or absence of muscular response, and details ofthe motor response, intensity of neuroestimulation, depthof needle insertion, motor block and success of the tech-nique were evaluated. We consider valid motor responsesthose distal over the foot, and invalid those proximal overthe posterior aspect of the thigh and gluteus maximus. Atthe same time we evaluate the same approaches over 6different new born cadavers in the university of our city.Distance from the needle to the nerve, and depth of theinsertion were registered.

RESULTS: One tibial motor response was obtained in L,five in W, one in C and five in R. One peroneal response



in L, two in C and one in R. Only in one case combinedmotor response of tibial and peroneal was obtained in R.The rest of approaches obtained proximal motor respon-ses or was absent. The depth of insertion of the needle was5 ± 2 cm, 0,18 mA the minimal intensity of stimulationand 0,5 mA the máximum. Evaluating the same 4 diffe-rent approaches in new born cadavers, only the Rucci‘sapproach inserted the needle over the sciatic nerve in allcases, And Winnie’s approach in all but one.

CONCLUSION: This study shows, in instance, that theRucci’s approach seems to be the most clinically success-ful approach and less needle depth must be required in thepediatric patient. The same results were confirmed incadaver section. Perhaps, this approach uses a very largenumber of anatomical landmarks sometimes difficult toidentify. The Labat’s classical approach modified by Win-nie shows similar success with bony landmarks easy toidentify. The depth of insertion of the needle was 5± 2 cm,been necessary the use of 50-100 mm isolated needles forall approaches.

P-31 PEDICLED GRAFT O F THE NERV U SCOMMUNICANS FIBULARIS. A N ATO-MICAL DESCRIPTION AND CLINICALAPPLICATION.

Carrera, A., Llusá, M., Pacha, D., Soldado, F.,Mendez, A.

Departamento de Anatomía y Embriología Humana.Facultad de Medicina. Universidad de Barcelona.

End-to-end nerve repair using autografts is a standarttechnique in microsurg e r y. The environment around thenerve graft site is important. Scar tissue should be avoid asmuch as possible to provide a good blood supply to thegraft site from healthy tissues, but this is not always possi-ble. In some specific cases it is useful to employ vascurari-zed nerve grafts. The technique is not easy and frequentlyis more aggressive than the standart free sural nerve graft.We present and anatomical description of the nervus com-municans fibularis around the posterolateral aspect of theknee and its microvascularization. Besides, we describe thes u rgical technique to use this nerve as a pedicled flap inorder to improve the vascularization and regeneration of thenerve. Finally our clinical experience with this technique infibular nerve lesions around the knee will be exposed.

P-32 SPECIFIC PATELLAR DISEASES. ANA-TOMOCLINICALBASES.

Spínola-Guirao, D.1, Guirao-Piñeyro, M.2,Guirao, M.2

1 Student of Physiotherapy (3rd year). San Antonio Ca-tholic University, Murcia. 2 D e p a rtment of A n a t o m yand Human Embry o l o g y, University of Granada.

An in-depth study was first conducted on the anatomyof the patella and its relationship with the different struc-tures that form the knee, as a basis for its clinical unders-tanding. Diseases that solely involve the patella were thendescribed, without taking into account involvement of thepatella by secondary knee diseases.

After consulting the literature, these diseases wereclassified into four groups: patellar fracture, patellar

luxation, recurrent patellar luxation and patellar chondro-malacia. For each group, an analysis was undertaken ofthe production mechanisms, predisposing factors, clini-cal symptoms and treatment, with special emphasis onp h y s i o t h e r a p y.

The following conclusions were drawn from theseanalyses:

1.- The patella is a bone exposed to a large number ofdegenerative and traumatic diseases.

2.- There is a wide consensus among different aut-hors except on the surgical approach to recurrent pate-llar luxation.

3.- All authors agree that a correct selection of surgi-cal treatment is essential for a good recovery.

4.- A wide variety of techniques are available.5.- The most important conclusion from the physiot-

herapeutic standpoint is that the intervention undergoneby the patient must be known before manipulations areperformed.

P-33 APPROXIMATION TO THE EVOLUTIVEPROCESS OF ANTERIOR CRUCIAT EL I G A M E N T A U TO G R A F T R E C O N S-TRUCTION THROUGH EMG.

P é rez, F. J .1, Villaplana, L.A.2, Serra, M.P. ,López, L.2, Igual, C.2

1 Departamento de Anatomía y Embriología Humanade la Universitat de València. 2 Departamento deFisioterapia de la Universitat de València.

IN T R O D U C T I O NA N D OB J E C T I V E S:The electromyographerof surface is a device that gathers the electrical activity thatis generated by the muscle. Its function is the neurophysio-logical evaluation of the activity neuromuscular.

The objective of our study is to value the increase ofthe potential of the action as a reflex of the improvementin the ability of contract of the muscle semitendinous andintern vast, after the application of an early protocol ofphysiotherapy in patients which are operated of a tornligament.

MATERIAL AND METHODS: The sample has been 8sportsmen with torn anterior cruciated ligament. An elec-tromyographic study was carried out in both inferiormembers of the muscle semitendinous, registered in theone canal, and of the intern vast muscle in the 2 canal. Ona first moment, in the injured member, we gather the acti-vity of both muscles in the maximum isometric contrac-tion of each one of them (stage 1). On a second moment,the activity of this muscles infunction walking along acorridor of 12 metres was being gathered. Finally, werepeat the process with the healthy muscle. The study wascarried out at 3 and 6 month after the operation.

RESULTS: The increase of the difference of potential, inmicrovolts, which is produced in the injured semitendi-nous muscle at 3 and 6 month after intervention of theligaments of anterior cruciated ligament, is of 12%. In thecase of the intern vast muscle, the increase of activity is of35%.

CO N C L U S I O N: The rapid protocol of physiotherapyproduces an increase of the muscular electrical activitywhich is measured in microvolts, objectived in the elec-tromyographer of surface, therefore, it proves a recupe-ration in the semitendinous muscle and in the intern vastmuscle in patients which are operated of a ligamento p e r a t i o n .



BIBLIOGRAPHY.PRESTON D, SAPHIRO B. Electromyography and neuromus-

cular disorders clinical-electro physiologic correlation. Boston:Ed. Butter Work Hinemann, 1990 (5): 170-171.

EDWARD F, DELAGI M, PEROTTO A. Anatomic guide of elec-tromyographer, 2ª Ed. USA: Springfield Illinois, 1981:195, 186.

SH E L B O U R N E KD, GR AY T. Anterior cruvciate ligamentreconstrucion and rehabilitation with autogenus patellar tendongraft followed by accelerated rehabilitation. A two to nine-yearfolloup. Am J Sports Med, 1997, 25:786-795.

P-34 AZYGOS LOBE. INCIDENCE A N DCHEST X-RAY CORRELATION.

Navia, P.1, Arráez, L.A.2

1 Hospital Ramón y Cajal, Madrid. 2 UniversidadComplutense de Madrid.

Abnormal fissures and lobes of the lungs are commonand usually insignificant. The azygos lobe consists of theupper right lung developing around the azygos vein. Itbegins during fetal life when the apical bronchus growssuperiorly medial to the arch of the azygos vein instead oflateral to it. As a result, the azygos vein comes to finish atthe bottom of a deep fissure in the superior lobe of theright lung, dragging along with it the parietal and visceralpleura; the vein remains in the substance of the lungs, butas the upper lobe develops inferiorly to superiorly, a dou-ble fold of visceral pleura (two layers of visceral pleuraand two layers of parietal pleura) develops. The fourlayers of pleura are then known as the azygos fissure, andthe lung tissue separated from the rest of the lung isknown as the azygos lobe. The azygos vein may simulatea pulmonary mass that often alarms physicians not fami-liar with its appearance. We have reviewed 1,000 PA chestx-ray films founding 6 cases of azygos lobe. The chest x-ray films checked owned to inpatients and outpatients ofthe Ramón y Cajal Hospital in Madrid.

P-35 A N ATOMIC VA R I ATIONS OF T H ES U P R A A O RTIC BODIES. STUDY V I AANGIO-TC AND ANGIO-RM.

Aparicio Bellver, L.1, Mata Escolano, F2, Mar-tínez Sanjuan, V.2, Casas Roman, E.1. SanchísGimeno, J.A.1

1 D e p a rtamento de Anatomía y Embriología Humanas.Universidad de Valencia. 2 E resa. Imagen Diagnóstica.

In the current work we have achieved a valuation ofthe arrangement and morphology of the supra aorticbodies, using radiological methods of high technology,and comparing the results, with the ones observed in theclassic anatomic literature, obtained by traditional anato-mic dissection.

Tables and graphic representations of the frequency ofapparition of the different variations of the supra aorticbodies in the radiological studies are included, and also acomparative study with the results of authors like Thom-son Nizankosky or Sobota.

MATERIALAND METHOD:Population:900 individuals 400 females and 500 males studied by

Angio-TC and Angio-Rm, valuating the disposition of thesupra aortic bodies.

Machines used:RN Sonata SIEMENS

Signa 1,5 and LX GETC Light speed ultra GERESULTSAND CONCLUSIONS:– The study achieved confirms that the results obtai-

ned by radiological techniques, are the same as the onesachieved by the classic dissection.

– The “typical” display of the supra aortic bodies is theone described classically as three bodies TBCD, ACI, ASIwith a frequency superior to 80%.

– The mayor part of the variations respond to modifi-cations of the left side of the aortic arch, being the TBDCthe most stable.

–Variations can be observed depending on the numberof bodies from 2 to 6, but there are also variations depen-ding on the type of body studied.

P-36 COMPARATIVE STUDY OFTHE ANATO-MIC VARIATIONS OF THE SUPRA AOR-TIC BODIES IN THE MASCULINE ANDFEMENINE POPULATION EVALUATEDVIAANGIO-TC AND ANGIO-RM.

Mata Escolano, F1, Martínez Sanjuan, V.1, Apa-ricio Bellver, L.2, Casas Roman, E.2, SanchísGimeno, J.A.2

1 Eresa. Imagen Diagnóstica. 2 Departamento deAnatomía y Embriología Humanas. Universidad deValencia.

In the current work we have achieved a comparativestudy of the frequency of appearance of the anatomicvariations of the supra aortic bodies in two populations,one of 500 men and another of 400 women, using radio-logical techniques of high technology like the Angio-Rmand the Angio-TC.

Tables of the frequency of apparition of the differentvariations of the supra aortic bodies in the radiologicalstudies of both populations are included and also a com-parative study and statistic valuation, by the T method ofStudent, of both populations.

MATERIALAND METHOD

Population:900 individuals 400 females and 500 males studied by

Angio-TC and Angio-Rm, valuating the disposition of thesupra aortic bodies.

Machines used:RN Sonata SIEMENS

Signa 1,5 and LX GETC Light speed ultra GERESULTSAND CONCLUSIONS

The study achieved confirms by the application of thestatistic T method of Student that “with a value >0.05, thatthere are no significant statistic differences between theresults obtained in both populations.

P-37 ANATOMIC APPROACH TO THE BARO-REFLEX SYSTEM: CONTROL OF HIGHBLOOD PRESSURE.

Arráez Aybar, L.A.1, López-Vidriero Tejedor,E.2, López-Vidriero Abelló, E.2


42 European Journal of Anatomy, 2 0 0 5 , vo l . 9 (Suppl. 1)

1 Dpto Anatomía y Embriología Humana II. FacultadMedicina. UCM. 2 Unidad de Hipertensión. HospitalGeneral Universitario Gregorio Marañón.

Since the work of Hering and De Castro in the begin-ning of the 20th century, there have been little advances inanatomy and physiology of the high blood pressure sys-tem wich is the main cardiovascular risk factor. In thiswork we try to sintetize and update the knowledge aboutthis complex system.

We have search in old references, phylosofal doctora-te thesis and pubmed bibliography using the key words:sinus caroticum, hering nerve, high blood pressure, baro-reflex.

The baroreflex arch begins at the sinus caroticum(mechanoreceptors) located in carotid bifurcation. It hastwo types of receptors. The afferent information runs upto the brain by the first extracraneal root of glosopharin-gey nerve (nerve of Hering). These afferences are integra-ted at the nucleus of tractus solitarius at the dorsal medu-lla with the suprasegmentus information. There areconections with the C1 area of ventral rostral nuclei of themedulla (vascular tone pacemaker) just in front of nucleusambiguous. From C1 leave efferences to the intermedio-lateral horn of spinal cord, from where there leave amieli-nic fibers to the blood vessels and suprarenal glands.There is also another efference from nucleus retrofacialisat the medulla to the heart via vagus nerve (Xcp).

This system works detecting high blood pressure atthe baroreceptors of carotid bifurcation and buffering it bydecreasing blood vessels tone and heart rate.

The baroreflex arch is a complex system wich is has avery important role in the control of high blood pressure.Further knowledge is needed to better understanding ofit´s anatomy and physiology.

P-38 RELATION BETWEEN OCULAR MOR-PHOMETRY AND INTRAOCULAR PRES-SURE.

Muñoz, M.A.1, Sanz, J.C.2, González, F.3

1 Dept. Anatomía y Embriología Humana I; E.U.Óptica; Universidad Complutense de Madrid. 2 Dep.Óptica; E.U. Óptica; Universidad Complutense deMadrid. 3 Clínica Universitaria de Optometría; E.U.Óptica; Universidad Complutense de Madrid

The measure of the intraocular pressure (IOP) is fun-damental in the clinical practice and basing on her it iscarried out decisive diagnoses in the valuation and the tre-atment of important ocular pathologies for what it isimportant a precise determination of their value. Theyhave been carried out many studies in order to determinethe factors or parameters oculars that could act like ele-ments that influence in the variation of the values of theintraocular pressure. In this work we have studied therelationship between the intraocular pressure and the ocu-lar morphometrics values such like the axial length,thickness of lens, depth of the anterior chamber, vitreousbody and the corneal radius. So, we have analyzed thevalues of the intraocular pressure obtained by means nocontact tonometry and the biometrics values obtained bymeans of a ultrascan biometer and in different refractivegroups. This way we studied great part of the ocular para-meters that are related, in order to determine the opticaland physiological properties of the ocular system and to

determine the paper that they play in the variations of theintraocular pressure. We have checked in our results that,in groups refractive of middle and moderate myopia, theintraocular pressure is related with the increase of the cor-neal radius (r= 0.38; p= 0.0001). However, this relations-hip doesn’t occur in the rest of the refractive consideredgroups (hyperopia, emmetropia and low myopia). Neitherrelationship between the pressure exists neither with therefractive error neither the axial length in spite of the factthat these last two parameters yes they are related to eachother. Therefore, the relationship between the value of theocular pressure and the corneal radius in the middle andmoderate myopias it seems to be consequence of thestructure biomechanics of the cornea. It doesn’t seem thatthe rest of studied factors have a significant influence,such like the increase of the axial length or the refractiveerror. We believe therefore, that it would be necessary tokeep in mind these results in the measures of the intrao-cular pressure.

P-39 CLINICAL SIGNIFICANCE OF OBTURA-TOR VESSEL VARIATIONS.

Bernal-Mañas, C.M., González-Sequeros, O.,Fernández-Villacañas, M., Moreno-Alarcón, C.

Department of Human Anatomy, School of Medicine.University of Murcia. Spain.

Abdominal aortic bifurcation is found usually at L2-L3, originating more distally both internal and externaliliac arteries. The obturator artery branches from theanterior stem of the internal iliac artery, but occasio-nally branches from another stem or from the externaliliac artery.

The number of variations of Obturator artery origin,increases from 11.11% to 69%, according to different aut-hors, and the origin from the external iliac artery is about2.4%. There is no enough information about how thevariant obturator artery divides after passing the obturatorcanal. Different studies have been done about terminalbranches of external or internal iliac artery but there are afew data in the case of veins, that usually finished in theinternal iliac vein. The existence of an abnormal obturatorartery does not involve an abnormal obturator vein or viceversa.

In the case we show, both obturator artery as obtu-rator vein branches, bilaterally, from iliac vessels.Obturator artery branches from the inferior epigastricartery and walk about 1/3 inner face of pubic arch andsupplies the muscles obturator internus and rectusabdominis but after passing the obturator canal theobturator artery divides, as usual, into the anteriorbranch to the adductor muscles and the posterior branchto the hip joint.

Because of the frequency of obturator vessels varia-tions, we think is very important to know its anatomicalrelations, not much referenced in textbooks of anatomy,respect to vessels or nerves such as inferior epigastric ves-sels, obturator nerve, inguinal ligament or femoral ves-sels. This anatomical relations are reviewed in severalmedical actuations on pubic or inguinal region, such asanterior surgical approach of the hip joint, pelvis fracture,repairing of femoral, inguinal and obturator hernias, linfa-denectomy of common iliac artery or external iliac artery,etc.



P-40 CONSEQUENCES OF REFLUX ON THESTRUCTURE AND THE CYTO S K E L E-TAL PROTEINS OF VARICOSE SAPHE-NOUS VEINS.

Mompeó, B., Ortega, F., Pérez, L.

Departamento de Morfología, Universidad de LasPalmas, Gran Canaria, Spain.

OBJECTIVES: To know if the presence of reflux in dif-ferent segments of the great saphenous vein producesmodifications in the structure, in the micro-vasculariza-tion or in the expression of cytoskeletal proteins of thevenous wall.

MATERIAL AND METHODS: 42 sections of sahenousveins, obtained from 20 patients undergoing surgery forvaricosities, were used in the study. Sectors with and wit-hout reflux were selected. To determine the sectors withreflux, the patients were under an eccodopler study befo-re surgery. It was possible to locate the levels of venoussufficiency and insufficiency with the system. The sec-tions obtained were processes for Weigert stain andimmunohistological determination of von Willebrand fac-tor, Actin and Desmin. The structure of the wall, the pre-sence and distribution of cytoskeletal proteins and microvessels were studied.

RESULTS: The sections with and without insufficiencydid not show great differences in the same patient. Thedifferences found were restricted to zones where therewas a dilation of the venous wall. In those places of dila-tion there were decreased internal layers of smooth mus-cle cells when they were compared with other zones in thesame section. The vasa-vasorum were located up to thecircular smooth muscle cells in the media layer in thecompetent segments. When there was insufficiency, thevessels were up to the intimal layer. Only in the dilationzones there was a decrease in the desmin protein, whichaffected the smooth muscle cells of internal and circularlayers.

CONCLUSIONS: In varicose veins there are structuralmodifications in the venous wall before reflux. After thereflux is produced, there are a lost of smooth muscle cellswhich belong to the layers nearer the venous lumen in theplaces with dilation.

P-41 THE RISK OF INJURY TO POSTERIORCIRCUMFLEX HUMERAL A RT E RYVA R I ATION. CLINICAL C O N S E Q U E N-CES.

Miguel, M. , Cuní, L., Buxeda, M., Bosch, A.,González, L., Aldirra, S., de Anta, J.M., Göt-zens, V.

Area de Anatomía y Embriología Humana (Campusde Bellvitge). Facultad de Medicina. Universidad deBarcelona.

IN T R O D U C T I O N: The risk of injuring important anato-mic structures of the proximal part of the humerus ispossible in different techniques used. If these structurespresent an anatomic variation the risk could increase. Inthe shoulder the posterior circumflex humeral artery wasan important artery. It supplied the posterior and middleparts of the deltoid muscle. Normally, this artery arisesfrom the lateral border of the third part of axillary’sa r t e r y. The aim of the study is present the variation of

posterior circumflex humeral artery for its clinical ands u rgical implications.

MATERIAL AND METHODS: In a woman’s dissectedcadaver we observed an unusual course of the posteriorcircumflex humeral artery.

RE S U LT S: In the woman’s dissection the axillar nerveruns in the quadrilateral space but the posterior circum-flex humeral right artery arises under the teres majormuscle, medial to the long head of the triceps brachialm u s c l e .

CONCLUSION: The course of the posterior circumflexhumeral artery is important in order to prevent their dama-ge in case of surgery involving this area or to correctothers pathologies as for examples its compression.

Biomechanics

P-44 DEVELOPMENT AND ASSESSMENT OFA FINITE ELEMENT MODEL OF THE C3ARTICULAR PROCESS.

Lapuebla, A.1, Herrera, M.2, Cervera, J.1, Jimé-nez, A.1

1 Departamento de Mecánica de los Medios Conti -nuos y Teoría de Estructuras. Universidad Politécni -ca de Valencia. 2 Departamento Interuniversitario deÓptica, sección de Alicante. Laboratorio de Anato -mía, Biomecánica y Embriología Humanas. Univer -sidad de Alicante.

Articular processes (AP) of upper and middle cervicalcolumn have been involved in load weight-bearing func-tions. Currently, finite element (FE) analyses are used toassess the stress and strains upon bones. The aim of thisstudy is to assess the role of cervical AP in supportingloads using a FE model to whom different load cases wereapplied. For this purpose, a highly-accurated FE model ofthe AP from the C3 vertebra was developed. The modelwas analysed under various load cases and boundary con-ditions simulating the load transmission in the C2/C3 jointin an effort to understand stress transmission in the trabe-cular bone tissue up to the cortical walls of the AP.

Anatomic data for the FE mesh geometry were acqui-red from a 25-years-old volunteer. C3 vertebra was scan-ned using CT scan at slice intervals of 1.5 mm; twenty-one consecutive scans were taken. The cross-sectionalshapes of the transverse slices were digitised and stackedautomatically to obtain a 3-D model of the C3 vertebra.AP was isolated and submitted to commercial FE softwa-re MSC. NASTRAN 2004 R2. The model used 4188 4-noded thetraedra elements with 8210 nodes. Linear staticmaterials were considered for bone. Young moduli valueswere 10000 and 450 MPa for cortical and trabecular bonerespectively. Poisson’s ratio was 0.41 for both.

A normal force was applied upon the whole articularsurface. Even the boundary conditions designed, a zone ofhigh stresses corresponding to the anterior wall of thesuperior articular process (SAP) was observed. When anuniaxial force was applied on the anterior one-third of thearticular surface (AS), high peaks were recorded in theanterior wall of the SAP. On the other hand, uniaxial for-ces applied on the posterior one-third of the AS show astress distribution in the posterior zone of SAP, inferiorarticular process and neural arch.

The results of the study give experimental support to adifferential stress transmission within the C3 AP, depen-


44 European Journal of Anatomy, 2 0 0 5 , vol . 9 (Supp l. 1)

ding of the joint movements executed. Some evidence isgiven that abnormal loads exerted upon the SAPcould ini-tiate the progression to osteoarthritis of the joint surfaces.

P-45 GAINS IN ISOKINETIC MUSCLESTRENGTH IN THE ELDERLY FOLLO-WING SIX-MONTHS OF POWER TRAI-NING.

Slocker, A., Gómez Martín, E., Carrascosa, J.,Aguado, S., Gómez Pellico, L.

Universidad de Alcalá.

The increase in the elderly population in recent deca-des has brought with it a significant increase in the eco-nomic and health care demands placed on the SpanishHealth System. One of the most frequent conditions inthis age group which is caused by the deterioration of thelocomotor system is gonarthritis. We thought that preven-ting an increase in this condition would improve the qua-lity of life of the subjects and therefore decrease the needfor medical assistance among this population.

We therefore conducted a study of the muscularstrength provided by the flexor and extensor muscles ofthe knee joint with isokinetic muscular dynamometry in ahealthy population of 14 men and 26 women between theages of 52 and 75. The study was conducted before andafter a six-month power training period. The variable inpeak torque in the right knee was analysed at speeds of60º/s and 120º/s. Our results showed significant increasesin the peak torque in both sexes and at both speeds in bothflexor and extensor muscles.

The conclusion reached is that muscular strength trai-ning leads to significant increases in the strength of theknee joint and therefore improves quality of life in theelderly.

P-46 THE BEHAVIOUR OFISOKINETIC MUS-CULAR STRENGTH IN THE ELBOWJOINT IN AN ELDERLY POPULATIONSAMPLE FOLLOWING PHYSICALTRAI-NING.

Carrascosa, J., Slocker, A., Gómez Martín, E.,Aguado, S., Gómez Pellico, L.


Manual tasks among the elderly are indispensablefrom both a physical and psychological point of view toprevent disabilities which can deteriorate the quality oflife and place a strain on the health care system. Thesemanual tasks are only possible if the elbow muscle worksproperly. We conducted a study of the flexor-extensormuscles of the elbow both before and after a six-monthtraining period of using isokinetic dynamometry (biodex).The sample was composed of a population of 40 healthysubjects (14 men and 26 women) between the ages of 52and 75 on whom functional evaluations were conductedusing isokinetic muscular dynamometry.

The study had a rigorous isokinetic protocol for theelbow joint, with shoulder alignment with 30% abductionand 45% flexion. The peak torque variables were analysedand the whole work at speeds of 60º/s and 120º/s for eachextension and flexion movement.

The results showed increases in both peak torque andtotal work in the extensor muscle group of the elbow joint,although these results could not be demonstrated in theflexor muscle group. We concluded that power trainingexercises in the elderly are beneficial to this population,clearly improving both physical and mental aspects.

P-47 INCREASE IN THE ARTICULAR TRA-JECTORY OFTHE KNEE FOLLOWING APERIOD OF PHYSICAL POWER TRAI-NING IN ELDERLY WOMEN.

Gómez Martín, E., Slocker, A., Carrascosa, J.,Aguado, S., Gómez Pellico, L.


The debilitation of the knee joint with age is wellknown. Consequently, we thought that by specificallytraining the strength provided by the knee’s flexor-exten-sor muscles would improve the articular position at thetime of maximum mechanical solicitation, thus avoidingunnecessary overloading of certain articular points. Wetherefore conducted a study of the articular position at thetime of maximum force on the knees of 26 healthy womenbetween the ages of 52 and 75 using isokinetic dynamo-metry before and after a six-month period of physicalpower training.

This study statistically demonstrates that the peak tor-que angle is smaller 6 months after the power training, butonly during extension. In other words, peak torque appearsduring the articular trajectory, which therefore translatesinto an improvement of the speed and quality of the mus-cular contraction of the knee in the extensor musculargroup in women. These results could not be statisticallydemonstrated during flexion, perhaps because it is the wea-kest muscular group. In conclusion, the improvement of thearticular position of the knee is accompanied by a signifi-cant increase in articular equilibrium and stability, decrea-sing reaction time and thus avoiding joint injuries and falls.

P-48 STRESS PATTERN IN CHILD´S FEETUNDER LOADING.

Panchón, E.1, Sánchez, F.2, Panchón, A .1,Herrera Lara, M.1

1 Universidad de Alicante. 2 Universidad MiguelHernández de Elche .

In the bibliography there are not many studies abouttransmission of forces through child’s feet that deal withthe use of three-dimensional models in which the dimen-sions used are in line with a half-real model obtained froma children sample.

The true points of stress and the way the compressionforces affect to the constituent elements of the plantarvault, in a global and a spatial point of view, are not well-known either. This question has a special importancebecause the footwear annexes the force transmissionthrough the anatomic structures and because the shoe-manufacturers consider child and adult’s footwear ashomothetic images. Moreover, it is known that anthropo-metrics mean measures of people change with the courseof time, this makes necessary a periodic updating of thetables of variables that define child’s feet.


45European Journal of Anatomy, 2 0 0 5 , vo l. 9 (Suppl . 1)

The aim of this work is to define the nomograme ofchild’s feet (right and left) for both genders, referred tothree different ages: 5,7 and 9 years-old.

In order to determinate the magnitudes that define thevault plantar morphometry it has been used an optic deviceof topographic moiré. With such device we obtain three-dimensional digital images and after we process the imagesobtained with a informatic program designed especially forthis work, which permits to measure longitudinal and angu-lar magnitudes, and the coordinates (x, y, z) of every pointof foot surfaces. This permits to calculate the analytic func-tion representatives of the former mentioned surfaces.

Later, using the mean values obtained, we make scalemodels to study their biomechanical behaviour with pho-toelastic interpherometry. This way, systems of isoclinicfringes are employed to determine the orientations ofprincipal stress axes. Also, singular points, includingpoints of zero stress, are determinated and we map injurerisk areas of child’s foot.

P-49 D I S TA L PHALANX OF THE HALLUX:D I S C R I M I N A N T FUNCTIONS AND UNI-VA R I ATE METHODS FOR SEX DETER-M I N AT I O N .

Monreal Redondo, D.G.1, Fernández Cama-cho, F.J.2, Hernández Fernández, L.M.2, Sán-chez Sánchez, J.A.2, Fernández-Baíllo, R.4

1 Departamento de Ciencias Morfológicas y Fisiolo -gía, Universidad Europea de Madrid. 2 SegundaCátedra de Anatomía Anatomía, Universidad deAlcalá. 3 Departamento de Medicina Legal y Toxico -logía, Universidad Complutense de Madrid 4 Investi -gador proyecto MAPACI.

Our aim was to obtain a reliable method for sex deter-mination using the distal phalanx of the hallux when otheruseful bones, as the pelvic and cranial bones, are not avai-lable. Nine linear measurements and 3 angles of the distalphalanx of the hallux were taken in 47 amputated lowerlimbs. Demographic and pathological conditions of thesample were known by clinical history. With the univaria-te analysis we could detect statistically significant diffe-rences of means for 7 linear variables; values were higherin males. Fisher’s stepwise discriminant analysis permit-ted to obtain an optimal discriminant function, which,with only 4 variables (total length, distal height, proximaltransversal curved length and valgus deviation angle),correctly identified sex for 95.7% of the sample, either inmatrix or jackknife classifications.

Musculoskeletal System: bone regeneration; art h r i t i s ;o s t e o p o rosis; muscular d i ff e rentiation; muscular d a m a g e

P-50 BONE REGENERATION THROUGH THEAPPLICATION OF P.R.P. AND BIO-OSSTO OSTEOPOROTIC RABBITS. A PILOTSTUDY.

Clemente de Arriba, C.1, Torres, J.2, Fernán-dez-Tresguerres, I.2 Blanco Jeréz, L.2

1 Universidad de Alcalá de Henares. 2 UniversidadRey Juan Carlos .

In surgical treatments the need occasionally arises toregenerate bone in patients whose bone regenerationcapacity is reduced, as occurs in osteoporosis. The objectis either to correct an osseous defect or to obtain greaterbone thickness to permit the placement of an implant. Todo so, “biofill materials” are used, which often includebone growth stimulating factors. One of the most com-monly used factors in odontostomatology due to the factthat it is easy to obtain and to use is P.R.P. (platelet-deri-ved growth factor). In this study we assumed that fillingan osseous cavity with an aloplastic biomaterial compo-sed of bovine hydroxyapatite (Bio-Oss®) impregnatedwith P.R.P. would cause osseous growth similar to thatobtained with the use of the biomaterial without the P.R.P.

To do so we used adult osteoporotic rabbits dividedinto two groups: (PRP+Bio-oss group and Bio-oss onlygroup).

Through a sagital incision, cavities measuring 1 cm indiameter were formed in the animals parietal bones whichmade it possible to stabilise two titanium cylinders, oneon each side of the sagital suture.

In the PRP+Bio-oss group the titanium cylinderswere filled with P.R.P+Bio-oss in the right parietal bonewhile that of the left was filled with PRP o n l y. In the Bio-oss group, the titanium cylinder of the right parietal bonewas filled with Bio-oss while the left side received nograft at all.

The animals were sacrificed four weeks alter the sur-gery and the study fragments were obtained. The sampleswere set in formol and included in methylmatacrilate.

Histomorphometry was performed, evaluating thebone area of the cavity created using MIP 4.1 image-analysing software.

The results showed that the combination of PRP+Bio-Oss achieves a higher percentage of osseous regenerationthan when Bio-Oss is used alone, although the differenceis not significant.

P-51 OSSEOUS RESPONSE TO DIFFERENTOSTEOPOROSIS TREATMENTS. A DEN-SITOMETRIC STUDY.

Clemente de Arriba, C.1, Aguado Henche, S.1,Rodríguez Tórres, R.1, Salazar, V.2, Gómez-Pellico, L.1

1 Universidad de Alcalá de Henares. 2 UniversidadComplutense de Madrid .

We know that the loss of bone mineral density is asso-ciated with the advanced age and also with the most fre-quent and well-known alteration of osseous tissue: osteo-porosis. There is a close relationship between the decreasein oestrogen which occurs in menopause and the higherfrequency of osteoporosis in this group of women. Advan-ced age brings with it not only a decrease in oestrogen butalso a decrease in the growth hormone (GH).

We assumed the variability of bone behaviour basedon the influence of these factors. To evaluate it, we took asample of Wistar female rats. We maintained a controlgroup and ovariectomies were performed on the rest ofthe rats at three months of age. After 18 months, the ova-riectomised rats were divided different groups: one groupthat received no treatment; another group to which subcu-taneous GH was administered; a third group to whichestradiol valerianate was administered subcutaneouslyand a fourth group which received a combination of the



two. To study the effects on the tibia, after 2.5 months oftreatment the rats were sacrificed at the age of 24 months.The bone was extracted and dual photon x-ray absorptio-mety was used to perform densitometry (DMO) on theproximal portion of the tibia including the metaphysiaryand proximal diaphysis region. Our results show DMOdifferences between the values of the control group andthose of the untreated ovariectomised rats and betweenthese and the ones that later received treatment. However,there are no statistically significant differences betweenthe DMO of the three treated group. Finally, when com-paring the values of the control group and the treatedgroups, the fact that no significant differences were founddemonstrates that all of the treatments recovered theDMO in the previously ovariectomised rats to similarlevels as those obtained in the control group of rats of thesame age.

P-52 C L I N I C A L - H I S TO L O G I C A L A N A LY S I SOF DIFFERENT MODELS OF GUIDEDBONE REGENERATION PRIOR TO THEE M P L A C E M E N T O F D E N TA LIMPLANTS. STUDY O F D I F F E R E N TBONE REGENERATION TECHNIQUES.

Pérez de la Cruz, M.A.1, Guadilla, Y2, Alonso,A.2, De Pedro Moro, J.A.3, Collia, F.1, Carras-cal, E.1

1 Dept. of Human Anatomy and Histology, Fac. ofMedicine of Salamanca. 2 Periodoncia, Fac. of Medi -cine of Salamanca. 3 Dept. of Surgery, Fac. of Medi -cine of Salamanca.

Since the eighties, the successful replacement of lostnatural teeth by means of analogous elements integratedinto tissues (dental implants) has been one of the best cli-nical advances in dentistry. The introduction of dentalimplants as a therapeutic alternative to dental replacementhas been documented in standard situations, with percen-tages of clinical success close to 100%. However, suchsuccess rates decrease in situations of bone deficit due tothe development of periodontal disease and traumaticlesions, which lead to both tooth loss and loss of the alve-olar bone and surrounding soft tissues, in turn eliciting adeficiency in the alveolar ridge and thereby hinderingemplacement of the dental implant. In light of this, it hasbecome necessary to obtain bone supports that will be suf-ficiently robust to guarantee the fixation of dentalimplants.

Different techniques have been implemented to achie-ve this, ranging from guided bone regeneration (GBR) tografts of both autologous and allogenic bone (frozen,dehydrated and lyophilised).

More recently, evidence has appeared that GBR maybe achieved, and even accelerated (Anitua, 2004), byusing autologous growth factors in what is termed plate-let-rich plasma (PRP) or growth factor-rich plasmaGFRP) and different techniques have been employed toachieve this. The aim of the present work was thus tocompare the different bone regeneration techniques usinglyophilised bone (2 cases), autologous bone (2 cases)plasma rich in GFRP or a combination thereof (20 cases).To accomplish this, we performed radiological and clini-cal studies in selected patients undergoing regenerativesurgery, followed at different times by a biopsy and histo-logical study to assess bone maturation. Although all the

cases studied developed bone that was suitable forimplantation, the histopathological trend varied, since inthe cases in which lyophilised bone was used fragments ofthis persisted at the time of biopsy, showing signs ofdecalcification with low cellular activity around them.

Funded by MAT 2004-01654.

P-53 REACTION OF BONE TISSUE A F T E RLESION. A HISTOLOGICALAND MORP-HOMETRIC STUDY O F THE A L B I N ORAT HINDLIMB.

Pérez de la Cruz, M.A.1, Criado, A.3, AboyLorenzo, J.A.3, Vazquez Lasa, B.2, San Roman,J.2, De Pedro Moro, J.A.3, Collia, F.1, Carrascal,E.1

1 Dpto. of Anatomy and Histology Human. Fac. ofMedicine of Salamanca. 2 Inst. of Science and Tecno -logy of Polim. CSIC. Madrid. 3 Dpto of Surgery. Fac.of Medicine of Salamanca.

Osteoporosis related to age, menopause or disuse isone of the major problems in clinical medicine. Here weused a model of neurectomy, or compression of the scia-tic nerve, in rats divided into four groups and sacrificed at2,4,6,8,12,16 and 24 weeks, dissecting out the femur andtibia, whose diaphyses were studied after embedding inplastic, transversally sectioned, and stained using theGoldner and von Kossa techniques, then performing thecorresponding histological and morphometric studies.From the histological point of view, there was a strikingperiosteal reaction associated with scant osteoclastic acti-vity. The cortical initially displayed a large increase inintracortical porosity but, later on, this regressed and therewas also an initially strong endomedullar osteoblasticactivity, which also decreased with time. Morphometri-cally, the most relevant observation was a decrease in thefemoral cortex area of the neurectomized part in compari-son with the contralateral part.

Funded by MAT 2004- 016 54.

P-54 M E S E N C H Y M A L STEM CELLS FROMS Y N O V I A L MEMBRANE REPAIR INVITRO A RTICULAR CARTILAGE OFO S T E O A RTHRITIC PATIENTS. PRELI-MINARY RESULTS.

Fuentes, I.1, Gimeno, M.2, López-Armada,M.J.2, Lema, B.2, Sánchez, M., Caramés, B.2,Ramallal, M.2, Sanjurjo, L.2, De Toro, F.1, Blan-co, F.2

1 Universidad de A Coruña. 2 Unidad de Investiga -ción del Hospital Juan Canalejo de A Coruña.

Mesenchymal stem cells (MSC) are defined as pluri-potent progenitor cells with the ability to repair damagedtissues.

The purpose of this study is to determine if humanMSC from synovial membrane repair cartilage of osteo-arthritis patients.

Cells extracted from synovial membrane of osteoarth-ritic patients were positives for characteristics markers of



MSC (CD29, CD44, CD73, CD90, CD117, CD166, PAX-7 and STRO-1) and negatives for hematopoietic markers(CD34 and CD45). Cartilage discs of osteoarthriticpatients were obtained at autopsy from femoral heads of 3donors (mean 65 years). The discs were divided in twogroups. In Group I, only culture media was deposited overthe cartilage surface, while in Group II 200.000 MSC sus-pended in 10 ml of culture media were deposited over thesurface of osteoarthritic cartilage discs treated with fibro-nectin (45 minutes at room temperature). Discs were incu-bated for 4 and 8 weeks at 37oC in a humidified atmosp-here of 5 percent CO2/95 percent air. Culture mediaconsisted of chondrogenic medium with 33,3 ng/ml trans-forming growth factor (TGF)-beta3 and 10 ng/ml basicfibroblast growth factor (FGFb). Frozen sections wereprocessed for standard histologic staining (hematoxylin-eosin, safranin O, Masson trichrome) and immunostainingwith monoclonal antibodies against type I and II collagen,integrin subunit beta1, chondroitin-4-sulfate and keratansulfate.

Repair was only observed in the cell-treated cartilages.After 4 culture weeks, the cartilage surface did not showconsistent signs of repair. After 8 weeks culture the carti-lage surface was covered by a neo-synthesized tissue thatconsisted of several layers of rounded cells. This tissueshowed a high cellularity, a moderately regular surface,and zones of excellent integration with the native cartila-ge. Furthermore, the neo-synthesized tissue showed nostaining for safranin O (indicating low contain in prote-oglycans). Immunostaining for type I collagen and cha-racteristic markers of articular cartilage (type II collagen,chondroitin-4-sulfate, keratan sulfate and integrin subunitbeta1) were positive.

These dates suggest that the MSC from synovial mem-brane of osteoarthritic patients can have the ability torepair osteoarthritic cartilage.

P-55 C O R R E L ATIVE RADIOLOGICAL, HIS-TO L O G I C A L AND MORPHOMETRICSTUDY OF HUMAN FEMUR HEADS.

Pérez de la Cruz, M.A.1, Martín A.P.3, Salva-do, M. 3, De Pedro Moro, J.A.3, Vazquez Lasa,B.2, San Roman, J.2, Collia, F.1, Carrascal, E.1

1 Dpto. of Anatomy and Histology Human. Fac. ofMedicine of Salamanca. 2 Inst. of Science and Tecno -logy of Polim. CSIC. Madrid. 3 Dpto of Surgery. Fac.of Medicine of Salamanca .

Currently, the two diseases with the greatest social andeconomic repercussions within the field of traumatologyare arthrosis and osteoporosis. These pathologies mainlyaffect the proximal end of the femur. This load-bearingjoint must adapt to the stress it must support through acomplex system of trabecular bone.

The aim of the present study was to gain furtherinsight into these two diseases, both histomorphometri-cally and radiologically, and compare the results of bothanalyses.

During 2000-2003, we collected 100 femur headsfrom patients receiving a hip replacement. From thesesamples, we obtained a central slice that included the hardcore and the zone of the round ligament, embedding themin methacrylate. We then performed Goldner and vonKossa trichrome stainings and analysed -histomorphome-trically- the osteoid area and the trabecular volume of the

histological sections. From the complete samples offemur head we obtained X-ray images for assessment ofthe Singh Index (degree of osteoporosis).

Significant differences (p<0.05) were obtained regar-ding sex and pathology in the peripheral area of the oste-oid, the trabecular bone volume, both central and periphe-ral, and the radiological Singh Index. Differences in thecentral trabecular volume and the Singh index were onlyfound as regards the age variable.

Women, subjects who had broken a hip and older indi-viduals showed a lower percentage of bone, less osteoidsubstance and a pathological Singh Index.

Funded by MAT 2004- 01654.

P-56 ANTHROPOLOGICALIDENTIFICATIONOF THREE CASTILIAN PRINCES.

Gil, J.A.1, De Paz, F.J.1, Pastor, J.F.1, Barbosa,M.1, Gutiérrez, B.1, Arroyo, E.2

1 University of Valladolid. 2 University Complutenseof Madrid.

The contents of the sarcophagus of the Castilian prin-ces Don Alfonso was given to the Museum of Anatomy ofthe University of Valladolid in order to carry out an anth-ropological study.

The following results were obtained after proceedingto the separation of the different individuals, the makingof an inventory of the remainings, the determining of theages, the estimation of the sexes, the radiological stu-dies, the histological study of the soft parts and the DNAs t u d i e s :

INDIVIDUAL 1: According to the stages of dental erup-tion and the measurements of the long bones the agewould be about 3 years old. All the bones show morpho-logy absolutely compatible with normality. No alterationson the skin or subcutaneous tissue were observed. No pro-per material for a DNA study could be extracted.

IN D I V I D U A L 2: Thanks to the good conservation ofthe orbital and nasal regions, and even counting on theretraction due to dehydration, a certain grade of hyper-telorism was obvious. According to the stages of dentaleruption and the measurements of the long bones theage would be about 2 years old. The ends of the longbones showed a certain grade of widening, being espe-cially obvious on the distal ends of femurs and on theproximal ends of tibiae. In these same places the bonecortical was thinned. The scanner showed mummifiedocular structures in the right socket. No alterationswere found on the processed skin remainings. Only atooth sample was suitable for DNA studies, and it con-firmed the presence of the “Y” chromosome. The alleleDSY389-II*27 revealed a slightly higher value in 3consecutive experiments. The obtained halotype had alow enough frequency to be used as a potential markerall throughout the descendant generations from kingSancho IV.

INDIVIDUAL 3: According to the stages of dental erup-tion and the measurements of the long bones the agewould be about 10 months old. All the bones show morp-hology absolutely compatible with normality. No altera-tions on the skin were observed. No proper material for aDNAstudy could be extracted.



P-57 BONE COLLECTIONS OFTHE MUSEUMOF ANATOMY OF THE UNIVERSITY OFVALLADOLID.

Gutiérrez, B.1, Pastor, J.F.1, De Paz, F.1, Barbo-sa, M.1, Díez, M.2, Barbosa, E.1

1 University of Valladolid. 2 Fauna Recovery Centreof Valladolid. Junta de Castilla y León.

The bone collection of the Museum of Anatomy of theUniversity of Valladolid dates from 1856 when the studiesin Medicine were re-established in Valladolid. In just afew years it already had hundreds of skulls and completeskeletons cleaned in the modern facilities and use almostexclusively for teaching purposes. Since the sixties twomain factors influenced on the decreasing of the bonematerial: On the one hand the diminishing on corpses, andon the other hand the students overcrowding, which madethe material to faster deteriorate.

The contents of these collections are the skeletonsor parts of them. This way every vertebrate group isr e p r e s e n t e d .

The unbuilt skeletons are mainly used as scientificcollections, for studies in anatomy (human and animal),compared anatomy, anthropology, paleobiology, arqueologyand forensic osteology. In these kinds of collections a gooddating of the specimens is very important and also the exis-tence of type specimens. The skeletons built in the anatomi-cal position are used in teaching collections or for exhibits.The osteological material constitutes a valuable source ofinformation, for, apart from the morphostructure, it can pro-vide genetic material, so important for modern studies ont a x o n o m y, population ecology, molecular biology and foren-sic techniques. The optimum use of the human and animalmaterial requires facilities and staff, and also the establish-ment of a biological material compilation net, signing agre-ements with cemeteries, zoos and fauna recovery centres.

Since 1987 a job of increasing the human osteologycollection is being carried out. It has to main objectives, onthe one hand to get quality material for the pregrade practi-cal classes and on the other hand the establishment of a refe-rence collection for anthropological research. There are atthe moment 238 human complete skeletons with age, sexand origin known, 460 crania and also post-cranial bones.

Of recent creation is the collection for compared oste-ology, used mainly for research purposes, although it isalso used for display and teaching purposes. There are, sofar, 3,266 skeletons, including fish, amphibians, reptiles,birds and mammals. We would like to underline the orderPrimate, including 87 different species within the collec-tion so far.

A good cataloguing, computerization and storage arefundamental in order to keep the specimens readily acces-sible for their examination.

Scientific examination on morphology is more andmore demanded and an interinstitutional collaboration isneeded in order to make a proper use of the specimens andto optimize the generated biological material, creating,apart from the bone collections, others of hides, eggs,organs, histological material and genetic material.

P-58 BIOMETRIC AND ORAL HEALTH DATAC O M PARISON BETWEEN A R C H A E O-L O G I C A L AND MODERN IBERIANPOPULATIONS: THE “ENCANTADES DEMARTÍS” CAVE.

Ta l l ó n - Walton, V1, M a n z a n a re s - C é s p e d e s ,M.C.1, Neves-Ferreira, F2, Agustí-Farràs, B.3,Serra-Renom, I.1, Carvalho-Lobato, P.1,Miguel-Pérez, M.I.1, Götzens-García, V.1

1 Unidad de Anatomia y Embriologia Humana-Bell -vitge, Universidad de Barcelona. 2 Facultade deMedicina Dentaria, Instituto Superior de Ciencias daSaùde-Norte, CESPU, Gandra, Portugal. 3 Universi -dad de Girona.

The aim of this study is to describe the biometric dataand the oral pathology signs present in the teeth and theosseous fragments found in the “Encantades de Martis”cave, compared with the data obtained from the presenttime population of the Iberian Peninsula. The cave wasused as a burial site during 2500 years, from the Neoliticuntil the Roman Empire. The archaeological materialsfound in this cave were not, unfortunately, properly classi-fied, making its study quite difficult. 4274 teeth have beenidentified from the 4325 teeth found, from which 3877were definitive and 397 deciduous. Moreover, 774 osseousfragments, 170 maxillae and 604 mandibles, were studied.

75 permanent teeth were selected, following two crite-ria: dentary type and absence of deforming lesions and ofabrasion. The teeth were measured for all dimensions(crown length, rooth length, total length, mesio-distal dia-meter, vestíbulo-lingual diameter) and their crown morp-hological index, crown strenght index and crown module,as well as the Bolton index were calculated. These datawere compared with those previously obtained in modernpopulations of the iberian peninsula.

The results obtained show that the archaeologicalpopulation teeth were smaller than the teeth from themodern population. According with our previous results,some dental measurements are statistically correlated withthe height of the individual. A comparative study of thelength of the femoral bones stated that the individualsburied were significatively shorter than our present popu-lation. This could be the reason of the differences both indental size and in dental volume indexes observed betwe-en the two populations.

The osseous fragments studied do not differ signifi-cantly in shape from those from the modern population.Size measurements have been limited by the fragmentationof the bone structures. Only a 1,64% of the teeth werea ffected by caries, situated in areas that suggest the conco-mitant presence of periodontal disease. Abrasion is a cons-tant finding in the teeth, both the isolated and those stillanchored in bone structures, either permanent or deciduous.These data indicate that the ancient inhabitants of the areawere healthy, and that their diet was rich in fruits, vegeta-bles and fiber, allowing a good self-cleaning of the teeth.

P-59 EXPRESSION OF MAJOR HISTOCOM-PAT I B I L I T Y CLASS I ANTIGENS ISM O D U L ATED BY P - G LY C O P R O T E I NT R A N S P O RTER: IMPLICATIONS INMUSCULAR DIFFERENTIATION.

Prados, J.1, Melguizo, C.2, Carrillo, E.1, Velez,C.2, Caba, O.1, Marchal, J.A.3, Boulaiz, H.3,Rodríguez, F.3, Martínez, A.3, Fernández, J.E.1,Suárez, I.1, Rama, A.R.1, Álvarez, L.1, Maded-du, R.4, Aránega, A.1

1 Universidad de Granada. 2 Universidad de Almería.3 Universidad de Jaén. 4 Universidad de Sassari.



Muscle differentiation may be influenced by modula-tion of resistance genes expression (mdr1) which are ableto induce changes of myosin and to generate the appari-tion of myofilamentous material which is considered aclear evidence of myogenic differentiation. On the otherhand, the activity of this gen may be involved in the con-trol of the MHC class I expression. This relationshipcould be explained by the homology found between theproduct of the mdr1 gene, termed P-glycoprotein (P-gp),and the putative peptide transporters TAP1 and TAP2.Because of the amount of the MHC class I on the cell sur-face is related to the quantity and quality of peptides in theendoplasmic reticulum, transport of protein fragmentsfrom the cytosol to the endoplasmic reticulum by P-gpmay modulate HLA expression. Interestingly, modulationof HLA class I antigens expression has also been relatedto the degree of differentiation in some cell types. Usingembryonal rhabdomyosarcoma (RMS) samples and celllines as an experimental model to study myogenic diffe-rentiation, we have examined the resistance mediated bymdr1 and its correlation with the HLAclass I expression.Analysis by PCR indicated that positive RMS to mdr1show higher HLA class I expression than those whichwere negative to mdr1 PCR. To demonstrate if the mdr1gene expression in these cells modifies the MHC molecu-les expression, we generated two resistant RMS cell line(A-204-1 and 2) using actinomicyn D. FACScan analysisof both cell lines showed an increase of mdr1 and HLAclass I expression. Moreover, the blockade of P-gp inRMS cells using a non-toxic treatment with verapamil,caused a significant decrease in the HLA class I expres-sion. Our results confirming that the product of mdr1modulates the expression of HLAclass I antigens in RMScells. However, further investigations will be necessary todetermine the mechanism by which the development ofMDR mediated by mdr1 can modulate HLA class Iexpression. Our findings may be relevant to understandthe myogenic differentiation process and may have someapplication in RMS immunotherapy against tumor-asso-ciated antigens presented by HLA class I molecules.

P-60 MUSCULAR DIFFERENTIATION PRO-CESS: ANALYSIS IN RHABDOMYOSAR-COMA CELLS.

Prados, J.1, Melguizo, C.2, Carrillo, E.1, Vélez,C.2, Caba, O.1, Marchal, J.A.3, Boulaiz, H.3,Rodríguez, F.3, Martínez, A.3, Fernández, J.E.1,Suárez, I.1, Rama, A.R.1, Álvarez, L.1, Aránega,A.1

1 Universidad de Granada. 2 Universidad de Almería.3 Universidad de Jaén.

Understanding the molecular mechanisms that controlmuscle gene expression is an important requisite to deve-lope therapeutic strategies that correct muscle dysfuntion.Rhabdomyosarcoma (RMS) which derives from striatedmuscle, is an in vitro model to analyse the myogenic dif-ferentiation. Our analysis of these cells demonstrate thatmyogenic differentiation program involves to separatephases; a biochemical differentiation with the expressionof nonmuscle and muscle specific gene and a morpholo-gical differentiation in which the mononucleated myo-blasts fuse to form multinucleated myotubes. Treatmentof RMS with DMSO results in an increase of desmin anda decrease of vimentin expression with a rapid accumula-tion of actin, tropomyosin and alpha-actinin. This structu-ration of the cytoskeleton is involved in modulating signal

trasduction and in the morphological changes duringcellular differentiation. At the same time, a biochemicalmodulation of the muscle isoenzyme of creatin kinase(CK-MM, MB and BB) expression happened. In this pro-cess, transcriptional factors that are expressed exclusivelyin skeletal muscle are decisives. So, our results show thata decrease in Id, a negative regulator of MyoD, induce theactivation of muscle CK expression. However, MyoDfamily may not represent the only mechanism for activa-tion of the myogenic program. These factors present anamino acid segment which shares significant homologywith the members of the c-myc family. In RMS, we haveobserved modulation of c-myc expression during the pro-cess of myogenic differentiation whereas levels of N-mycshowed no modulation. The exact mechanism throughwhich these factors activate their target genes is unknown.On the other hand, using low concentrations of actinomy-cin D, we have obtained an ultrastructural myogenic dif-ferentiation in RMS cells that showed cytoplasmic pro-jections, contact between cell membranes and formationof myotubes. By electron microscopic, RMS cells contai-ned increased numbers of cytoplasmic organelles, lipidvesicles, intermediate filament and, the main indicator ofcomplete myogenic differentiation, the presence of nume-rous myofilaments which formed well-defined bundles(Z-bands). In conclusion, myogenesis follows a sucessionof developmental stages, each one characterized by dis-tinct morphological events and patterns of gene expres-sion. RMS cell lines are an in vitro model to analyse theprocess of myogenesis.

P-61 DETECTION OF MUSCLE DAMAGE BYTHE DETERMINATION OF S E R U MALPHA-ACTIN LEVELS.

Martínez-Amat, A.1, Marchal, J.A.1, Carrillo,E.2, Boulaiz, H.1, Rodríguez-Serrano, F.1, Caba,O.2, Prados, J.C.2, Padial, P.3, Melguizo, C.4,Vélez, C.4, Aránega, A.2

1 Dept. Ciencias de la Salud, Universidad de Jaén. 2

Dept. Anatomía y Embriología Humana, Universidadde Granada. 3 Dept. de Educación Física y Deporti -va, Universidad de Granada. 4 Dept. Neurociencias yCiencias de la Salud, Universidad de Almería.

Intense physical exercise is associated with the deve-lopment of muscle injuries. Their severity and evolutionare variable, depending on the intensity, duration, andtype of exercise carried out. To date, enzymes (CK, LDH,MDA) and proteins (troponin and myoglobin) in serumhave been used as markers of skeletal muscle damage.However, all currently available parameters present limi-tations in their sensitivity, specificity, and/or release-time,and they do not clearly reflect structural muscle damage.The skeletal muscle protein a-actin was investigated inthe serum of subjects with severe skeletal muscle damagein order to assess its utility as a reliable predictive markerof muscle contractile dysfunctions.

METHODS: Serum samples were obtained from 33healthy controls and 33 patients with serious skeletal mus-cle damage, defined by a total CK value of > 500 IU/L(Rosalki method). Troponin I, T and myoglobin concen-trations were determined by immunoassay and a-actinconcentrations by Western blot and densitometry.

RESULTS: The mean serum concentration of a-actin incontrols and patients with skeletal muscle damage was600.9 and 1968.51 ng/mL, respectively, a statistically sig-nificant difference. The sera of the patients with muscle

50

damage showed higher concentrations of a-actin than oftroponin or myoglobin. No significant difference in tropo-nin I levels was observed between the groups.

CONCLUSIONS: According to these results, a-actin wasthe most significant skeletal muscle damage markeranalyzed and may be an ideal candidate for the earlydetection of skeletal muscle damage in sportspeople.

P-62 M O N O L AYER CELL C U LTURE A N DGENERATION OF AUTO-INDUCED TIS-SUE FROM HUMAN HYALINE A N DFIBROUS CARTILAGE SAMPLES

Fàbregas, P.J.1, Rubiralta, X.2, Farré, J.2, Agui-lera, J.M.3, Barrachina, J.3, Cairó, J.J.2, Gòdia,F.2, Nebot-Cegarra, J.1

1 Unitat d’Anatomia i Embriologia. Universitat Autò -noma de Barcelona. 2 Departament d’EnginyeriaQuímica. ETSE. Universitat Autònoma de Barcelona.3 Hospital Asepeyo Sant Cugat del Vallés.

In synovial joints, confronted articular surfaces ofbones are coated by hyaline cartilage. Hyaline articularcartilage is avascular, alymphatic, and aneural, and itsnutrition mainly depends on the synovial fluid. The onlycells present in hyaline cartilage are chondrocytes, whichare responsible for the synthesis and maintenance of theextracellular matrix.

Joint cartilage differs from other tissues in its respon-se to injury. Due to its avascularity, the natural healingcapability is poor. Although there have been manyattempts to treat cartilage injuries, no treatment has pro-duced long-lasting hyaline cartilage.

Autologous condrocyte implantation (ACI) is a formof tissue engineering that is being increasingly used totreat damaged articular cartilage. The success of ACIdepends to a large extend in the proper tissue differentia-tion, the architecture of the final tissue, the mechanicalproperties and its integration in the recipient tissue.

In the present work, fragments of human meniscus andjoint cartilage were obtained by means arthroscopic metho-dology as result of the treatment of diverse knee patholo-gies. Tissues were disgregated by collagenase and pronaseand then cells were cultured in monolayer in order to expandcell population. Because three-dimensional structure is keyin the development of the morphofunctional cellular pattern,we centrifuged cultured cells allowing the formation of afine cellular lamina in the flat bottom of a tube constituting,in this way, an artificial three-dimensional tissue.

After different growing periods, either monolayer cul-tures or laminas were fixed in 4% paraformaldehyde andstained with Masson’s trichrome and safranin O techni-ques in order to analyze the cell characteristics and theextracellular matrix production. The presence of hyalinecartilage markers Col-2 and Sox-9 was analyzed bymeans immunohistochemistry. Different cell morpholo-gies and grades of intracellular secretion were observed inmonolayer cultures depending on the cells source: menis-cus or joint cartilage. Laminas displayed a compact dis-position, adequate cell viability and diverse cellular pat-terns from fibroblastic to more chondrocitic.

Laminas constitute a first step in the generation of animplantable tissue that can potentially restore damagedhyaline cartilage; next steps include the use of biopoly-mers and the addition of specific growth factors in the cul-ture medium.

Cancer, Proliferation, Apoptosis

P-63 PROGNOSTIC VALUE OF RT-PCR TYRO-SINASE DETECTION IN PERIPHERALBLOOD OF MELANOMA PATIENTS.

Carrillo, E.1, Prados, J.1, Marchal, J.A.2, Bou-laiz, H.2, Melguizo, C.3, Vélez, C.3, Martinez,A.2, Rodriguez-Serrano, F.2, Caba, O.1, Suarez,I.1, Fernández, J.E.1, Alvarez, L.1, Aránega, A.1

1 Universidad de Granada. 2 Universidad de Jaén. 3

Universidad de Almería.

The prognosis for malignant melanoma (MM) hasbeen related to different factors such as tumor thickneess,clinical stage. The risk of metastasis has been related tothe presence of tumoral cells in peripheral blood.

For detecting tumour cells in melanoma patientsreverse transcriptase-polymerase chain reaction (RT-PCR) has been used to identify tyrosinase, a key enzymein melanin biosynthesis in the circulation of patients withdisseminated disease.

We report results of the detection of tyosinase by RT-PCR in peripheral blood samples from 58 MM patientswith all clinical stages (I, II, III and IV). The aim of thisstudy was to determine the clinical relevance of this pro-cedure, exploring the relationship between presence oftyrosinase in peripheral blood of MM patients and theirclinical stage and prognosis.

Blood samples were obtained from 58 consecutivelyselected MM patients at time of primary tumour diagno-sis or at relapse. All stage I-IV patients were visited every4 months during the first 2 years after the diagnosis andevery 6 months thereafter.

We used RT-PCR assay to detect tyrosinase mRNA i nthe patients with MM. Atwo-tailed Fischer’s exact test wasused to assess the association between RT-PCR results andclinical stage. Relationships among RT-PCR results, clinicalstage and overall survival were evaluated in all patients.

The results of the RT-PCR assay for tyrosinase wererelated to two prognostic markers used to evaluate thesestumors: clinical stage and thickness. Positive PCR resultswere more frequent in primary tumors measuring > 4mm(83%) than in thinner tumors (1.1-4.0mm, 74%; < 1.0mm, 23%) (p= 0.005).

After a median follow-up of 24 months (range, 3-48months), statistically significant correlation between RT-PCR results and recurrence (p<0.05) and clinical stage III( p<0.05) was found. Analysis of stage III tumours alone todetermine the prognostic value of melanoma cell presencein peripheral blood found 24-month overall survival to be70% in the RT-PCR negative group versus 10% in the posi-tive group (p<0 . 0 2 ) . The prognostic va1ue of the detectionof circulating melanoma cells may be especially relevant instage III patients, whom the RT- PCR positivity seems todefine a group of patients with a high risk of recurrence.

P-64 DETECTION OF CIRCULATING TUMORCELLS IN PATIENTS WITH MELANOMABY RT-PCR: CORRELATION WITH CLI-N I C A L S TAGE, TUMOR T H I C K N E S SAND HISTOLOGICALTYPE.

Carrillo, E.1, Prados, J.1, Marchal, J.A.2, Boulaiz,H .2, Vélez, C.3, Melguizo, C.3, Rodriguez-Serra-no, F.2, Martinez, A .2, Caba, O.1, Fernández, J.E.1,Suarez, I.1, Alvarez, L.1, Aránega, A .1



1 Universidad de Granada. 2 Universidad de Jaén. 3


For detecting tumour cells in melanoma patientsreverse transcriptase-polymerase chain reaction (RT-PCR) has been used to identify tyrosinase, a key enzymein melanin byosynthesis, in the circulation of patientswith disseminated disease.The tyrosinase gene is activelyexpressed only in melanocytes cannot be detected in thecirculation, the detection of tyrosinase RNA indicates thepresence of melanoma cells.

The prognosis for malignant melanoma (MM) hasbeen related to different factors such as tumor thickneess,invasion level and histological tumor type although thesefeatures are of limeted value.

We studied the detection of melanoma cells using RT-PCR in the peripheral blood of 58 patients with MM.Healthy subjects or non melanoma patients were used asnegative controls. As a positive control, we used a lymphnode metastasis from a patient with MM.

Blood sample were collected in tubes containing ede-tic acid (EDTA), was centrifuged and the plasma was dis-carded of the serum. Our primers ME-1 (5´TTG GCAGAT TGT CTG TAG CC) and ME-2 (3´ AGG CAT TGTGCA TGC TGC TT) amplify a 284 bp fragment of thetyrosinase. a- actin primers spannig an intron were devi-sed from the gene and used to check the integrity of theRNA samples for RT-PCR.

The primers were 5´ATC ATG TTT GAG ACC TTCAA3´ and 5´CAT CTC TTG CTC GAAGTC CA3 ´, pro-duce a 248 pb fragment of a- actin. We used RT-PCRassay to detect tyrosinase mRNA in the patients affectedof melanoma. For statiscal evaluation the Pearson ?2 testand Mann-Whitney test was used to correlate the PCRresults with clinical stage, tumor thickness and histologi-cal type.

We tested peripheral blood samples from 58 patientswith MM in different stage for the presence of tyrosinasetranscripts by clinical stage the distribution was the pri-mary tumor was classified as nodular melanoma (NM, n=19), superficial spreading melanoma (SSM, n= 30), acro-lentiginous melanoma (ALM, n= 7) or lentigo malignamelanoma (LMM, n=2). No tyrosinase mRNAwas detec-ted in any of the healthy control patinets and with othermalignancies.

The results of the RT-PCR assay for tyrosinase wererelated to two prognostic markers typically used to eva-luate theses tumors: clinical stage and thickness. PositivePCR results were more frequent in primary tumors mea-suring > 4mm (83%) than in thinner tumors (1.1-4.0mm,74%; < 1.0 mm, 23%) (p= 0.005). No statistical correla-tion was found between the PCR results and histologicalappearance of the primary tumor.

P-65 MORPHOLOGICAL CHANGES, MODU-LATION OF CELL CYCLE, AND APOP-TOSIS IN HUMAN BREAST C A N C E RINDUCED BY A NOVEL HIGHLY LIPOP-HILIC 5-FLUOROURACIL DERIVATIVE.

Boulaiz, H.1, Marchal, J.A.1, Suárez, I.2, Cam-pos, J.3, Carrillo, E.2, Rodríguez, F.1, Caba, O.2,Martínez, A.1, Prados, J.2, Madeddu, R.4, Mel-guizo, C.5, Vélez, C.5, Aránega, A.2

1 Department of Health Sciences, University of Jaén,Spain. 2 Basic Cardiovascular Research Section,

Department of Human Anatomy and Embryology,School of Medicine, University of Granada, Spain. 3

Department of Pharmaceutical and Organic Che -mistry, Faculty of Pharmacy, University of Granada,Spain. 4 Department Anatomy, University of Sassary,Italy. 5 Department of Health Sciences, University ofAlmería, Spain.

Breast cancer is among the most common tumoursaffecting women. The morbidity and mortality rates forthis type of cancer have prompted a search for novel the-rapeutic strategies, such as the use of novel antitumourdrugs that are less toxic to normal tissues and more speci-fic to malignant cells. The fluoropyrimidines, especially5-fluorouracil (5-FU), are antimetabolite inhibitors of denovo purine and pyrimidine syntheses. They have playedan important role in standard chemotherapy protocols fora range of solid tumours, including breast and colorectalcancers. The antitumour activity of 5-FU is dependent onthe ability of the drug to bind and inactivate the enzymethymidylate synthase (TS). However, the intravenousadministration of 5-FU in patients with metastatic breastcancer is highly toxic to normal cells and present majortoxicities that include mucositis and hand-foot syndrome.One approach to overcome this toxicity of 5-FU is toconstruct prodrugs with lower hydrofobicity and citotoxi-city, preferentially activated in cancer cells.

The research reported investigates antitumour activity,the cell cycle arrest and apoptotic properties of novellipophilic benzene-fused seven-membered 5-fluorouracil(5-FU) analogs in comparison to 5-FU on MCF-7 humanbreast cancer cells.

In the present investigation we demonstrate the highantitumour properties of three novel lipophilic benzene-fused seven-membered 5-FU analogs (ESB-786B, ESB-252A and ESB-928B) by apoptosis induction and cellcycle arrest in MCF-7 breast cancer cells.

Our results indicate a dose-dependent activity againstMCF-7 and IC50 values in the low micromolar range. Thenovel derivatives slow down the cell cycle progression, asindicated by the decrease in the %S and the increase in the%G0/G1. Moreover, because a larger proportion of cells (>60%) is been recruited into early apoptosis and DNAstrand breaks are produced in the MCF-7 cells, a cell linewhere the induction of DNA fragmentation is very diffi-cult, we suggest that novel 5-FU derivatives can be consi-dered as specific apoptotic inducers. These experimentalfindings provide evidence of specific antitumour activityof these new substances having the 5-FU moiety andwarrant further evaluation in in vivo models of breast can-cer for future clinical applications.

P-66 BONE A N ATO M I C A L L O C A L I Z AT I O N SO F THE META S TASES IN BREASTC A N C E R .

Aparicio Bellver, L.1, Ruiz Simón, A.2, SantosCores, J.2, Martin García, I.2, Marhuenda Flui-xa, A.2, Cervera Deval, J.2, Casas Roman, E.1

1 Department of Human Anatomy and Embryology.University of Valencia. 2 Foundation Oncology Insti -tute of Valencia (IVO).

The bone invasion by a malignant tumour is due, witha lot more frequence, to the metastasis than to the primarytumours, being the breast cancer one of the tumours thatwith more frequency metastasize in the bone.


52 E uropean Journal of Anatomy, 2 0 0 5 , vo l. 9 (Supp l. 1)

The metastases dissemination of the breast cancer canoccur in several organs, but the bone is one of the mostfrequent localizations, which occurs in a 73% of patients.

The importance of the metastases localization in thebone is basically the pain and the pathological fractures,in addition to other metabolic complications as the incre-ase of calcium.

The morphologic standard of these injuries can belitic, blastic o mixture.

The diagnosis of the bone metastasis is done by x-raybeing complementary the explorations with simple Rx,TAC and RM, also the bone scan with tenecium 99 candetect more precociously injuries and direct towards theinjured areas the selective x-ray studies. When these sig-nals are unique they are not always metastasis and there-fore force a diagnosis with other techniques and even abiopsy. However if the isotopic receptions is multiple ishighly suspicious of metastases.

Within the skeleton regions more affected are the spi-nal column (88%), the pelvis (64%), the skull (43%), theribs (46%), and the inferior (39%) and superior (17%)limbs.

In the limbs, the outstanding localizations are the longproximal bones (femur and humerus), being the distalregions very sporadically affected.

In different series the frequency of metastases in thebones of the carpal or of the foot is very low and sometimes are confused the symptoms with other pathologies.

We present the skeleton regions more frecuently aff e c-ted in metastatic breast cancer and two innusual metastasiscases in the astragalus, calcaneus and metatarsal. Bothpatients had other areas of metastases localizations in spi-nal column and pelvis. Both received palliative antialgictreatment with radiotherapy with very good local response.

When painful distal exists in patients affected by bre-ast cancer, we can not exclude the presence of metastases;therefore we will have to do radiological studies to exclu-de this localization.

P-67 A P O P TOSIS AS TA R G E T IN HUMANGENE T H E R A P Y: MORPHOLOGICALCHANGES AND MODULATION OFGROWTH MARKER EXPRESSIONINDUCED BY G E F GENE IN BREASTCANCER CELLLINE.

Boulaiz, H. 1, Prados, J.2, Marchal, J.A.1, Mel-guizo, C3, Carrillo, E.2, Martínez, A.1, Rodrí-guez, F.1, Caba, O.2, Vélez, C.3, Álvarez, L.2,Fernández, J.M.2, Aránega, A.2

1 Department of Health Sciences, University of Jaén,Spain. 2 Basic Cardiovascular Research Section,Department of Human Anatomy and Embryology,School of Medicine, University of Granada, Spain. 3

Department of Health Sciences, University of Alme -ría, Spain.

Nowadays numerous research fronts exist in the fightagainst cancer that attempt to overcome the main obsta-cles of standard therapeutic approaches. New therapeuticstrategies are required to overcome the limitations of con-ventional breast cancer treatment. Suicide gene therapyoffers a potential approach to this type of tumour, sincesystems based on the use of prodrugs may present somedrawbacks related to toxicity, drug release and bioavaila-bility. This lead to the development of strategies based on

the use of toxic genes that do not need a prodrug to beeffective in tumor cells. The gef gene has cell-killingfunctions in Escherichia coli and does not depend on theuse of a prodrug for its action, making it an attractive tar-get for suicide gene therapy.

We created a gef-overexpressing human breast cancercell line (MCF-7TG) by transfecting the gef gene underthe control of a pMAMneo promotor. Dexamethasone-induction of gef gene expression in MCF-7TG cells pro-duced a significant decrease in Ki-67 expression, which isa known proliferation marker. In addition, Annexin-V-FITC and propidium iodide assays showed the presenceof apoptotic cell death, which was confirmed by scanningelectron microscopy. The most significant finding was thepresence of “craters” in the cell membrane, as previouslydescribed in other apoptotic breast cancer cells.

In conclusion, the present data demonstrate that theinduction of g e f gene expression in breast cancer MCF-7cells induces a decrease in the expression of Ki-67, anantigen related with the proliferation rate of these tumourcells, and produces a process of cell apoptosis characteri-zed by major modifications in the cell morphology. A l t-hough the mechanism has yet to be established and furt-her research is required, these results suggest that the gefgene is a potentiel candidate for novel gene therapy stra-tegies against breast cancer cells. This approach would besimpler than other suicide suicide gene therapy modelsystems because no prodrug is required. Furthermore, theuse of selective transcriptional control sequences of g e fmay offer the gene therapist additional tools of greatp o t e n t i a l .

P-68 D I F F E R E N T I ATION OF THE INTESTI-NAL EPITHELIUM MEDIATED BY EXO-GENOUS NUCLEOSIDES.

Rodríguez-Serrano, F.1, Marchal, J.A.1, Bou-laiz, H. 1, Caba, O.2, Martínez, A.1, Prados, J.2,Carrillo, E.2, Suárez, I.2, Melguizo, C.3, Vélez,C.3, Fernández, J.E.2, Ríos, A.2, Aránega, A.2

1 Universidad de Jaén. 2 Universidad de Granada. 3


In the Lieberkühn intestinal crypts, it’s carried out acellular dynamic process that allows the epithelial reno-vation. The progenitors cells, derived from stem cellslocated in the base of the crypts, migrate towards theintestinal villus. During this course takes place the diffe-rentiation, morphologic and functionally, that it reacheshis maximum degree in the apex of the villus, where thecells are finally flaked.

We present a study where we evaluate the effects thatproduce the exogenous nucleosides on the enterocytic dif-ferentiation. For it, intestinal epithelial cell line IEC-6 hasbeen chosen, that is derived from rat jejunal crypts. T h ebehavior of IEC-6 line in cell culture is resembled to whichexperience the enterocytes in vivo. These cells present ini-tially undifferentiated phenotypic characteristic, but whenthey get cellular confluence begin to differentiate themsel-ves, and finally they are flaked in the culture medium. T h enucleosides were administered in two mixtures, constitutedby thymidine (NTmixture) or uridine (NU mixture), toget-her with guanosine, cytidine and inosine, at a final concen-tration of 100mM of each nucleoside.

The confluent cultures treated with the nucleosidesmixtures, had a similar microscopic appearance, where



we could find structures characteristic of diff e r e n t i a t e dIEC-6 cells, whereas the control group, treated with aculture medium without nucleosides, lacked them. Onthe other hand, the villin expression, a structural markerof enterocytic differentiation, was bigger in both groups,N T (7.17 ± 0.65%, p<0.01) and NU (6.76 ± 0.59%,p<0.01), regarding the control group (2.76 ± 0.27%).These results of the marker expression were corrobora-ted by Western Blot. Finally, the quantification of thetotal protein amount of the different cultures, gave a big-ger amount in the groups NT (128 ± 4%, p<0.01) andNU (136 ± 9%, p<0.01), respecting to the control group(100 ± 5%).

As a conclusion, the exogenous nucleosides stimulatethe differentiation of intestinal epithelial IEC-6 cells. Inaddition, the behavior of the used mixtures, NT and NU,is similar in this process.

P-69 ACTION OF EXOGENOUS NUCLEOSI-DES ON INTESTINALEPITHELIALPRO-LIFERATION.

Rodríguez-Serrano, F.1, Marchal, J.A.1, Martí-nez, A.1, Caba, O.2, Boulaiz, H.1, Prados, J.2,Carrillo, E.2, Suárez, I.2, Vélez, C.3, Melguizo,C.3, Ríos, A.2, Álvarez, L.2, Aránega, A.2

1 Universidad de Jaén. 2 Universidad de Granada. 3


Several studies point out to the exogenous nucleosidesas important elements for the normal development andgastrointestinal maintenance. Thus, the absence in the dietof these compounds has been related with a height reduc-tion of the intestinal villus, a smaller content of RNA,DNA and smaller rates of protein synthesis.

The objective of our study is focused in evaluating theeffect of two nucleosides mixtures, constituted by thymi-dine (NT mixture) or uridine (NU mixture), together withguanosine, cytidine and inosine, on the proliferation of anestablished intestinal epithelial cell line denominatedIEC-6, coming from rat jejunal crypt.

P a r a d o x i c a l l y, our results show deep differences bet-ween both mixtures, on the proliferation of IEC-6 cells,at a final concentration of 100mM of each nucleoside.The NT mixture, increased the cell content of the cultu-res over a 51.89% with respect to the control withoutnucleosides (p<0.01), whereas NU mixture reduced theproliferative capacity over a 25.39% (p<0.01). T h ee ffects produced by the NT mixture could be associatedto a reduction of the percentage of cells located in thesynthesis phase and to an increase in phase G2-M of thecell cycle. On the other hand, the antiproliferativeaction of uridine in NU mixture, didn´t associate neitherto morphologic changes, nor to variations in the cellcycle phases, nor to a modification of the viabilitylevels, necrosis or apoptosis, nor to the acquisition of abigger degree of cellular diff e r e n t i a t i o n .

These data indicate that intestinal epithelium is sensi-tive to the presence of exogenous nucleosides, being ableto produce an increase of the cellular proliferation. Howe-ver, the consideration of the uridine at 100mM as a situa-tion of physiological range seems an error, reason why inulterior assays it must be considered the administration ofa lesser amount of the nucleoside.

P-70 THE PERITO N E A L MESOTHELIUM CO-VERING THE INFUNDIBULUM IN T H ES O W: ASCANNING ELECTRON MICROS-C O P Y S T U D Y.

R e c reo, M.P.1, Arceiz, E.2, Marco, M.A.3,Yaniz, J.4

1 Dpto. de Anatomía e Histología Humanas. 2 Dpto deAnatomía Patológica, Medicina Legal y Forense yToxicología. 3 Dpto. Microbiología, Medicina Pre v e n -tiva y Salud Pública. Facultad de CC de la Salud y elD e p o rte. Huesca. 4 Dpto. de Producción Animal. E.U.Politécnica de Huesca. Universidad de Zaragoza.

This study was undertaken to describe the surface fea-tures of the peritoneal mesotelium covering the infundi-bulum and the transition between oviductal and mesothe-lial epithelium in the sow, and to compare them with thosepreviously described in the cow (Yániz et al, 2000). Theexternal infundibulum of 12 cyclic sows was examined byscanning electron microscopy. Tissue specimens 0.5 to 1cm2 were taken from the infundibulum, at a point adjacentto the free margin and another 1-2 cm distal. The pieceswere fixed in 2.5% glutaraldehyde, postfixed in 1%osmium tetroxide, dehydrated and subjected to critical-point drying using liquid CO2 substitution. The dried spe-cimens were coated with gold and examined and photo-graphed in a Zeiss DSM 940 scanning electronmicroscope at 15 kV. As in the cow, the oviductal mucosaexceeded the free margin in the external side of the infun-dibulum, forming a continuous band measuring 0.25 to 1cm in width. This oviductal epithelium showed cyclicalvariations with a predominance of ciliated cells during thefollicular phase. Differences with the cow include thetransition between the mucosa and serosa, which wasabrupt in the cow and gradual in the sow, with oviductalepithelium penetrating between mesothelial cells, bothindividually or in groups forming islets. Mesothelial cellsshowed also morphological peculiarities in the sow: theywere flat or slightly prominent and were covered either byshort microvilli with low density, allowing observation ofcell plasma membrane, or by a dense mat of medium/longmicrovilli, making it difficult to appreciate the limits bet-ween cells. Additionally, the presence of solitary cilia inmesothelial cells was frequently observed in transitionalareas. In the mesothelial side numerous bulbous processeson the exposed surface of mesothelial cells were fre-quently detected, suggesting a secretory-like activity. Thiswork describes the peritoneal mesothelium covering theinfundibulum in the sow, showing numerous peculiaritieswhen compared with observations previously performedin the cow.

Embryology

P-71 PROTEOMIC A N A LYSIS OFTHE G A L L U SG A L L U S E M B RYO AT S TAGES 13H H, 21H H,2 9H H AND 40H H O FD E V E L O P M E N T.

Agudo Garcillán, D., Díaz Gil, G., GómezEsquer, F., Delcán Girádlez, J., Santamaría Her-nández, B., Palomar Gallego, Mª.A., LinaresGarcía-Valdecasas, R.

Área de Anatomía y Embriología Humana, Departa -mento de Ciencias de la Salud III, Facultad de Cien -cias de la Salud, Universidad Rey Juan Carlos.


54 European Journal of Anatomy, 2 0 0 5 , vo l. 9 (Suppl. 1)

The chicken embryo is one of the most widely usedexperimental model organisms and has been for manyyears the most advanced model organism suitable forexperimental embryology. Moreover, the chicken repre-sents the model system which, permitting experimentalintervention in ovo, most resembles other higher vertebra-tes. As such, it represents an important complement tomouse model systems (Gregory, C. et al, 1998).

Measuring gene expression at the protein level ispotentially more informative than mRNA analysis. Incontrast with the genome, which is essentially static, aproteome is highly dynamic. Processes such as differen-tiation, cell activation, disease or invasive infections canall significantly change the relative protein repertory.

In order to understand the molecular mechanismsunderlying the normal and abnormal development of thechicken, we used two-dimensional electrophoresis (2-DE)to construct a proteome reference map of different stagesof development (Hamburger, V. and Hamilton H. L. 1992)(Kawakami, Y. et al, 2003).

Proteins were separated by isoelectric focusing oninmobilized pH gradient (IPG) strips, and by 11% sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Protein identification was done by peptidemass fingerprinting with matrix assisted laserdesorption/ionization-time of flight-mass spectrometry( M A L D I - TOF-MS) (Agudo et al, 2004).

In all, 4 stages of embryonic development were com-pared (stages 13, 21, 29 and 40), and were detected about300 spots that appears in different stages, or in differentamounts in each studied stage.

These maps will be update continuously and will serveas a reference database for several investigators, studyingchanges at the protein level under different physiologicalconditions. These results suggest that the proteomic appro-ach is valuable for the study of the embryonic development.

P-72 B M F, A P R O A P O P TOTIC “BH3-ONLY ”PROTEIN IDENTIFIED DURING CHIC-KEN EMBRYOGENESIS.

G ó m e z - E s q u e r, F., Díaz-Gil, G., Santamaría, B.,Agudo, D., Delcán, J., Palomar, M.A., Linares, R.

Universidad Rey Juan Carlos (Madrid).

Apoptosis, a process critical for sculpting organsduring development and ensuring homeostasis throughoutlife, has been conserved during evolution. The “BH3-only” proteins are proapoptotic members of the Bcl2-family that share with their relatives only the short BH3domain (9-12 amino acids), which allows their interactionwith prosurvival Bcl-2 family members to trigger apopto-sis. Genetics experiments have demonstrated that they areessential for initiation of programmed cell death andstress-induced apoptosis and are highly conserved in spe-cies as distantly related as C. elegans and H. sapiens.However outside the mammals, just a few reports about“BH3-only” have been published. Recently, a draft geno-me sequence of Gallus gallus has been published, provi-ding a new perspective on vertebrate genome evolutionand consolidating the chicken as an important modelorganism that bridges the evolutionary gap between mam-mals and others vertebrates. In this context, we report herethe identification and characterisation of BMF fromGallus gallus. The amino acid sequence of chicken BMF

show 61%, 43% and 60 % identity to human, mouse andrat BMF, respectively. The very high interspecies conser-vation of BH3 domain (100% identity) and DCL2-bindingmotif (100% identity) is consistent with an involvementof theses regions in the functional activity. Exogenousexpression of BMF caused apoptotic cell death in two dif-ferent cell lines and mRNABMF expression was detectedalong all whole embryo and tissues at different stages ofdevelopment. In addition, a novel isoform has been detec-ted (sBMF: short BMF). The sBMF amino acid sequencewas identical to the BMF except for a deletion of 75 aa(from 31 to 106 aa), including the DCL2-binding motif.The developmental expression pattern and the ability toinduce apoptosis of this isoforms were also examined.

P-73 P R O A P O P TOTIC BH3 ONLY B C L - 2FAMILY MEMBERS IN GALLUS GALLUS,A MODELORGANISM FOR INVESTIGA-TION OF EMBRYONIC DEVELOPMENT.

Díaz-Gil, G., Gómez-Esquer, F., Agudo, D., San-tamaria, B., Delcan, J., Palomar, M.A., Linares, R.

Dpto. Ciencias de la Salud III. Área de Anatomía yEmbriología Humana. Universidad Rey Juan Carlos.Alcorcón (Madrid).

Apoptosis is an evolutionary conserved process invol-ved in proper embryonic development, tissue homeostasisand also in the pathogenesis of a variety of diseases. TheBcl-2 family is an important regulator of apoptosis thatcan be grouped into subfamilies of pro-survival and pro-apoptotic molecules. Inducers of apoptosis membersinclude BH3-only proteins. These proteins have been sug-gested to play an important role in initiating mitochon-dria-mediated apoptosis. At least eight “BH3-only” havebeen identified in mammals, whereas in C. elegants, a sin-gle “BH3-only” protein, EGL-1, is required for the initia-tion of all developmentally programmed deaths. From thecomplexity of the mammals to the simplicity of the inver-tebrates, other organisms exist in which apoptosis mecha-nisms are clearly conserved. From an evolutionary stand-point, the chicken is well positioned model organism toprovide an intermediate perspective to understand a pro-cess as complicated as apoptosis. The recent publicationof a draft genome sequence of Gallus gallus, togetherwith the accessibility of chick embryos, consolidating thisorganism as an important model mainly for developmentexperimentation.

To identify chicken orthologs of mammalian BH3-onlygenes, we search at the TIGR Chicken genome database forsequences that encode conserved BH3 domain. The searchrevealed three different ORFs with a putative BH3 onlyregion (70% homology with mammalians BH3 domains).Two of then correspond to Bim and Bid human orthologs.The other one, named Bcr, correspond to a new BH3-onlywith no mammalians orthologs. The proapoptotic activityof chicken BH3-only proteins was demonstrated by rapidinduction of apoptosis after transiently transfection in HeLaand MCF-7 cell lines. The mRNA expression of chickenBH3-only were detected along different embryo stages andtissues examined suggesting a role for these proteins in theregulation of cell life and death in various types of cellsduring the developmentall process.

These results indicated the functionality of BH3-onlymammalian orthologs in chicken and the expression of



these genes during the embryonic development providinga valuable genetic tool and preparing the way for the stu-dies of their roles in the development and even in disease.

P-74 FETAL DEVELOPMENT OF HORNER’SMUSCLE.

Peces Peña, M.D.1, De la Cuadra Blanco, C.1,Ruiz Mezcua, J.R.2, Merida Velasco, J.R.1

1 Universidad Complutense de Madrid. 2 UniversidadAlfonso Décimo El Sabio

In the study of 18 human fetuses ranging from 38-137mm CR development we have tried to analyse and syste-matise the development of the Horner’s muscle.

At the beginning of the fetal period, 9t h week of deve-lopment, we observed myoblasts at palpebral level whichformed the primordium of the orbicular muscle of the eye-lids. At the 10t h week, coinciding with the appearance oflumen in the lacrimal system, muscular fibres around thelacrimal canaliculi were observed. Alax mesenchyme wasinterposed between the muscular fibres and the canaliculi.

The presence of the lacrimal portion of the orbicularmuscle dorsal to the lacrimal sac was observed from the12th week onward. The fibres of Horner´s muscle ran dor-sal to the lacrimal sac, surrounded the canaliculi and werecontinuous with the fibres of the orbicularis oculi muscle.At the 13 th week, the mesenchyme which surrounded thecanaliculi and lacrimal sac condensed and the direct ten-don of the medial palpebral ligament appeared on theanterior lacrimal crest. The reflex and direct tendon of themedial palpebral ligament was observed at the 15th weekof development.

We concur with Fernández Valencia and Gómez Pelli-co (1990) in that the fibres of Horner´s muscle are notfixed in the lacrimal sac but run dorsal to it, while the pre-tarsal fibres of the orbicular of the eyelids are fixed in themesenchyme which surrounds the the lacrimal sac.

Shinolara and col. (2001) point out that the fibres of Hor-ner´s muscle reach the external palpebral ligament. Howe-v e r, we believe that Horner´s muscle is a clearly diff e r e n t i a-ted part of the orbicular muscle of the eyelids and althoughits deep portion surrounds the canaliculi, its superficial fibresare continuous with the orbicular muscle of the eyelids.

P-75 EFFECTS OF PULSED MAGNETICFIELDS ON EYE DEVELOPMENT.

Roda-Moreno, J.A., Roda-Murillo, O., Pas-cual-Morenilla, M.T., López-Soler, M.

Universidad de Granada.

Little is known about the harmful action of magneticfields on eye development. Fertilized eggs of Gallusdomesticus were exposed during incubation to a harmonicsignal of 10mT intensity and 50 or 100 Hz frequency,acting for 1 s with a pause of 0.5s. Embryos were dividedinto two groups. One was exposed to pulsed magneticfield for only the first 15 days of incubation and the otherthroughout incubation (21 days). Embryos were extractedat 15 and 21 days of development. The following parame-ters of the eye were studied by ultrasound: anteroposteriordiameter of cornea, anterior chamber of eye and antero-posterior diameter of lens and eye.

At 15 days of incubation, the anteroposterior diameterof the lens alone was significantly (p< 0.01) larger inembryos exposed to a 10mT 50 Hz field than in controls.In embryos exposed to a 10mT 100 Hz field, the anteriorchamber of eye was significantly smaller (p< 0.05).

At 21 days of incubation, the group exposed to a 10mT50 Hz field for 21 days had significantly (p<0.05) larg e ranterior chamber of eye and anteroposterior diameter ofcornea versus controls. In the group exposed to 10mT 1 0 0Hz field, the anterior chamber of eye and the anteroposteriordiameters of cornea and eye were significantly smaller thanin controls (p<0.05; p<0.01 and p<0.05, respectively).

In the group exposed for only the first 15 days of incu-bation, only the anteroposterior diameter of the eye sho-wed a significant difference versus controls (p< 0.05).

These findings indicate that pulsed magnetic fields of100 Hz affect the development of the eye.

P-76 PULSED MAGNETIC FIELDS A N DEMBRYONIC DEVELOPMENT.

Roda-Murillo, O., Roda-Moreno, J.A., López-Soler, M., Pascual-Morenilla, M.T.

Universidad de Granada.

The possible effects of magnetic fields on embryonicdevelopment have been well studied. However, whereassome authors found no alterations in embryos exposed tolow-frequency pulsed magnetic field, others reported thatthis exposure produced embryonic malformations.

We exposed fertilised Gallus domesticus eggs duringtheir incubation to pulsed magnetic fields of 10mT inten-sity and 50 or 100 Hz frequency, acting for 1 s with apause of 0.5 s.

Embryos were extracted from both groups at 45 h ofincubation and somite count and development stagingwere performed.

At 15 days of incubation, further extractions weremade to assess the somatic weight and development stage(third-finger measurement). Also at 15 days of incubation,half of the exposed eggs were moved to another incuba-tor, removing them from the action of the magnetic fields.

In exposed embryos extracted at 45 h of incubation,there were significantly fewer somite pairs versus controls(p<0.05); somatic weight and development stage of embr-yos extracted after 15 days of exposure to 50 Hz fieldwere significantly higher versus controls (p<0.05).

At 21 days of incubation, the group exposed to a 50Hz field throughout incubation had a lower somaticweight versus controls (p<0.05), while those subjected toa 100 Hz field significantly differed from controls both insomatic weight and development stage (p<0.01 andp<0.05, respectively).

In the group removed from exposure at 15 days, therewere no statistical differences with controls in somaticweight or development stage.

Our findings support the hypothesis that magneticfields act on embryonic development.

P-77 TGF-b1 AND CELLDEATH IN THE PALA-TAL MIDLINE EPITHELIAL SEAM ANDDENTAL KNOT.

Barrio, C., Del Río, A., González, I., Trinidad,E., Murillo, J., Izquierdo, C., Amorós, M., Gar-



cillán, B., Resel, E., López, M., Martínez-Sanz,E., Martínez-Álvarez, C.

Dpto. Anatomía y Embriología Humana, I. Facultadde Medicina. Universidad Complutense de Madrid.

During embryonic development, cell death is a crucialprocess that allows the correct morphogenesis of severalo rgans, such as the palate and the tooth. T G F -b1 is a growthfactor highly involved in the appearance of cell death, butalso of cell survival, differentiation and the production ofextracellular matrix molecules. All these events take placeduring the palatal midline epithelial seam (MES) and den-tal knot formation and disappearance. In order to gaininsight on the role that T G F -b1 can play during the deve-lopment of these structures, we aimed to compare in themthe presence of T G F -b1 and cell death. We labeled embryo-nic day 14.5 mouse gelatin sections with an antibodyagainst T G F -b1, or with T U N E L (terminal deoxynucleo-tidyl trasnsferase-mediated d-UTP nick-end labeling) orwith both, and observed the sections using both an opticand confocal microscope. Our results show temporospatiald i fferences in the expression of T G F -b1 in the MES, andthat its highest expression is, as for the dental knot, stri-kingly related to the presence of cell death. We thereforesuggest that T G F -b1 is likely having a role as cell deathinductor in these structures, although experimental in vitroanalysis are needed to confirm our suggestion.

This work has been supported by Fondo de Investiga -ción Sanitaria, ref.: PI 03/0185, and Comunidad Autóno -ma de Madrid, ref.: GR/SAL/0539/2004.

P-78 ROLE OF THE MEDIAL EDGE EPITHE-LIUM EXTRACELLULAR MATRIX INTHE ORIGIN OF THE CLEFT PALATEPRESENTED BY TGF-b3 NULL MUTANTMICE.

Martínez-Sanz, E., Resel, E., Trinidad, E.,Izquierdo, C., López, M., Garcillán, B., Amo-rós, M., Del Río, A., Barrio, C., Martínez, M.,Murillo, J., Martínez-Álvarez, C.

Universidad Complutense de Madrid. Spain.

TGF-b3 null mutant mice show all the possible kind ofcleft palate, from complete to submucosa, depending onthe strain of mice where the gene is targeted, thus impl-ying different degrees of ahdesion/fusion of palatal shel-ves. Our aim has been to analyse the presence/distributionof several extracellular matrix and adhesion molecules intwo strains of TGF-b3 null mutant mice to investigatetheir importance in the process of palatal shelf adhesionand fusion. We have performed immunohistochemistrywith antibodies against fibronectin, laminin, colagens IVand IX, a5 and b1 integrins and ICAM-1 on heads of embr-yonic day (E) 14.5 Albino Swiss and C57 wild type andTGF-b3 null mice. In addition, we have performed E13.5palate cultures, treated or not with TGF-b3 or treated withblocking antibodies against fibronectin or the integrins a5and b1. Our results show differences in the presence of themolecules analysed between the wild type and null mutantpalates, more severe in the C57 (the one having completecleft palate) than in the Albino-Swiss strain (where thecleft palate is incomplete). The addition of TGF-b3 res-cues the normal presence of these molecules. The use ofblocking antibodies against fibronectin and the a5 integrin

results in a statistically significant decrease of the adhe-sion and fusion of the palatal shelves. We therefore con-clude that the presence of some extreacellular matrix andahdesion molecules is very important to palatal shelfadhesion/fusion and that is part of the pathogenesis of thecleft palate presented by the TGF-b3 null mice.

This work has been granted by the Comunidad Autó -noma de Madrid (Ref. GR/SAL/0539/2004) and Fondo deInvestigación Sanitaria (PI030185).

P-79 EXCESS OF TGF-b1 AND GENESIS OFTHE CLEFT PA L ATE PRODUCED INTHE ABSENCE OF TGF-b3.

Murillo, J., Barrio, C., Del Río, A., Trinidad,E., Izquierdo, C., Amorós, M., Garcillán, B.,López, M., Resel, E., González, I., Martínez-Sanz, E., Martínez-Álvarez, C.

Departamento de Anatomía y Embriología HumanaI. Facultad de Medicina. Universidad Complutensede Madrid.

T G F -b1 is a growth factor highly involved in both cellproliferation and death. In the mouse, T G F -b1 is observedin the epithelium covering the tips of palatal shelves(medial edge epithelium –MEE–) and in the midline epit-helial seam (MES) that forms when they contact in the mid-line, but is weakly seen in the underlying mesenchyme.H o w e v e r, in the T G F -b3 null mutant mouse, that bear cleftpalate, T G F -b1 is strikingly increased, both in MEE andmesenchyme. There is a great decrease in the presence ofdead cells in the MEE of these mice with respect to the wildtype, and cell death is crucial to the MES disappearanceand the final fusion of palatal shelves. The aim of this workwas to determine whether excessive T G F -b1 is able todecrease MEE cell death during in vitro palatal fusion, whatwould give it a role in the persistence of the MES observedin those T G F -b3 null mice that have incomplete cleft pala-te. We have performed embryonic day 13.5 paired palatalshelf cultures, adding or not a supraphysiological doses(–400 ng/ml– physiological doses: 10 ng/ml) of T G F -b1 t othe culture medium. After 18 hours, cultures were fixed andembedded in paraffin. Sections were labeled with T U N E L( T d T mediated dUTP Nick End Labeling) to detect deadcells in the MES. Using a Zeiss Axioplan 2 Microscope, theMES area of each culture was measured and T U N E Lp o s i-tive cells present per area were counted. Acquisition of theimages and statistical treatment were performed by usingthe MetaMorph 5.07 and SPSS 12.0 programs respectively.Our results show a significant (p < 0.01) decrease in thenumber of dead cells in the MEE of T G F -b1 treated cultu-res related to controls. We have also found significantdecrease of the MES formed, what suggests that the excessof T G F -b1 may also reduce palatal shelf adhesion.

This work has been supported by Fondo de Investiga -ción Sanitaria, ref. PI 03/0185, and Comunidad Autóno -ma de Madrid, ref.: GR/SAL/0539/2004.

P-80 TOWARDS A NON SURGICAL TREAT-MENT FOR CLEFT PALATE?

Resel, E.1, Alonso-Bañuelos, C.2, Lagarón, E.3,González-Meli, B.4, Martínez-Sanz, E.1, Trini-



dad, E.1, Izquierdo, C.1, Amorós, M.1, López,M.1, Garcillán, B.1, Martínez-Álvarez, C.1

1 Universidad Complutense de Madrid. 2 HospitalUniversitario Gregorio Marañón. 3 Hospital Doce deOctubre. 4 Hospital Infantil Universitario Niño Jesús.

Cleft palate is a congenital malformation whose currenttreatment includes several surgical procedures throughoutthe patient’s life. We are trying to find an alternative met-hod to surg e r y. Our hypothesis is that once early orthopediahas helped the approache of the cleft palate edges, the i ns i t u sinthesis of extrabone could allow their total contact inthe midline. Inmediately, the chemical removal of the epit-helium covering these edges (medial edge epithelium–MEE–) would permit the contact of the underlying con-nective tissues and the adherence of the cleft palate tips,thus resembling a normal palate.

OBJECTIVES: 1) To explore the possibility of inducingin vitro with BMP new osteogenic cells from the fibro-blasts located at the edges of the cleft palate. 2) To remo-ve the MEE and to achieve the subsequent adhesion of theunderlying connective tissue.

METHODS: We used as experimental models the cleftpalate presented by TGF-b3 null mouse and small frag-ments of tissue obtained during surgery from the edges ofhuman cleft palates. First we placed heparin beads soakedwith BMP2 below the MEE of embryonic day 18 (E18)TGF-b3 null mouse cleft palates and cultured these speci-mens. The presence of induced osteogenic cells was iden-tified by immunolabelling with an anti-osteocalcin anti-body. We then removed the MEE from both E18 TGF-b3null mouse cleft palates and fragments from human cleftpalates with a trypsin solution, followed by apposition ofthe opposing underlying tissues.

RESULTS: 1) The fibroblasts located at the edges of thecleft palate can be induced to form osteogenic cells. 2) Itis possible to remove the MEE covering these edges andto achieve subsequent adhesion of the underlying tissues.

CONCLUSION: These in vitro results allow to hypothe-sise that a future non surgical treatment for the cleft pala-te is possible.

Supported by CAM (GR/SAL/0539/2004) and FIS(PI030185)

P-81 EFFECTS OFPROINSULIN A D M I N I S T R A-TION ON CARDIOGENESIS IN DEVELO-PING CHICK.

Lopez-Sanchez, C.1, Mansilla, A.2, Hernandez-Sanchez, C.2, Garcia-Martinez, V.1

1 Human Anatomy and Embryology, Faculty of Medi -cine, University of Extremadura, Badajoz. 2 Depart -ment of Cell Biology and Development, CIB-CSIC,Madrid.

Proinsulin gene expression regulation and functionduring early embryonic development are remarkably dif-ferent from those found in postnatal organisms. Duringdevelopment, apoptotic cell death is temporally and spa-tially regulated during proliferative stages of neurogene-sis, and embryonic proinsulin is presumably an endoge-nous protective factor. Here we characterize a newproinsulin mRNAvariant generated by retention of intron1 in the 5’ untranslated region. Inclusion of intron 1 didnot affect nuclear retention or cytoplasmic decay but it

inhibits proinsulin translation almost completely. Thenovel proinsulin mRNAisoform expression was develop-mentally regulated and tissue-specific. The proportion ofintron retention increased from gastrulation to organoge-nesis, and was highest in the heart tube and presomiticregion, whereas it could not be detected in pancreas. Weimplanted proinsulin-coated beads into the lateral limit ofthe precardiac region of chick embryos. Proinsulin led toformation of ectopic tissue encircling the bead. In situhybridization showed that this tissue expressed VMHC1,a marker of differentiating cardiac myocytes, later restric-ted to ventricular myocytes. These effects were observedregardless of the distance of the bead to the host heart,relatively distant or near. Moreover, the ectopic tissuecontained cells positive by immunohistochemistry forMF20, a myosin heavy chain normally expressed in mus-cle cells in the myocardium of the major chambers. Proin-sulin beads did not induce expression of other mesoder-mal markers such as Shh, specific to the axial mesoderm,or paraxis, specific to lateral mesoderm. These resultsindicate that proinsulin induced ectopic expression of car-diac lineage markers and that it could thus play a crucialrole in cardiogenesis. Proinsulin did not act as an initialinductor of non-specified mesoderm, which later coulddifferentiate into cardiac mesoderm. These results are inaccordance with previous studies in which insulin inducesterminal cardiac differentiation. We propose that regula-ted unproductive splicing and translation is a mechanismfor regulating proinsulin expression, according to specificrequirements in developing vertebrates.

P-81b DEVELOPMENT OF CHICK CARDIOM-YOCYTES: MODULATION OF I N T E R-MEDIATE FILAMENTS BY ALL-TRANSAND 13-CIS RETINOIC ACID.

Vélez, C.1, Melguizo, C.1, Prados J.C.2, Mar-chal, J.3, Carrillo, E.2, Boulaiz, H.3, Fernández,J . E .2, Martinez, A .3, Rodriguez-Serrano, F.3,Suárez, I.3, Caba, O.2, Alvarez, L.2, Aránega, A.2

1 University of Almería. 2 University of Granada. (3)University of Jaén.

Retinoids, the active metabolites of vitamin A, regula-tes complex gene networks involved in vertebrate morp-hogenesis, growth, cellular differentiation, apoptosis andhomeostasis. Early heart development is known to be sen-sitive to retinoid. Retinoic acid (RA) provides a criticalsignal to allow progression of heart developmento to adultphenotype. The molecular mechanism by which RAexertits biological function involves specific binding to intra-cellular proteins and interaction with nuclear receptor tomodulate the transcription of specific genes. Although theinfluence of retinoids on cardiac morphogenesis has beendescrived previously, the effect of retinoids on cardiom-yocyte differentiation and gene expression during deve-lopment is largely uncharacterized. In order to test theeffect of all-trans RA (ATRA) and 13-cis RAon cardiom-yocyte differentiation, we studied the level and the subce-llular compartimentalization of intermediate filamentsdesmin and vimentin in cultures of chick embryo car-diomyocytes obtained from Hamburger and Hamilton´s(HH) stage 22, 32 and 40 embryos. Fertile white leghornchicken eggs (Shaver Star cross 288) were incubated at38.5°C in a humid atmosphere. Hearts were ablated whenthe embryos had reached HH22, HH32 and HH40. Chick



cardiomyocyte were isolated and treated during 24 and 48h with ATRAand 13-cis RAto a final concentration of 10-6 M in the growth medium. Sodium dodecyl sulfate-gelelectrophoresis, immunoblotting and fluorescense activa-ted cell sorter analysis (FACScan) were used to quantifythe effects of retinoids on cultures of chick cardiomyocy-tes during development. In general, the retinoids increa-sed the concentration of desmin and reduced of vimentinin cardiomyocytes cultured at all three stages of develop-ment. The effects of RAwere time-dependent. Our resultsshown a marked decrease in vimentin levels at HH22 cellscultured for 24 and 48h in both, cytoplasmic (CP) andcytoskeletal (CS) compartments. The greatest increases indesmin concentration ocurred in more mature stages(HH40) cells exposed to retinoids. We found that after48h of RA treatment the increases in desmin levels werelarger in the CS than in the CPfraction. These findings arefurther evidence that RA plays a modulating role in thereorganization of cytoskeletal proteins during the processof cardiomyocyte maturation.

P-82 A N A LYSIS OF THE EMBRY O L O G I C A LD E V E L O P M E N T O F THE CHICKENLIVER USING T W O - D I M E S I O N A LR E F E-RENCE MAPS.

Delcán Giráldez, J., Santamaria Hernandez,B., Agudo Garcillán, D., Díaz Gil, G., GómezEsquer, F., Palomar Gallego, M.A., LinaresGarcía-Valdecasas, R.

Facultad de Ciencias de la Salud, Universidad ReyJuan Carlos, Alcorcón, Madrid.

Liver development needs to be analysed in depth formedical and scientific reasons because it is a very interes-ting embryological process. In order to begin to unders-tand the molecular mechanisms underlying the normaland abnormal development of the chicken, we used two-dimensional electrophoresis (2-DE) to construct a proteo-me reference map. Proteins were separated by isoelectricfocusing on inmobilized pH gradient (IPG) strips, and by11% sodium dodecyl sulfate-polyacrylamide gel electrop-horesis (SDS-PAGE) gels. 2-DE reference maps are veryuseful for these studies because most of the main proteinscan be identified.

The chicken (Gallus gallus) embryo is one of the mostwidely used experimental model organisms and has beenfor many years the most advanced model organism suita-ble for experimental embryology. Moreover, the chickenrepresents the model system which, permitting experi-mental intervention in ovo, most resembles other highervertebrates. As such, it represents an important comple-ment to mouse model systems.

The liver 2-DE reference map compared with thecomplete embryo 2-DE reference map, let us identifiedprotein not present in the complete embryo map. Thisanalysis allowed us to identified 1800 new spots in thechicken embryo proteomic map.

When we compare2-DE liver reference map of the sta-ges 30 and 38 we observed differences in the proteins pre-sent at these two stages possibly owing to liver develop-mental processes. The differences observed between liverand complete embryo chicken maps could be due to liverspecific proteins. This possible result could be very usefulin order to study metabolic paths involved in the liverembryological development.

P-83 THE URORECTA L S E P TATION COM-PLEX OF THE HUMAN EMBRYO.

Nebot-Cegarra, J.1, Fàbregas, P.J.2, Sánchez-Pérez, I.2

Universidad Autónoma de Barcelona, Facultad deMedicina: 1 Unidad de Anatomía y de Embriología; 2

Unidad de Bioestadística.

In order to analyze the patterns of cellular prolifera-tion both in the mesenchyme of the urorectal septum(URS) and in the adjacent territories (posterior urogenitalmesenchyme, anterior intestinal mesenchyme and cloacalfolds mesenchyme), as well as their contribution to theprocess of cloacal division, a computer-assisted methodwas used to obtain the nuclear area of 3874 mesenchymalcells from camera lucida drawings of nuclear contours ofselected sections of human embryos (Carnegie stages(CSs) 13-18). Based on changes in the size of the nucleusduring the cellular cycle, we considered proliferating cellsin each territory to be those with a nuclear area over the75th percentile and from them we determined by embryoand territory the rate of proliferating cells that has beenused as comparative parameter. The URS showed increa-sing cell proliferation, with proliferation patterns thatcoincided closely with cloacal folds mesenchyme, andwith less overall proliferation than urogenital and intesti-nal mesenchymes. Furthermore, at CS 18, we observedthe beginning of the rupture in the cloacal membrane;however, no fusion has been demonstrated either betweenthe URS and the cloacal membrane or between the cloa-cal folds. The results suggest that cloacal division dependson a morphogenetic complex where the URS adjacentterritories could determine septal displacement at the timethat their mesenchymes could be partially incorporatedwithin the proliferating URS.

P-84 D I F F E R E N T I ATION AND EMBRY O N I CDEVELOPMENT IN THE DIFFERENTIA-TION-BASED CANCER THERAPY.

M a rchal, J.A.1, Boulaiz, H.1, Campos, J.2,Caba, O.3, Rodríguez-Serrano, F.1, Martínez-Amat, A.1, Madeddu, R.4, Carrillo, E.3, Prados,J.C.3, Melguizo, C.5, Vélez, C.5, Fernández,J.E.3, Aránega, A.3

1 Dept. de Ciencias de la Salud, Universidad de Jaén.2 Dept. de Química Farmacéutica, Universidad deGranada. 3 Dept. Anatomía y Embriología Humana,Universidad de Granada. 4 Dip. Sci. Biomed.–Sez.Anat. Istol., Universitá di Sassari (Italy). 5 Dept.Neurociencias y Ciencias de la Salud, Universidadde Almería.

Differentiation is a complex multistep process of cellspecialization that begins with the installation of a geneticprogramme, named determination, specific for a cell line-age. Terminal differentiation is the end stage of this pro-cess where the cells irreversibly lose their proliferativec a p a c i t y. In embryonic development, diff e r e n t i a t i o nbegins with early determination processes on the cleava-ge divisions in the egg reaching intermediary levels of dif-ferentiation. Finally, after amplification divisions, eachcell lineage mature to specialized cell types and tissues.The knowledge of the mechanisms involved in differen-tiation and malignant transformation has allowed thesearch of alternative routes for antitumoural therapy that



does not imply cellular death. In this study we tackle dif-ferent aspects of differentiation from a view point ofembryonic development, the relationship with the neo-plastic transformation and the application of this know-ledge to the anticancer differentiation therapy, usingagents that re-enter the tumoural cells into the normalpathway of development.

We present a series of natural and pharmacologicalagents, enhancers of differentiation used in clinical trialsor recently proved in experimental assays. Our resultsindicate that the use of low doses of antineoplastic drugscan induce therapeutic differentiation in some humantumours and the synthesis of new differentiative drugsfrom well-known chemical structures allowed the lesse-ning of the adverse cytotoxicity. Analogues of purines andpyrimidines inhibit the growth of tumour cells, interferein the synthesis and metabolism of nucleic acids acting onkey enzymes for the synthesis of DNAand induce cell dif-ferentiation. We used specific markers of normal muscu-lar differentiation to decide if these compounds led to there-entry of the tumoural cells into the normal pathway ofdevelopment. These 5-FU derivatives induced myogenicdifferentiation in the rhabdomyosarcoma human cells notinducing the multidrug resistance phenomenon, indicatingthat this type of tumour may be amenable to treatment bydifferentiation therapy.

We conclude that the better understanding of the mole-cular mechanisms responsible for the multistage processof differentiation, embryonic development and neoplastictransformation, will be allowed a more rational clinicalapplication of differentiation cancer therapy.

Nervous System

P-85 TGF-BETA FAMILY DURING NEURULA-TION AND DEVELOPING SPINALCORD.

Rabadán, M.A., Mecha, M., Alonso, V., Pérezde Miguelsanz, J., Maestro, C., Blanco, M.J.

Universidad Complutense de Madrid.

Development of the nervous system involves a serie ofmorphogenetic steps leading to the formation of highlyorganized structures such as the brain and spinal cord. Thetransforming growth factor (TGF-bs) is involved in theregulation of crucial processes through the regulation ofproliferation, cell movements along the radial glia andneuronal differentiation. It has been reported that TGF-b2and -b3 are present in glial cells and along developingtracts, being considered as the “neural” TGF-bs isoforms,while TGF- b1 is normally restricted to the meninges andcoroid plexus. However, some functions attributed toTGF-b1 like cell-cycle control, neuron survival, extrace-llular matrix formation and differentiation, support thehypothesis that this factor might have important rolesduring the formation of the central nervous system (CNS).We have studied the expression of the three isoformsduring neurulation and early stages of spinal cord deve-lopment (E7.5- E13.5 d.p.c.). Our results indicate that theexpression of TGF-betas shows a temporal and spatialdynamic pattern. During neurulation, T G F -b1, -b2 and -b3proteins were present in the neuroepithelium, althoughT G F -b3 showed the weakest immunoreactivity (IR). In thespinal cord, the three isoforms were detected on diff e r e n tspatial localization. As expected, T G F -b2 and T G F -b3 sho-

wed overlapping areas of expression at the basal plateregion of the spinal cord, the ventral commissure, dorsalfuniculus, ventral root fibers and fibers at the marginallayer (E13.5). The expression pattern of TGF-b1 is detec-ted quite homogeneously within the spinal cord at E11.5,while at E13.5 the periventricular region, and the half dor-sal zone of the spinal cord showed more intense IR. TheTGF-b1 protein is observed at the region where prolifera-tion and migration of neuroblasts occur as detected byBrdU staining. However, the pattern of TGF-b2 and -b3 iscompletely different, related to axons and with the fibercomponent of the spinal cord mantle. Together with theknown biological activities of TGF-bs, we propose thatthese factors are important mediators of cell-cell interac-tion during development of the CNS and that the presen-ce of these isoforms might be related to migration of neu-ral cells on a glial substrate.

P-86 BRAIN DEVELOPMENT AND TGF-BETAFAMILY.

Mecha, M., Rabadán, M.A., Alonso, V., Peña,A., Blanco, M.J.

Universidad Complutense de Madrid.

The transforming growth factor isoforms (TGF-b1, -b2and b3) are the most abundant cytokines in the central andperipheral nervous system. A high degree of overlappinghas been described in the expression pattern of T G F -b2and -b3 in the telencephalic cortex, mesencephalon andcerebellar anlage, detected along peripheral nerves, inradial glial cells and along central nervous system (CNS)axon tracts. In contrast, TGF- b1 expression seems to beconfined to the meninges and coroid plexus in the unlesio-ned nervous system. According to their expression pattern,it has been proclamed that a possible role in the control ofcell division is excluded since no one of the isoformsseems to be detectable in the periventricular zones throug-hout the CNS. However, studies in vitro suggest that T G F -bs play a role in proliferation although positive or negati-ve effects depend on culture conditions. We propose tostudy the pattern of T G F -b1, T G F -b2 and T G F -b3 proteinexpression detected by immunostaining on free-floatinggelatin sections, a system that provides mainly two advan-tages: it avoids epitope retrieval neccessary in paraffin sec-tions, and detect weak immunoreactivity due to the use of50 mm thick vibratome slices. We have studied mousebrains of embryonic stages ranging from E11.5 to E14.5.In our experimental system, T G F -b1 immunoreactivity(IR) was observed in the ventricular zone in the prosen-cephalon, mesencephalon and rombencephalon. The IRseems to be similar to the proliferation pattern observed byBrdU staining. The T G F -b2 isoform is expressed in ahomogenous and intense pattern in the CNS, with peak ofexpression in the eye lens, the pallium/subpallium of thetelecephalic cortex and some encephalic regions whered i fferentiation is more advanced (intermediate and marg i-nal zones). T G F -b3 is clearly visible at the fibers of thecephalic marginal zone. In summary, all the three isoformscan be detected in the embryonic nervous system, with ad i fferential and dynamic expression pattern. In addition,we have found a striking, not yet reported, complementarypattern of expression between these two isoforms. In thiss t u d y, we analyse the possible functions of T G F -bs in theCNS during mouse embryogenesis.



P-87 A D J U S T M E N T O F THE NUMBER OFCELLS DURING V E RT E B R ATE RETI-NALNEUROGENESIS.

Mayordomo, R., Chavarria, T., Hidalgo Ruiz,S., Lineros, M.C.

Universidad de Extremadura.

During the development of the retina a high number ofcells need to be adjusted to get a normal and funtionalstructure in the adult nervous system. Apoptosis seems tobe one of the major mechanism involes in this specificselectión of funtional neurons for the mature nervious sys-tem to work. The prevalence of programmed cell deathaffecting connecting neurons during late neural develop-ment is well established. On the other hand, the incidenceof apoptosis during neurogenesis on proliferating neuroe-pithelial cells and recently differentiated neuroblasts hasonly recently been recognized.

Using the terminal tranferase deoxiuridine-triphospha-te nick end labelling (TUNEL) as a thechnique and theembryonic chick retina as a model system we have desig-ned several in vivo experimental interferences with natu-rally occurring cell death. In ovo treatment with caspaseinhibitors resulted in decreased apoptosis and increasednumber of retinal ganglion cells.

We then approached the cell death naturally affectingganglion cell generation and found that more than 50% ofrecently born ganglion cells die within 6-9 hours aftertheir last S-phase. While these results demonstrate anearly phase of neurogenic cell death of similar magnitudethan the later occurring neurotrophic cell death, the criti-cal developmental and cellular processes that underlie thisearly phase of cell death during neurogenesis are stillpoorly understood.

P-88 THE CHICKEN CIRCUMVENTRICU-LAR LAT E R O S E P TA L AND PREOPTICORGANS ARE FORMED ALONG THE STRIATO PA L L I D A L M O L E C U L A RB O U N D A RY.

Bardet, S.M., Cobos, I., Puelles, E., Martinez-de-la-Torre, M., Puelles, L.

Departament of Human Anatomy and Psychobiology.School of Medicine. University of Murcia. Campus ofEspinardo. 30071 - Murcia.

The lateroseptal organ (LSO) is a circumventricularspecialization present in the avian and reptilian telen-cephalon (Kuenzel y van Tienhoven, 1982). It has apopulation of liquor-contacting neurons which are V I Pimmunoreactive. Another neighboring circumventricu-lar organ is that of the preoptic recesso. Recent datasuggest that the LSO is associated to extraretinal photo-reception, insofar as its neurons express molecules ofthe opsin family and their activity changes with expo-sure to light. Although according to its name the LSOwould seem to lie in the septum, actually only a smallportion of it has this localization, whereas the larg e s tpart extends below the lateral ventricle and lies in itslateral wall, associated to the ependymal region of stria-tum or pallidum. Its position within the subpallium hasnot been investigated precisely, although this representsa prerequisite for any causal embryological interpreta-tion of the LSO.

To this end, we studied the topography of the chickenLSO relative to the striatopallidal complex, delineatedm o l e c u l a r l y. It is known that during development the sub-pallium generally expresses D l x family genes (in both stria-tal and pallidal portions) and N k x 2 . 1 signal (only in thepallidum and preoptic area), as a substrate for production ofvarious GABAergic neurons. Another subpallial histogene-tic zone, the anterior entopeduncular area (AEP), shows, inaddition to D l x and N k x 2 . 1 , signal of the gen sonic hedge-hog (S h h), a pattern apparently associated to production ofthe cholinergic neurons of the basal forebrain.

Using immunohistochemical and in situ hibridizationtechniques, we mapped calbindin (which identifies earlyon the LSO), the genes Nkx2.1, Nkx2.2, Shh, Dlx5 and theprotein BEN. The results jointly define with precision thetopography of the chicken LSO in the subpallial telencep-halon, show for the first time a group of distal neuronsassociated to the LSO, and provide embriological eviden-ce that this organ actually forms throughout its lengthalong a striatal band parallel and in contact with the mole-cular striatopallidal boundary, as defined by the limit ofNkx2.1 expression. This band is continuous with an extra-telencephalic counterpart along which the preoptic cir-cumventricular organ develops. Accordingly, there seemsto exist a common molecular background for the forma-tion of these two ventricular organs.

P-89 NEURONAL ORIGIN OF PALLIAL ANDS U B PA L L I A L AMYGDALAR SUBDIVI-SIONS.

Legaz, I., García-López, M., Abellán, A., Medi-na, L.

Dept. of Human Anatomy, Faculty of Medicine, Uni -versity of Murcia, Spain.

The amygdala is a very complex structure, located inthe ventrolateral telencephalon and involved in a largevariety of functions, such as control of homeostasis andemotional behavior. The anatomical and functional com-plexity of the amygdala is partially explained by its mul-tiple developmental origins in several histogenetic com-partments (Puelles et al., 2000). Thus, the nuclei/areas ofthe amygdala derive from different sectors of pallium orsubpallium, each one expressing a different combinationof developmental regulatory genes. The complexity of theamygdala is also related to the existence of tangential cellmigrations in the telencephalon, mainly from the subpa-llium to the pallium. The aim of this study has been toinvestigate in mouse the neuronal origin of pallial andsubpallial amygdalar subdivisions, and analyze the contri-bution of tangential migrations to their complexity. Forthat, we prepared organotypic cultures of mouse embryo-nic telencephalon (embryonic day 14.5), and labeled cellclones in different telencephalic ventricular areas, usingthe fluorescent cell trackers CMTMR or CMFDA. Theresults on cell migrations were compared to the differentmolecular domains of the telencephalon, based on expres-sion patterns of the genes Tbr1, Dlx5, Nkx2.1, Lhx6, Lhx7,Gbx1 and Shh. Our results offer further support for themulticompartmental organization of the amygdala, andprovide new data on the specific subpallial origins ofamygdalar nuclei. Thus, the central amygdala originatesin the caudal ganglionic eminence, whereas the postero-dorsal part of the medial amygdala and a related part ofthe extended amygdala originate in the medial ganglionic



eminence plus the anterior entopeduncular area of thesubpallium. In contrast, the basolateral complex of theamygdala originates in the ventrolateral pallium. Further,our results provide evidence for the existence of tangen-tial cell migrations from the subpallium to the pallialamygdala (including the basolateral amygdalar complex).Immigrant neurons of the pallial amygdala appear to inte-grate as different subtypes of GABAergic interneurons,modulating the activity of principal neurons. Finally, wepresent novel evidence indicating the existence of dorso-ventral tangential migrations in the amygdala, furthercontributing to the high complexity of this structure.

Sponsors: MCYT-FEDER (BFI2003-06453-C02-02),Séneca Foundation (PB/50/FS/02), and Health InstituteCarlos III-Red CIEN.

P-90 STUDIES ON THE DEVELOPMENT OFTHE ECTOMAMILLARIS NUCLEUS INTHE CHICKEN.

Félix, E.1, Pérez, L.1, Puelles, L.2

1 Universidad de Las Palmas de Gran Canaria. Dpto.Morfología. 2 Universidad de Murcia. Dpto. de Ana -tomía Humana y Psicobiología.

The Ectomamillaris nucleus (EM) or the nucleus ofthe basal optic root (nBOR), isa diencephalic caudoven-tral nucleus that is located under the Tecto-thalamic tractin reptils (Martinez de la Torre, 1985) in the prosomere 1(Puelles, 1995), but in chicks the EM seems exceed theSinencephalon- parencephalic limit.

This nucleus is very important in the analysis of opticflow and the generation of the optokinetic response, rea-son why we studied its embryiologic origin in the chicken.

In the present study we´ve used chick embryos from 8(HH34) to 19 (HH45) days of incubation. Brains weresectionated in the horizontal plane at 100 mm and then wehave compared parallel series dyed with a mixture ofThionine–Neutral Red with Acetylcholinesterase (AChE),the acetylcholine degradation enzyme.

Expression of AChE starts around embryonic day 6 insome structures and follows mainly increasing time-cour-ses in the chick brain.

P-91 MOLECULAR PROFILING INDICAT E SAVIAN BRANCHIOMOTOR NUCLEIINVADE THE HINDBRAIN ALAR PLATE.

Ju, M.J.1, Aroca, P.2, Luo, J.1, Puelles, L.2,Redies, C.1

1 Institute of Anatomy, University of Duisburg-EssenMedical School, D-45122 Essen, Germany. 2 Dept.Human Anatomy and Psicobiology, School of Medi -cine, University of Murcia, 30100 Murcia, Spain.

It is generally believed that the spinal cord and hind-brain consist of a motor basal plate and a sensory alarplate. We now have molecular markers for these territo-ries. The relationship of migrating branchiomotor neuronsto molecularly defined alar and basal domains was exa-mined in the chicken embryo by mapping the expressionof cadherin-7 and cadherin-6B, in comparison to geneticmarkers for ventrodorsal patterning (Otp, Pax6, Pax7,

Nkx2.2, and Shh) and motoneuron subpopulations(Phox2b and Isl1). We show cadherin-7 is expressed in acomplete radial domain occupying a lateral region of thehindbrain basal plate. The cadherin-7 domain abuts themedial border of Pax7 expression; this common limitdefines, or at least approximates, the basal/alar boundary.The hindbrain branchiomotor neurons originate in themedial part of the basal plate, close to the floor plate.Their cadherin-7-positive axons grow into the alar plateand exit the hindbrain close to the corresponding afferentnerve root. The cadherin-7-positive neuronal cell bodieslater translocate laterally, following this axonal trajectory,thereby passing through the cadherin-7-positive basalplate domain. Finally, the cell bodies traverse the molecu-larly defined basal/alar boundary and move into positionswithin the alar plate. After the migration has ended, thebranchiomotor neurons switch expression from cadherin-7 to cadherin-6B. These findings demonstrate that a spe-cific subset of primary motor neurons, the branchiomotorneurons, migrate into the alar plate of the chicken embr-yo. Consequently, the century-old concept that all primarymotor neurons come to reside in the basal plate should berevised.

P-92 MRI IMAGES OF THE MEDIAL TEMPO-R A L LOBE IN POSTMORTEM FIXEDHUMAN BRAINS: NEUROANATOMICALCORRELATION.

Insausti, A.2, Insausti, R.1, Cabada, T.4, Mansi-lla, F.3, Cortés, F.3, Cros, T.3, Ciriza, M.4, Olier,J.4, Tuñón, T.5, Pro, P1, Mohedano, M.1, Arroyo,M.M.1, Artacho, E.1, Marcos, P.1, Martínez, A.1

1 Human Neuroanatomy Laboratory, Sch. of Medici -ne, UCLM, Albacete. 2 Unidad de Investigación(Lab.Regeneración), Servicio Navarro de Salud,Pamplona. 3 Servicio de Radiología Complejo Hos -pitalario Universitario de Albacete, Albacete. 4 DptoRadiología, Hospital de Navarra, Servicio Navarrode Salud, Pamplona. 5 Dpto. Anatomía Patológica,Hospital de Navarra, Servicio Navarro de Salud,Pamplona.

The anatomical detail provided by modern neuroima-ging techniques open new perspectives on the correlation ofanatomical structures that, because of their saliency, is rela-tively easy to identify with a certainty close to the neuroa-natomical histological detail. We, and others, have reportedd i fferent strategies to segment structures within the medialtemporal lobe in order to assess volume variations inpatients with various pathologies, mainly A l z h e i m e r’s dise-ase and other dementias, and temporal lobe epilepsy.

We report the findings obtained in the post-mortemfixed human brains that were encased in 1.5% A g a r, andexplored with a Siemens 1.5T and with a Philips Intera,also of 1.5T. The sequence used was FLAIR 3D,TR:6000, TE:353, TI:2200, 1mm thickness and FOV260. After completion of the MRI examination, the brainswere freed from the Agar encasing and sectioned in thecoronal plane (orthogonal to the AC-PC line) into 1 cm.thick slabs. The medial temporal lobe was dissected by asection that encompassed from the endorhinal sulcus tothe inferior limiting sulcus in the insula, in such a waythat the whole temporal lobe was separated for sectio-ning. Every slab of the temporal lobe was cryoprotectedwith 10 and 20% glycerol solutions containing


62 European Journal of Anatomy, 2 0 0 5 , vo l . 9 (Suppl. 1)

2%DMSO and sectioned at 50 mm in a sliding microtomeprovided with a freezing unit. A one-in ten series wasimmediately mounted onto subbed slides. The nineremaining sections in the series were stored in a cryopro-tectant solution and stored at 4ºC. The series was stainedwith thionin for cytoarchitectonic evaluation of themedial temporal lobe structures.

MRI images were of enough good quality as to off e ra good contrast between grey and white matter in T2. A l lof the major landmarks in the medial temporal lobe wererecognizable, and additional details are offered as to thedistances and border definition between neighbouringcytoarchitectonic fields. In conclusion, this proves avery powerful and useful technique for evaluation ofMRI-Neuroanatomical correlation in the medial tempo-ral lobe, thereby simplifying the interpretation of patho-logical images obtained in patients affected with A l z h e i-m e r’s disease, medial temporal lobe epilepsy and otherd i s e a s e s .

P-93 THE CONNECTIONS OF THE NUCLEUSINCERTUS IN THE RAT AND ITS RELA-TION IN THE HIPPOCAMPA L T H E TARHYTHM.

Teruel, V.1, Nuñez, A.2, Cervera, A.1, Perez-Villalba, A.1, Olucha-Bordonau, F.1, Ruiz-Tor-ner, A.1

1 Departamento de Anatomía y Embriología Huma -na, Facultad de Medicina. Universidad de Valencia.2 Departamento de Morfología, Facultad de Medici -na, Universidad Autónoma de Madrid.

The hippocampal theta rhythm is generated by thepacemaker activity of the medial septum-diagonal band ofBroca neurons (MS/BDv). These nuclei are influenced bybrainstem structures that modulate theta rhythm. Thenucleus incertus in the dorsal tegmental area, has beendescribed as the origin of an extensive projection to pro-sencephalic structures, MS/BD among others. The aim ofthe present work is to determine whether the nucleusincertus contributes anatomically and physiologically tothe activation of the hippocampal theta rhythm.

In the present study, anterograde tracer injections in thenucleus incertus showed terminal-like labeling in a wides-pread pattern of subcortical structures. These results ser-ved like target for retrograde tracer injections in order toextend and confirm the efferent connections of the nucleusincertus. These data led us to conclude that there is awidespread system of ascending projections arising fromthe nucleus incertus to the median raphe, mammillarycomplex, hypothalamus, habenula, amygdala, entorhinalcortex, medial septum, and hippocampus. Many of the tar-gets of the nucleus incertus were involved in arousalmechanisms including the hippocampal theta rhythmicity.

The anatomical evidences of the possible relation bet-ween nucleus incertus and theta oscillations were confir-med through physiological experiments. The hippocam-pal field activity was recorded in urethane anesthetizedrats. Electrical stimulation of the nucleus incertus evokedtheta rhythm in the hippocampus, and decreased theamplitude of the delta waves. No high frequency activity(>10 Hz) was spontaneously observed during the electri-cal stimulation of the nucleus incertus, suggesting that thenucleus incertus selectively induced hippocampal thetarhythm without altering the general arousal state. Conse-

quently, the nucleus incertus may be a relay station bet-ween brainstem structures and the MS-DBB in the controlof the hippocampal theta rhythm.

P-94 QUANTITATIVE CHANGES OFSEROTO-NERGIC FIBERS IN THE RAT CINGU-LUM BUNDLE AFTER MDMA ADMINIS-TRATION.

Artacho-Pérula, E.1, Hernández-Sansalvador,M.1,2, Arroyo-Jimenez, M.M.1, Martínez-Mar-cos, A.1, Marcos-Rabal, P.1, Aguirre, N.3, Fre-chilla, D.3, Del Río, J.3, Insausti, R.1

1 Laboratorio de Neuroanatomía Humana, Facultadde Medicina, Universidad de Castilla-La Mancha. 2

Hospital Universitario de Albacete. 3 Departamentode Farmacología, Facultad de Medicina, Universi -dad de Navarra.

MDMA (Ectasy) is a common drug of abuse, particu-larly among young adults, that is neurotoxic to the seroto-nergic system. In order to assess this phenomenon, thechange in the number of serotonergic fibers of the cingu-lum bundle in MDMA treated Wistar rats was quantified.We used 17 rats in three experimental series: controls(n=7), and two MDMAtreated groups (15 mg of MDMAin saline per kg) and either one week (n=5) or four weeks(n=5) survival. The quantitative study was performed attwo levels of the cingulum bundle (anterior and posteriorportions) with the CAST-Grid system for estimation of thenumber of profiles (5-HT immunolabeled fibers) per areaQA. Data were pooled for rostral and caudal levels and forright and left hemispheres. 5HT-positive fibers showed atortuous course, they were fine in diameter and containedsmall varicosities. The fiber density per area of cingulumbundle showed a statistically non-significant 19% decrea-se one week after MDMA administration compared tocontrols. Four weeks survival group showed a decrease of55% in the occurrence of 5HT positive fibers (p<0.001).In Wistar rats the 5HT positive fiber decrease (possiblydue to toxic damage) was protracted in time, and it mighthappen, not only at the terminal site, but somewhere alongthe axonal pathway, excluding cell bodies of the raphenuclei, according to other studies. In summary, the sus-ceptibility of the serotonergic pathway in Wistar rats afterMDMA administration was highly significant at fourweeks survival.

P-95 OREXINERGIC CONNECTIONS TO THEORAL PONTINE RETICULAR NUCLEUSIN THE RAT. IMPLICATION IN SLEEP-WAKE STATES.

Rodrigo-Angulo, M.L., Garzón, M.

Departamento de Anatomía, Histología y Neurocien -cia. Facultad de Medicina. Universidad Autónomade Madrid. Spain.

Orexine (hypocretine) is a neuropeptide located inneurons of the perifornical region of the posterior hypot-halamus that has been implicated in promoting arousal incats and rats. Degeneration of orexine-containing hypot-halamic neurons in humans induces narcolepsy. On theother hand, it has been described that the cholinergic sti-



mulation of the oral pontine reticular nucleus (RPO) in therat induces paradoxical sleep (PS). The aim of presentcommunication is to investigate the possible influence ofthe orexinergic hypothalamic neurons on the pontine PS-induction neurons, in the rat. Under general anaesthesia,five animals received stereotaxic injections of the retro-grade tracer cholera-toxin B subunit (CT) in the RPO PS-induction region by means of a 500 nl Hamilton syringe.After seven days survival time, animals were perfused andtheir brains frozen sectioned at 40µm in consecutive seriesto be processed for immunostaining with 1:2000 goat anti-orexin antiserum (Santa Cruz Biotechnology) and forrevealing CT using a silver enhancement kit (Amersham)procedure. One parallel series was devoted to Nissl stai-ning to delimit the different nuclei and anatomical structu-res. The study of histological material revealed the pre-sence of orexine immuno-positive neurons in the lateralhypothalamic area, dorsomedial hypothalamic nucleusand perifornical nucleus according with data previouslydescribed. A l a rge number of neurons retrogradely labe-lled with CT was detected mainly ipsilateral but also con-tralateral in the ventral, dorsolateral and medial hypotha-lamic areas extending also to zona incerta. An importantnumber of neurons displaying both CT labelling and ore-xine immunostaining were located ipsi and contralaterallyin dorsolateral hypothalamic areas and around the fornix,being less abundant in posterior hypothalamic area. Ourresults demonstrate the existence of projections to the pon-tine PS-induction region from several hypothalamic areasand a consistent orexinergic connection arising in neuronsmainly located in dorsolateral hypothalamic and periforni-cal nuclei. These results suggest that degeneration orhypofunction of this orexinergic projection in narcolepticsubjects could underlie pathological alternation of the dif-ferent phases of the sleep-waking cycle.

Supported by Grants DGICYT BFI2003-00809 andCAM GR/SAL/0188/2004

P-96 VENTRAL PALLIDAL INNERVATION OFTHE RAT MESOPONTINE TEGMENTUMAND ITS RELAT I O N S H I P TO CHOLI-NERGIC NEURONS.

Pérez-Lorenzo, E., Mengual, E.

C e n t ro para la Investigación Médica A p l i c a d a(CIMA), Universidad de Navarra.

The downstream projection of the ventral pallidum(VP) to the mesopontine tegmentum is part of the cortico-striato-pallido-pontine circuits, which are altered in someneurodegenerative diseases such as Parkinson’s disease orHuntington’s chorea, as well as in neuropsychiatric disor-ders such as schizophrenia.

Pharmacological, physiological and lesion studies inrats have reported an effect of the VPon the activity of thepedunculopontine tegmental nucleus (PPTg), thus provi-ding indirect evidence of putative VP-PPTg projections.Anatomical studies carried out so far, however, have notclearly shown ventral pallidal terminal fibers in the PPTgto support the physiological and pharmacological fin-dings. In order to analyse in detail the ventral pallidalinnervation of the rat mesopontine tegmentum, smallinjections of the sensitive anterograde tracer biotinylateddextran amine were performed in different rostrocaudaland mediolateral portions of the ventral VP by iontopho-

resis. The precise localization of the injections within theVP was neurochemically assessed by means of doublelabeling with Leu-enkephalin, a specific marker of the VP.The subsequent anterograde labeling in the mesopontinetegmentum was analyzed throughout the rostrocaudalextension of PPTg. In addition, the relationship betweenthe terminal fibers and the cholinergic neurons in PPTgwas studied by means of double labeling using the vesi-cular acetylcholine transporter (VAChT).as a cholinergicmarker. Significant anterograde labeling was found in themesopontine tegmentum, although its density varied inrelation to the size of the injection. Most of the fibers wereterminal fibers displaying putative synaptic boutons.Though at low magnification the terminal fields were dor-somedially located relative to PPTg cholinergic neurons,at higher magnification, several varicose fibers wereintermingled among the cholinergic neurons. Our resultsclearly demonstrate the VP-PPTg projection in the rat,thus providing the anatomical substrate for the functionalmodulation of PPTg neurons from the basal forebrain.

(Funded through the “UTE project CIMA”).

P-97 TOPOGRAPHIC LOCATION AND CIR-CANNUALEVOLUTION OFTHE SYNAP-TIC BODIES IN THE PINEALGLAND OFTHE ALBINO RAT.

Martínez Soriano, F., Hernández Gil de Teja-da, T., Valverde Navarro, A.A., Sanchis Gime-no, J.A., Tomás Aguirre, F.

Departamento de Anatomía y Embriología Humanas.Universitat de Valencia.

The synaptic bodies of mammalian pineal gland arepoorly understood ultrastructural organelles into the cyto-plasm of the pinealocytes. Previous studies in the rabbithave shown that these organelles are rather heterogeneousin shape, decrease in number during the light phase andincrease in number at dark phase. It has also found pro-nounced season-related differences as well as differencesrelated to pineal regions.

No studies are currently available on seasonal changesin other species and it is unknown whether the biologicalrhythms are identical in the proximal, intermediate anddistal parts of the elongated pineal.

Our study was carried out on 120 male albino rats keptunder natural lighting conditions in order to examinenumerical variations of the different types of synapticbodies in the proximal, intermediate and distal regions ofpineal glands procured at different time points of 24-hourcycle and in the four annual seasons.

In the present study rod-like, sphere-like and triangu-lar synaptic bodies were distinguished, being the first twotypes the most abundant. Results also displayed circadianchanges in the number of synaptic bodies with the lowervalues during light phase and higher ones at dark phase.We have also found pronounced season related differen-ces as well as differences related to pineal regions.

The circadian and seasonal results shown that day andnight length are structurally coded in the pineal gland bymeans of the number of synaptic bodies. The regional dif-ferences are probably related to the different origin of theinnervation: the sympathetic fibres enter to the gland dis-tally while the proximal fibres come from the central ner-vous system.



P-98 VASSOPRESIN, T Y R O S I N E - H Y D R O X Y-LASE AND DOPAMINE b-HYDROXYLA-SE DISTRIBUTION IN SUPRAOPTICAND ARCUATE NUCLEUS. A COMPARA-TIVE STUDY BETWEEN FARMER ANDCOST GOAT.

Castañeyra-Perdomo, A .1 , 2, Pérez-González,H .1, González-Marrero, I.2, Fernández-Rodrí-guez, P.2, Sánchez-Ortelano, C.2, Mosquera-Paz,G .2, Sánchez-González, C.2, Evora-Soto, N.2,Carmona-Calero, E.M.1, 2, Ferres-Torres, R.1

1 Departamento de Anatomía, Facultad de Medicina,Universidad de La Laguna, La Laguna, Tenerife,Islas Canarias. 2 Departamento de Biotecnología,Instituto de Investigación y Ciencias de Puerto delRosario, Fuerteventura, Islas Canarias.

The hormone vasopressin (A-V) is well known for itspressor and antidiuretic effects, is primarily synthesized inthe magnocellular neurons of the SPO and other hypotha-lamic structures. The SPO participate in the e salt-waterbalance thorough of A-V secretion, also the vasopressinappears to modulate the cardiovascular reflex control ofthe sympathetic nervous system. The (TH) and (DBH) arepresent in the catecholaminergic neurons located in thehypothalamus and brain areas related with the vascularregulation. The secretion of A-V by the SPO is controlled,at least in part, by angiotensinergic (AGII) inputs thatcome from the subfornical organ (SFO). The purpose ofthe present work is to study angiotensin-vasopressin sys-tem in the hypothalamus of the “majorera” goat (Caprahircus variation ), comparing the goat development in far-mer with goat that live in extreme condition of drinkingintake of the arid clime in Fuerteventura island cost.

We have used a total of six adult male and femalegoats divided in two groups: a farmer group (well fee-ding) and a cost groups (feeding in natural way in the aridclime of the cost). We have found the vasopressin-ir andTH-ir are decreased in the different hypothalamic nucleusof the cost goat, contrarily DBH immunoreactive is aug-mented in this goat. We could conclude that the decreaseof vasopressin-ir and TH-ir and the increase of DBH-ir inthe hypothalamus of the cost animal, could be an expres-sion of the water retention, since in this kind of goat thedaily drinking water is scarce.

P-99 DISTRIBUTION OF IL-6 AND nNOS IN VASOPRESSINERGIC AND CRF-ERGIC NEURONS OF THE RAT HYPO-THALAMUS.

Pérez Delgado, M.M.1, González, M.C. 2, Ran-cel Torres, N.1, Cruz Muros, I.1, Barroso Chi-nea, P1, Alonso Oramas, D.1, Abreu, P.2, Gonzá-lez Hernández, T.1

D e p a rtments of Anatomy 1 and Physiology 2, School ofMedicine, University of La Laguna, Tenerife, Spain.

Corticotropin-releasing factor (CRF) and arg i n i n -vasopressin (AVP) are the two major regulatory peptidesin the hypothalamus-pituitary-adrenal (HPA) axis. Parvo-cellular neurons of the paraventricular hypothalamicnucleus (PVN) are the main source of CRF in thehypophysial portal vascular system, after being releasedin the median eminence (Sawchenko and Swanson, 1990).

AVP, however, is synthesized in both magnocellular andparvocellular regions of the PVN, and in the supraoptic(SON) and suprachiasmatic (SCN) nuclei. Furthermore,CRF and AVP colocalize in parvocellular PVN neurons(Grino an Zamora, 1998) and secretory granules at thenerve endings in the ME (Gillies et al., 1982). Numerousstudies indicate that interleukin-6 (IL-6) and nitric oxide(NO) play regulatory roles on the HPA axis response todifferent kinds of homeostatic threats. IL-6 stimulatessynthesis and secretion of CRF and AVP at hypothalamiclevels, and is robustly expressed in the PVN and SPOnuclei, largely co-localized in these centres with AVP(Ghorbel et al., 2003). Nitric oxide synthase (nNOS), NOsynthesis enzyme, has been detected at different levels ofthe HPA axis. However, the involvement of NO in the axismodulation is still controversial. It is known the existenceof a biological master clock localized in the hypothalamicSCN, and circadian variations in plasma IL-6 levels havebeen recently described (Abreu et al., 2004).

The aim of the present study is investigate co-locali-zation of IL-6 and nNOS in CRF and AVP neurons of dif-ferent hypothalamic nuclei of the rat using immunohisto-chemistry and double staining immunofluorescence insham and colchicine intracerebroventricular injected ani-mals in order to a best visualization of the markers.

Our results show the different distribution of thesepeptides in the PVN, SPO and SCN hypothalamic nuclei,and the existence of two different populations of AVPer-gic cells, one containing CRF, but no IL-6, that project tothe median eminence, and another one that expresses IL-6, but no CRF, and project to the neural lobe of thehypophysis. This last one also coexpresses nNOS in mostof its neurons, suggesting that IL-6 and nNOS are invol-ved in AVP release from posterior lobe of the pituitary. Inthe SCN, we have detected a cell subpopulation expres-sing AVP and IL-6, and another expressing CRF.

P-100 COMPARATIVE STUDYOFnNOS IN THESUPRAOPTIC NUCLEI OF THE HYPOT-HALAMUS OF HYPOPHYSECTOMISEDRATS AS COMPARED WITH RATS SUB-JECTED TO STRESS.

Riesco López, J.M, Riesco Santos, J.M., Jua-nes Méndez, J.A., Rubio Sánchez, M., Blanco,E., Carretero, J., Sierra Benítez, M.E., PérezZaballos, M.T., Vázquez Rodríguez, R.

Dpto. Anatomía e Histología Humanas. Universidadde Salamanca.

Nitric oxide synthase is the enzyme responsible for thesynthesis of nitric oxide and its neuronal isoform (nNOS)can be detected in hypothalamic nuclei by immunocyto-chemistry. Accordingly, here we performed a study ofnNOS in the supraoptic nuclei of animals with opposingfunctional states and different from normal, such as survi-val at 40 days post-hypophysectomy and stress inducedby 4 hours of immobilisation, with a view to determiningthe neuronal behaviour of nitric oxide under such condi-tions. Adult male Wistar rats were used: untreated (n=5);hypophysectomised (n=5) and animals subjected to stress(n=5). All brains were fixed in 4% paraformaldehyde,embedded in paraffin and sliced in serial sections of 6mm. nNOS was detected in the hypothalamic neurons bysingle-label immunocytochemistry, streptavidin-biotinperoxidase, using anti-nNOS polyclonal antiserum raised


65European Journal of Anatomy, 2 0 0 5 , vol. 9 (Suppl . 1)

in sheep and diluted 1:15000. Together with the morpho-logical study we performed another morphometric analy-sis, calculating the cellular and nuclear areas of the immu-noreactive neurons. In the untreated animals, nNOS-immunoreactive neurons appeared, especially in the dorsalregion of the prechiasmatic subdivision of the supraopticnucleus, with a double immunostaining pattern: either asan intense immunoreaction covering both the perikaryonand the prolongations or as a weak reaction limited to theperikaryon. Hypophysectomy led to a decrease not only inthe number but also in the intensity of reaction of the neu-rons to nNOS. This appeared almost exclusively in the pre-chiasmatic subdivision, although it was also detected bothin the perikaryon and in the prolongations. Finally, stressinduced a qualitative and quantitative increase in thenNOS reaction in both subdivisions (pre- and retrochias-matic), with an intense reaction in the perikaryon and neu-ronal prolongations. The morphometric values of thisgroup of animals were significantly greater than any othersa n a l y s e d .

P-101 DOPAMINE b-HYDROXYLASE AND P73EXPRESSION IN THE ORGANUM VAS-CULOSUM OF THE LAMINA TERMINA-LIS AND SUBFORNICAL ORGAN. ASTUDY OF SHR AND WKY RATS.

Ferres-Torres, R.1, Carmona-Calero, E.M.1,2,Pérez-González, H.1, González-Marrero, I.2,Fernández-Rodríguez, P.2, Sánchez-Ortelano,C.2, Cabrera-Socorro, A., Castañeyra-Perdomo,A.1,2, Meyer, G.1

1 Departamento de Anatomía, Facultad de Medicina,Universidad de La Laguna, La Laguna, Tenerife,Islas Canarias. 2 Departamento de Biotecnología,Instituto de Investigación y Ciencias de Puerto delRosario, Fuerteventura, Islas Canarias.

The organum vasculosum of the lamina terminalis(OVLT) is a circumventricular organ located in the ante-roventral region of the third ventricle. The OVLT is richin neuropeptides such as angiotensin II and catecholami-nes. The subfornical organ (SFO) and OVLT are charac-terized by the absence of a blood-brain barrier. The SFOhas connections with the brain regions implicated in thecentral regulation of drinking, blood pressure and cardio-vascular function, among them the anteroventral region ofthe third ventricle and OVLT. The protein p73 is a mem-ber of a family of transcription factors, which also inclu-des p53 and p63. The p73 is present in developing neu-rons as a truncated isoform whose levels are dramaticallydecreased when sympathetic neurons apoptosis afternerve growth factor (NGF) withdrawal. Thereafter p73, isnecessary for survival and long-term maintenance of CNSneurons, including postnatal cortical neurons. The purpo-se of present work is study the expression of the dopami-ne b-hydroxylase (DBH) and p73 in the OVLT and theSFO and their variations in the ventricular dilatation andarterial hypertension.

Brains and cerebrospinal fluid (CSF) from controlWistar-Kyoto rats (WKY) and spontaneously hypertensi-ve rats (SHR) were used. The paraffin section containingthe OVLT and SFO were immunohistochemically proces-sed with anti-DBH and anti-p73. DBH and p73 band wereidentified in SFO extract by western blot.

We found that DBH was increased in the hypertensiveanimals only in the SFO while the increase of the p73expression was found in both SFO and OVLT of thehypertensive rats. Also the SFO extract band was markedwith anti-DBH and anti-p73 and the intensity of the reac-tion was higher in SHR than in the WHYrats. The presentresults and the fact that the deltaNp73 is essential for sur-vival of sympathetic neurons, would indicate that p73 inan essential survival protein in CNS catecholaminergicneurons, therefore there would be a correlation betweenp73, DBH and these cerebral centres implicated in car-diovascular regulation.

P-102 S U B F O R N I C A L ORGAN EXPRESSIONO F C E RTAIN PROTEINS FOUND INCEREBROSPINAL FLUID OFTHE SPON-TANEOUSLY HYPERTENSIVE RATS.

G o n z á l e z - M a r re ro, I.1, Carmona-Calero,E . M .1 , 2, Pérez-González, H.2, Ormazabal-Ramos,C .2, Pérez-García, C.G.2, Fernández-Rodríguez,P.1, Evora-Soto, N.1, Sánchez-González, C.1, Jam-brina, R.1, Castañeyra-Perdomo, A .1 , 2

1 Instituto de Investigación y Ciencias de Puer -to del Rosario, Fuerteventura, Spain. 2 Depar -tamento de Anatomía, Facultad de Medicina,Universidad de La Laguna.

The subfornical organ (SFO), like other circumventri-cular organs, is characterized by the absence of blood-brain barrier. The SFO has connections with the brainregions implicated in the central regulation of drinking,blood pressure and cardiovascular function, among themthe anteroventral region of the third ventricle. A l t e r a t i o n sin the Angiotensin II receptor have been described in theSFO of the SHR, moreover underdevelopment of theSFO and variations of the angiotensin II Receptor contentof the subfornical organ have also been found in hydro-cephalic animals The aim of this work was study the pos-sible role in the concomitance among hydrocephalus,arterial hypertension and the variation in subfornicalo rgans. Brains and cerebrospinal fluid (CSF) from con-trol Wi s t a r-Kyoto rats (WKY) and spontaneously hyper-tensive rats (SHR) sacrificed with chloral hydrate wereused. CSF and extract o were processed by western blot.Antiserum against proteins band of 141, 117 and 48 kDa(anti-B1, anti-B2 and anti-B3) were used in the immu-nohistochemical and Immunoblotting study of the sub-fornical organ and adjacent structures. The SFO, thirdventricle ependyma and choroideus plexus (CP) showedimmunoreactive material (IRM) for antibody against141kDa, 117 and 48 kDa proteins band The larg e ramount of the IRM was found in the SFO of the SHR.Several protein bands were marked mainly with anti-b3and an increase in the IRM was found in the SFO SHRextracts. Our results and the alterations observed by otherauthors in the SFO in hydrocephalic and hypertensiverats, support the possibility that these circumventricularo rgans, some proteins of the CSF and ventricular dilationcould be connected with the physiopathology of this typeof the hypertension.

Acknowledgments: This work was supported by Fun -dación Canaria de Investigación y Salud (FUNCIS) pro -ject nº 74/03



P-103 EFFECTS OFTHE HYPERTENSION ANDITS CAPTOPRIL TREATMENT ON THEADRENAL MEDULLA. A CATECHOLA-MINERGIC AND CHROMOGRANINSTUDY.

G o n z á l e z - Toledo, J.M.1, Carmona-Calero,E . M .1 , 2, González-Marrero, I.2, Fernández-Rodríguez, P.2, Pérez-González, H.1, Sánchez-González, C.2, Bermúdez, A.2, Sánchez-Caste-llano, C.2, Castañeyra-Perdomo, A .1 , 2,Ferres-Torres, R.1

1 Departamento de Anatomía, Facultad de Medicina,Universidad de La Laguna, Tenerife. 2 Instituto deInvestigación y Ciencias de Puerto del Rosario,Fuerteventura.

We investigated the alteration of tyrosine hydroxylase(TH), dopamine-beta-hydroxylase (DBH), phenylethano-lamine-N-methyltransferase (PNMT) and chromograninA (ChA) expression in the adrenal medulla of spontane-ously hypertensive rats (SHR) and its variation with thecaptopril treatment.

We have used a total of 20 male rat divided in fourgroups: a control group formed by five WKY rats, a con-trol treated group formed by five WKY rats treated withcaptopril, a hypertensive group formed by five SHR ratsand a hypertensive treated group formed by five SHR ratstreated with captopril.

In the adult adrenal gland, chromogranin A and T Hwere increased in the SHR, while dopamine beta-hydroxylase activity was diminished. The captopril tre-atment produce a decrease in IRM in the hypertensivegroup. The PNMT was also diminished in the hyperten-sive groups. These results are partially in concordancewith another works on the Chromogranin A m R N Aexpression where it was also elevated in the SHR adre-nal medulla, contrarily, in another rodent model ofhypertension, the hypertensive mouse (BPH/2), adrenalchromogranin Aand noradrenaline were diminished. Weconclude that over-expression of chromogranin A a n dthe alteration found in the different enzymes of catecho-lamines are a variable feature of mammalian genetich y p e r t e n s i o n .

P-104 INFUNDIBULAR CAT E C H O L A M I N E SVA R I ATIONS IN A RT E R I A L H Y P E R-T E N S I O N .

Pérez-González, H.1, Carmona-Calero, E.M.1,2,González-Marrero, I.2, Fernández-Rodríguez,P.2, Martín-González, C.2, Sánchez-Ortelano,C .2, Bermúdez, A .2, Sánchez-Ruano, A . G .2,Jambrina, R.2, Castañeyra-Perdomo, A.1,2

1 Departamento de Anatomía, Facultad de Medicina,Universidad de La Laguna, Tenerife, Islas Canarias.2 Departamento de Biotecnología, Instituto de Inves -tigación y Ciencias de Puerto del Rosario, Fuerte -ventura, Islas Canarias.

The infundibular area include the arcuate nucleus (AN)and median eminence (ME), that are brain structures clo-sely related with hypothalamic-hypophyseal axis and playan important roll in the cardiovascular regulation throug-hout of its catecholaminergic innervations which act on theangiotensin II and vasopressin release. The axons origina-

ting in these neurons pass through the internal zone of themedian eminence and reach the posterior lobe of thehypophysis. The secretion of this hormone is controlled, atleast in part, by angiotensinergic inputs from the subforni-cal organ (SFO) and reaches the hypothalamus.

We have used a total of 20 male rat of 10 and 15 weekold divided in two groups: a control group formed byWKYrats, a hypertensive group formed by SHR. We havefound TH and DBH immunoreactive material (IRM) inneurons and fibres of the AN, periventricular nucleus(PEV) and in fibres of the ME. At ten weeks old thehypertensive group shows TH- IRM slighter augmentedthan the control groups, moreover the hypertensive grouphas a greater number of fibres marked than the controlgroup, this fibres go into the PEV. Contrarily, at 15 weeksold the control group shows the amount of TH-IRM ele-vated and a greater number of marked cells than thehypertensive group. The DBH-IRM at ten week old wasmore intense in the control group. While the AN of theSHR groups at 15 weeks old showed the intensity of DBHimmunoreaction augmented in a 20%. The amount of THand DBH identifying by Western blot in the hypertensivegroup was greater than in the control group. These fin-dings indicate that catecholaminergic projections on theAN could be implicated in the changes of angiotensin andvasopressin secretion described in the SHR rats.

P-105 I M P L I C ATION OF A R O M ATASE ONPITUITARY FUNCTION, EVIDENCED BYWESTERN BLOT, IMMUNOHISTOCHE-MISTRY, IN SITU HYBRIDIZATION ANDAROMATASE-ANTAGONISTS.

Herrero, J.J.1, Rubio, M.2, Sierra, E.2, Blanco,E.2, Burks, D.J.3, Prieto, A.3, Pérez, E.1, Carre-tero, M.4, Carretero, J.2

1 Department of Surgery, Faculty of Medicine, Uni -versity of Salamanca, Spain. 2 Department of HumanAnatomy and Histology and Laboratory of Neuroen -docrinology, Institute for Neuroscience of Castilla yLeón, University of Salamanca, Spain. 3 Institute ofCellular Biology, Valencia, Spain. 4 Area of Physicsand Mathematics, Department of Educational Scien -ces, School of Educational Sciences, University Pon -tificia of Salamanca, Spain.

In previous studies we demonstrate the immunocyto-chemical expression of aromatase in pituitary cells. Theaim of this study is demonstrate the expression of mRNAfor the enzyme in pituitary cells by in situ hybridizationand correlate with immunocytochemical expression ofaromatase in the pituitary of male adult rats. In situ hybri-dization demonstrates that aromatase-mRNAis located inthe cytoplasm of 41% of pituitary cells and it correlateswell with immunocytochemical staining.

In order to determine the physiological relevance ofpituitary aromatase in the control of prolactin pituitarycells an immunocytochemical and morphometric study ofprolactin-positive pituitary cells was carried out on pitui-tary of adult male rats treated with fadrozole. After treat-ment, the cellular and nuclear areas of prolactin cells, aswell as the percentage of prolactin-positive cells and per-centage of proliferating-prolactin cells was significantlydecreased (p<0.01, in relation to untreated and controlanimals). Moreover, fadrozole decreases serum prolactinlevels. Our results suggest that, in physiological condi-



tions, aromatase P450 exert a relevant control on prolac-tin-cells, probably transforming testosterone to estradiolin the pituitary gland.

Supported by the FIS Spanish program No. PI0-21803

P-106 GONADAL STEROIDS REGULATE ARO-MATASE P450 EXPRESSION IN THE RATPITUITARY.

Pérez, E.1, Blanco, E.2, Rubio, M.2, Sierra, E.2,Carretero, M.3, Herrero, J.J.1, Burks, D.J.4,Carretero, J.2

1 Department of Surgery, Faculty of Medicine, Uni -versity of Salamanca. Spain. 2 Department of HumanAnatomy and Histology and Laboratory of Neuroen -docrinology, Institute of Neuroscience of Castilla yLeón. University of Salamanca. Spain. 3 Area of Phy -sics and Mathematics, Department of EducationalSciences, School of Educational Sciences, UniversityPontificia of Salamanca, Spain. 4 Institute of CellularBiology, Valencia, Spain.

Rat aromatase immunohistochemical expression isdifferent in male than in female adult rats. In order toanalyze in these differences are related to presence ofgonadal steroids, an study was carried out on pituitaries ofadult castrated and castrated and treated with gonadal ste-roids rats using immunohistochemistry, western blottingand in situ hybridization for Rat Aromatase P450. Rat aro-matase P450 mRNA was detected in the pituitary of maleand female rats. Sex-related variations of mRNA eviden-ce were observed, the mRNA signal was more abundantin males than in females; moreover the male pituitariesshowed more immunohistochemical positive cells thanfemales and by western blotting the enzyme was mostabundant in males than in females. With the three methodsassayed, ovariectomy elicited a considerable increase inthe reaction to aromatase in females; in male rats, castra-tion reduced the number of reactive cells, although thereaction persisted. Treatment with gonadal steroids aftercastration modified aromatase expression in the sense thatin testosterone-treated castrated males the expression ofaromatase was increased while in castrated females trea-ted with estradiol decreased. Our results demonstrate thesynthesis of aromatase in the pituitary and its immunohis-tochemical expression in the gland of adult rats and sug-gest that the expression of this enzyme is sex-dependentand can be modified by castration and gonadal steroidadministration. This in turn suggests that aromatase maybe involved in the regulation of adenohypophyseal cyto-logy by gonadal steroids.


P-107 COEXPRESSION OF A R O M ATA S E ,ESTROGEN RECEPTOR aAND DOPAMI-NERGIC RECEPTOR D2L IN HUMANPROLACTINOMAS.

Blanco, E.1, Rubio, M.1, Sierra, E.1, Herrero,J.J.2, Pérez, E.2, Santos-Briz, A.3, Carretero,M.4, Burks, D.J.5, Prieto, A.5, Carretero, J.1

1 Department of Human Anatomy and Histology,Laboratory of Neuroendocrinology of Institute of

Neuroscience of Castilla y León. Faculty of Medici -ne. University of Salamanca, Spain. 2 Department ofSurgery, Faculty of Medicine, University of Salaman -ca, Spain. 3 Department of Cellular Biology and Pat -hology, Faculty of Medicine, University of Salaman -ca, Spain. 4 A rea of Physics and Mathematics,Department of Educational Sciences, School of Edu -cational Sciences, University Pontificia of Salaman -ca, Spain. 5 Institute of Cellular Biology, ValenciaSpain.

In previous studies we demonstrate the immunocyto-chemical expression of aromatase in pituitary cells andprolactinomas in the rat, suggesting that aromatase couldbe involved in the genesis of prolactinoma in rodents. Theaim of this study is demonstrate the expression of theenzyme in the human prolactinoma cells by immunocyto-chemistry and in situ hybridization and the relation amongimmunocytochemical expression of aromatase and dopa-mine receptor D2L and/or estrogen receptor a in humanprolactinomas. Immunocytochemistry and in situ hybridi-zation demonstrate that aromatase is expressed in 100%of human prolactinomas, which express moreover estro-gen receptor a. Dopamine receptor D2L was expressed in98% of human prolactinomas. These findings demonstra-te the ability of human prolactinomas to bind dopaminethrough to dopamine receptor D2L and the synthesis ofthe aromatase in human prolactinomas and its functionalability to develop the estrogenic effects mediated byestrogen receptor a, suggesting that these effects could beinvolved in the growth of human prolactinoma.


P-108 C O R R E L ATION OF ESTROGEN RECEP-TOR COACTIVATOR AIB1, ESTROGENR E C E P TOR a, A R O M ATASE W I T HCELLULAR PROLIFERATION AND A P O P-TOSIS IN HUMAN PROLACTINOMA.

Sierra, E.1, Blanco, E.1, Rubio, M. 1, Carretero,M.2, Pérez, E.3, Herrero, J.J.3, Font de Mora, J.4,Burks, D.J.4, Carretero, J.1

1 Department of Human Anatomy and Histology,Laboratory of Neuroendocrinology of Institute ofNeuroscience of Castilla y León. Faculty of Medici -ne. University of Salamanca, Spain. 2 Area of Physicsand Mathematics, Department of Educational Scien -ces, School of Educational Sciences, University Pon -tificia of Salamanca, Spain. 3 Department of Surgery,Faculty of Medicine, University of Salamanca, Spain.4 Institute of Cellular Biology, Valencia Spain.

Although differential responses of hormone-depen-dent tumors to sex steroids could be strongly influencedby the relative amounts of co-activator protein AIB1,AIB1-expression and its correlation with aromatase,enzyme responsible of the local aromatization of testoste-rone to estradiol, and estrogen receptor a in these tumorsare not yet established. The aim of the present study wasto investigate the protein expression of the co-activatorAIB1 in human prolactinomas and compare this expres-sion with the expression of aromatase and estrogen recep-tor a. and with the cellular proliferation in these tumors. Ofa series of 87 adenomas studied, 56% showed high popu-lation of prolactin-positive cells and were classified asprolactinomas because they show hyperprolactinemia.Moreover, 80% of the adenomas were aromatase-positive



tumors. Interestingly, 100% of the prolactinomas werearomatase-positive tumors. Western blotting with anti-aromatase antibodies revealed a twice increase in expres-sion of aromatase in pituitary tumors as compared to nontumoral human pituitary gland. All prolactin- and aromata-se-positive tumors were positive for estrogen receptor a. andAIB1. Eighty one percent of prolactinomas were PCNA-positive tumors. Nuclear expression of AIB1 was predo-minant expression in PCNA-positive prolactinomas.Seven percent of prolactinomas non PCNA-positive sho-wed cytoplasmic reaction for AIB1 and were active cas-pase 3-positive. Our results demonstrate for the first timethe pituitary expression of AIB1 in prolactinomas andtheir correlation with the cellular proliferation of thetumor, suggesting the possibility that AIB1 could beinvolved in the growth of the tumor and an abnormallyhigh conversion of testosterone into estradiol mediated byaromatase in pituitary cells.


P-109 I N VO LV E M E N T O F THE A R C U AT ENUCLEUS DURING COMBINEDGROWTH HORMONE (GH) SECRETA-GOGUE STIMULATION IN THE STUDYOF THE PITUITARY GH RESERVE.

Jiménez-Reina, L.1, García Martínez, E.2,Rojas Ramírez, J.P.1, Cañete Vázquez, M.D.1,Bernal Valls, G.1, Cañete Estrada, R.2

1 Dpto. Ciencias Morfológicas. Facultad Medicina.Córdoba. Spain. 2 Unidad Endocrinología Pediátri -ca. Hospital Universitario Reina Sofía. Córdoba.Spain.

Combined use of growth hormone-releasing hormone(GHRH) and GH secretagogues has been proposed forstudying the pituitary growth hormone (GH) reserve.However, very little is known of the response of hypotha-lamic arcuate nucleus neurons to these stimuli. The pre-sent study sought to identify that response through analy-sis of neuron activation using c-fos protein (neuron“trigger”) activation and GH release prompted by combi-ned GHRH and GH-secretagogue stimulation, with andwithout somatostatin (SRIH) pretreatment.

A total of 120 female Wistar rats aged 28-30 days wereused. Half received SRIH and the other half saline, at -90minutes (SRIH-pretreated and saline-pretreated groups,respectively). At 0 minutes, both groups were given oneof the following stimuli: saline, GHRH, GHRP-6, GHRE-LIN, GHRH + GHRP-6, GHRH + GHRELIN (1 µg/Kgdose in all cases). Rats were slaughtered at -90, 0 and +90minutes, and blood samples were taken for RIA determi-nation of GH; brains were fixed in paraformaldehyde, fro-zen and sectioned for immunocytochemical determinationof c-fos protein.

Combined GHRH and GH-secretagogue stimulationenhanced GH release, both with and without SRIH pre-treatment. However, in isolated cases stimulus was enhan-ced by SRIH pretreatment.

In the arcuate nucleus, all GH-releasing stimuli (exceptG H R P 6) prompted neuron activation. SRIH pretreatmentalso prompted neuron activation in the arcuate nucleus;subsequent stimuli were unable to enhance this activity,with the exception of the GHRH + GHRP6 combination.

These data suggest that the maximum somatotropicresponse to combined GHRH and GH-secretagogue sti-mulation may be limited by the activation of somatostati-nergic-type neurons in the arcuate nucleus, which exerci-se ultrashort control over neuron GHRH activity.

P-110 INFLUENCE OF DIETARY FOLIC ACIDSUPPLEMENTATION ON CEREBELLUMFISSURATION IN YOUNG RATS.

Pérez-Miguelsanz, J.1, Alonso-Aperte, E.2, Par-tearroyo, T.2, Maestro de las Casas, C.1, Varela-Moreiras, G.2, Peña-Melián, A.1

1 Departamento de Anatomía y Embriología HumanaI. Facultad de Medicina. UCM. 2 Departamento deNutrición, Bromatología y Tecnología de los alimen -tos. Facultad de Farmacia. USP-CEU.

It is well known that low levels of folic acid (FA) causehiperhomocysteinemia, which modifies methylation proc-cess and development of neuronal network pattern secon-d a r y, as well as potentially vasculogenesis, resulting in cog-nitive dysfunction in the elderly. In addition, duringprenatal development, maternal availability of choline FAhas been suggested to influence on cerebral neuroblasts andangioblasts in mice at the end of development in mice.

The aim of this work is to evaluate the influence ofdifferent levels of FA supplemented diets on several brainstructures (e.g. cerebellum). Four groups of 10 each oldrats were fed with an pure amino acid diet: Group 0 (0 mgFA/kg diet), Group 2 (2 mg FA/kg diet, control), Group 8(8 mg FA/kg supplemented diet), Group 40 (40 mg FA/kgsupplemented diet), for four weeks.

Brains were fixed in 4% neutral tampon phormalin andprocessed for histologycal study. Serial sagital sections of 8mm thickness were performed. Sections were stained withK l ü b e r-Barrera, Nissl, Hematoxiline-Eosine, Bodian andmetianamine silver techniques. Inmunohistochemistry forglyal fibrilar acid protein in order to visualize Berg m a n ’ g l y aarrangement, and calbindin for Purkinje’s cells were done.

Macroscopical study of the cerebellums and distribu-tion and number of folia was normal in all groups. Howe-ver, Group 0 cerebellums presented significatuve largersize when compared with Group 40.

Microscopic analysis showed alterations on Groups 0and 40, with partial fussion of opposite slopes of primaand secunda fissures, appearance of early fissures in deve-lopment, going with piamater missing, ectopic islands ofgranular cells appearance in molecular layer and abnor-mal pattern of Bergman’s glya prolongations. The obser-ved pattern closely remains to the one that occurs whenrats are chronically fed with alcohol.

In conclusion, either no folic acid or highly supple-mented diets cause corticogenesis failure in cerebellum. Alater functional evaluation is needed to confirm if thesecerebellum cortical anomalies may have some influenceon several cognitive and behavioural marks under diffe-rent dietary treatments.

P-111 DISTRIBUTION OF C AT E C H O L A M I N EBIOSYNTHETIC ENZYMES IN THE RATDORSAL VAGALCOMPLEX.

Carmona-Calero, E.M.1,2, Gonzalez-Marrero,I.2, Fernández-Rodríguez, P.1,2, Perez-Gonzalez,



H.1, Sánchez-Gonzalez, C.1, Trujillano-Dorora-do, A.2, Jambrina, R.2, Mosquera-Paz, G.2, Cas-tañeyra-Perdomo, A.1,2, Ferres-Torres, R.1

1 Departamento de Anatomía, Facultad de Medicina,Universidad de La Laguna, Tenerife, Islas Canarias.2 Departamento de Biotecnología, Instituto de Inves -tigación y Ciencias de Puerto del Rosario, Fuerte -ventura, Islas Canarias.

The dorsal vagal complex (DVC) is formed by thenucleus tractus solitarii (NTS), the dorsal motor nucleusof the vagus nerve (DMV) and the area postrema (AP. TheAP function has been connected with the cardiovascularregulations. The nucleus tractus solitarii (NTS) and dorsalmotor nucleus of the vagus (DMV) constitutes sensoryand motor nuclei of the dorsal vagal complex, respecti-vely. The (DMV) is a relay centre for integrating centraland peripheral signals related to cardiovascular regula-tion. The NTS is the primary site for cardiovascular affe-rent fibres termination and it is innervated by catechola-minergic neurons. The present work quest more evidenceson the implication of the DVC contains catecholaminergicneurons in the arterial hypertension.

We have used 30 male rats, fed with a standard diet,sacrificed at 2, 4 and 12 postnatal months, divided in twogroups: Group 1 control Wistar-Kyoto rats (WKY) andGroup 2 spontaneously hypertensive rats (SHR). Paraffinserial coronal sections and DVC extract were performed.Antibodies to the catecholamines synthetic enzymes tyro-sine hydroxylase (TH) and dopamine-beta-hydroxylase(DBH) were used in the immunohistochemical and wes-tern blot analysis.

The TH and DBH material immunoreactive were loca-ted throughout the rostrocaudal and dorsoventral extent ofthe area postrema, as well as in neurons within the NTS.The distribution and the number of TH neurons were thesimilar as those DBH, except in the dorsal motor nucleusof the vagus. In the area postrema the number of neuronsimmunocytochemical labelled for TH was higher thanthose labelled for DBH. Thereafter, we conclude theincrease of the catecholamines in neurons of the DVC inthe hypertension, are influenced by the increase the AGIIlevel in the DVC caused by augment of gene expressionof the AT1 receptor in SHR rats.

P-112 MORPHOLOGY AND MORPHOMETRYOF THE INFERIOR LARYNGEAL NER-VES OFTHE RAT.

Pascual-Font, A. 1, Maranillo, E.2, Verdú, E.2,Brillas, A.2, León, X.2, Merchán, A.1, Sañudo,J.R.1, Valderrama, F.J.1

1 Dpto. de Anatomía y Embriología Humana I.Facultad de Medicina. Universidad Complutense deMadrid. 2 Unitat de Anatomia i Embriologia. Facul -tat de Medicina. Universitat Autònoma de Barcelona.

The inferior laryngeal nerves (ILN) of the left and theright sides, due to their dissimilar course around the aor-tic and subclavian arches (recurrent nerves), are of subs-tantial different length. This variation in extent, in plainterms of conduction velocity, could involve a significantgap in the time of action of the musculature controlled byeach nerve. Thus, several explanations have been addres-sed to elucidate the closest of the glottis at the same timedespite the different length of the nerves in both sides.

Therefore, we undertake this study with the aim ofanalyze and compare the length and fibre composition ofthe ILN, as a background for further studies in the func-tion of the laryngeal nerves.

Adult male Sprague-Dawley rats were used in thestudy. Previously to any experimental procedure, thefunction of the larynx was confirmed in each animal bymeans of laryngoscopy, in order to discard any animalwith abnormalities in the laryngeal function. In one group,the ILN were dissected from its origin to the entrance inthe larynx. Then, the nerves were measured in length. Inother group, the ILN was excised distally to its origin -below the caudal pole of the thyroid gland-, and the sam-ple was procesed in order to obtain plastic semithin sec-tions, later stained with toluidin. Morphometry of themielinated fibres present in the sections were developedand analyzed using “Visilog” software.

The averaged length of the ILN was 2.79 cm in theright side and 3.63 cm in the left side, i.e., 30% longer inthe left side.

The mean number of mielinic fibres was 182, with nosignificative differences by side. The calibre distributionpattern of the mielinic fibres was unimodal, with a maxi-mum distribution range between 2.5 and 7.0 micrometers.No differences by side were observed in the mean calibreof the fibres.

This work is supported by the Spanish FISS PI030035.

P-113 PROJECTIONS OF THE RAT LARYNGE-A L N E RVES. A N O V E L S T U D Y W I T HDEXTRANES.

Pascual-Font, A.1, Merchán, A.1, Muñoz, A.2,González, A.2, Sañudo, J.R.1, Valderrama, F.J.1

1 Dpto. de Anatomía y Embriología Humana I.Facultad de Medicina. UCM. 2 Dpto. de BiologíaCelular. Facultad de Ciencias Biológicas. UCM.

The knowledge of the precise connections of thelaryngeal nerves is of capital relevance for the larynxfunction comprehension. Since the beginning of the for-ties decade, several works have approached to the topic bymeans of distinct techniques including lesions in thebrainstem, sections of the nerves, and, more recently, tra-cing studies with a variety of neuronal tracers. From thepreceding studies, some discrepancies arise concerning tothe organization of the central motor nuclei of the laryn-geal intrinsic muscles and, in addition, less attention ispaid on the central distribution of the afferents formingthe laryngeal nerves.

We have used, for the first time in the literature for thismodel, dextranes as tracers for the superior (SLN) andrecurrent (RLN) laryngeal nerves. Dextranes, when usedin certain experimental conditions, are anterogradely andretrogradely transported, allowing, in the same specimen,the study of the central and peripheral projections of agiven nerve.

Dextranes of several molecular weights (3.000,10.000) were applied on the SLN and RLN of healthySprague-Dawley adult male rats. After a survival period,the larynx and brainstem were processed, in order toanalyze the central and peripheral distribution of the com-ponents of the nerves. The SLN appears to project exclu-sively on the cricotiroid muscle, and the central projec-tions are distributed within the nucleus ambiguus, the


70 European Journal of Anatomy, 2 0 0 5 , vol . 9 (Suppl. 1)

dorsal motor nucleus of vagus, and the nucleus of the soli-tary tract. The RLN provides innervation to the intrinsiclaryngeal muscles, except the cricotiroid, and, centrally, itappears to project exclusively on the ambiguus nucleus.This results open the possibility that, at least in the rat, theRLN does not convey afferent fibres of the infragloticterritories or that these afferent fibres could be send to theSLN by means of a connecting branch/es.

Our results, compared when the scarce literature onthis subject, open new insights on the neural systems con-trolling the laryngeal function.

This work is supported by the Spanish FISS PI030035.

P-114 KIT POSITIVE CELLS IN THE HUMANURINARY BLADDER.

Martínez-Ciriano, C. , Junquera, C., Castiella,T., Aisa, J., Lahoz, M., Azanza M.J.

Dpto. Anatomía e Histología Humanas. Facultad deMedicina. Universidad de Zaragoza.

Interstitial cells of Cajal (ICCs), which are foundthroughout the gut, are now known to be the origin ofpacemaker signals having a major role in the transmissionof signals from enteric neurons to smooth muscle cells.The discovery that ICCs in the gastrointestinal tract couldbe labelled with antibodies to the proto-oncogen c-kit,which codes for a tyrosine kinase c-kit receptor, led to thesuccessful characterization of ICCs in the gut. In factCajal (1904) had already deduced that these cells could bepresent in all those organs with autonomous innervation(blood vessels, glands and smooth muscle fibres). Theaim of the present work has been to investigate the distri-bution and morphology of c-kit positive ICCs in the nor-mal urinary bladder. We have used urinary bladders sam-ples from human necropsies (Hospital ClínicoUniversitario Lozano Blesa, Zaragoza). The samples wereprocessed for immunohistochemical (IC) methods (EnVision, Dako®), using antibodies against glio fibrilar aci-dic protein (GFAP) (specific of glia cells), enolase (speci-fic of neurons (NSE)) and CD 117 which reveals the c-kitreceptor on the ICCs membrane. Our results show ner-vous ganglia in the submucous layer next to the smoothmuscle bundles. Neurons somas have been observed inthe nervous trunks placed at the submucous layer. GFAPpositive reaction has been observed in the nervous trunkslocated in the submucous layer and inside the muscle fas-cicles. In the lamina propria it has been observed isolatedbig nuclei piriform cells. The identification of urinarybladder ICCs and their interrelationships with smoothmuscle fibres innervation will lead to the interpretation oftheir function in this organ.

P-115 WILLIAM HUNTER’S SPECIMENS OFTHE CENTRAL NERVOUS SYSTEM.

McDonald, S.W., Russell, D., Growney, C.

Laboratory of Human Anatomy, University of Glas -gow, Glasgow, Scotland, UK.

William Hunter, the famous 18th Century London ana-tomist, was born near Glasgow in Scotland and left hisanatomical collection to Glasgow University. T h i s

demonstration shows 9 of the remaining 76 of Hunter’sspecimens on the central nervous system. Hunter carriedout research into the placenta, lymphatics and bone butthe specimens illustrating the nervous system seem to bea teaching collection.

The specimens illustrated are:21.06 - The vertebral canal of a child.21.13 - The cranial dura mater.21.14 - Dried specimen showing the dura mater of a

child.21.17 - The external surface of the dura with the

superior sagittal sinus and its lacunae latera-les opened.

21.24 - Specimen of pia and arachnoid mater showingthe vascularity of the pia.

21.31 - Dissection of the brainstem showing the floorof the fourth ventricle.

21.45 - Cerebellum of a man who had been mad formany years before his death.

21.50 - Sagittal section of the brain of a young woman.21.54 - The olfactory nerves running from the olfac-

tory epithelium to the cribriform plate.21.61 - The cauda equina dissected.The numbers and descriptions are quoted from the

Museum Catalogue.In a lecture (c1770), Hunter said it was highly pro-

bably that we would always remain ignorant of how thenervous system worked. In his lectures, Hunter describedthe meninges but gave little detail of the internal featuresof the brain. He did, however, cover the corpus callosum,corpus striatum, fornix, septum lucidum, thalamus andchoroid plexus. In one set of notes he is recorded assaying, “I have shown you the parts of the brain in ageneral way so that you are able to describe the sites ofdisease, and understand the anatomical language for thevarious parts of the brain: as to the use of these parts neit -her I nor anyone else knows it. One of the sets of notes(c1770) contains a remarkable insight – that there mightbe a link between the nervous system and electricity thathad recently been discovered.

P-116 CONTRIBUTION OF GEOMEDIA PRO-F E S S I O N A L T E C H N O L O G Y TO T H EC O M P I L ATION OF D O C U M E N TA LBRAIN MAPS.

Juanes, J.A.1 , 2, Prats, A .3, Riesco, J.M.1 , 2,Lagándara, M.L.4, Blanco, E.1,2, Rubio, M.1,2,Carretero, J.1,2, Vázquez, R.1,2

1 Universidad de Salamanca. 2 Instituto de Neuro -ciencias de Castilla y León. 3 Universidad de Barce -lona. 4 Desarrollos Informáticos ABADÍA. Madrid.

We describe an Informatic Aplication (IT) develop-ment that has allowed us to capture, edit and manage spa-tial data by means of the interrelationship among differentstandard industrial databases, including Microsoft Accessand Oracle SDO/SC.

As reference images we used axial sections from theVisible Human Project (National Library Medicine, Bet-hesda, Maryland, USA). The sections were obtained at aresolution of 2048 x 1216 pixels by 24 bits of colour perpixel and a separation between slices of 1mm. However,each section was stored in a single file compressed inUnix form and, once decompressed, affords a native



image of 512 x 512 pixels in size and with 16 bits/pixel.The collection of sagittal and coronal sections was accom-plished by means of the so-called re-slicing technique.

Vectoring and segmentation of each brain structureallows the collection of three-dimensional reconstructionsof these, after which they are assigned textures, semi-transparencies, etc.

Guided by a defined system of stereotaxic coordinates,based on the atlases of Tailarach and Tournoux (1988), theinformation was introduced into the databases. This offersa true georeferenced information system (GIS) of thehuman brain.

Geomedia Professional is a last-generation ITtool for thecompilation of maps, allowing a smooth link-up and inte-gration with other standard programs such as Visual basicand Visual C++, Power Builder, Delphi, Excel, or Foxproamong others. Also, this tool supports simultaneous accessto different GIS databases, and with different formats, suchas MGE, FRAME, A r c Vi e w, MGE Segment manager, MGEGeoData manager, and MAPINFO, among others.

The convergence of data joined by the association ofdifferent articulated databases is of great interest in thefield of neuroanatomy since it allows the simultaneousmanagement of abundant interrelated information.

TAILARACH J and TOURNOUX P (1988). CoplanarStereotactic Atlas of the Human Brain Thieme. Stuttgart.

P-117 IMPLEMENTING A NOVEL TOOL FORULTRADISECTION OFTISSUES.

Leis, O., Bulnes, S., Bengoetxea, H., Azkona,G., Argandoña, E.G., Lafuente, J.V.

Universidad del País Vasco/ Euskal Herriko Unibert s i -tatea. Dpto. Neurociencias, Laboratorio de Neuro c i e n -cia Clínica y Experimental (LaNCE), Grupo 15887.

Introduction: Antibody arrays are being proposed as auseful tool for identification and simultaneous quantifica-tion of proteins. Samples from topographically selectedareas can identify specific regional proteic profiles.

MAT E R I A LA N DM E T H O D S:Extracting of proteins from theselected tissue area was performed using special lysis buff e rand after several incubation times with the antibody array,we studied the protein profile of 35 samples of diff e r e n tbrain tissues; 5 from healthy individuals (CTRL), 15 belon-ging to low grade astrocitoma (LGA) and 15 from glioblas-toma (GBM), using a 19 antibody array (Bcl-2, Caspase-1,Caspase10, E2F1, E-Cadherin, EGFR, HSP-70, Integrinalfa 5, Integrin beta 3, MGMT, MMP-9, P21waf1/cip1, p53,PTEN, Rb (p110), VEGFR1, VEGFR2, ZO-1 and A c t i n ;Hypromatrix). Quantification of inmunopositive spot areashad been done by Image J computer software.

RESULTS:We found differential protein expression bet-ween CTRL and GBM groups, and between CTRL andLGA; those differences increased between the two studiedtumoral stages for almost all the proteins included in theassay and analysed results by a univariated varianza SPSSsoftware analysis; obtaining significant diff e r e n c e sp<0.05, for the expression of Caspase-1, Caspase10, E-Cadherin, p53, VEGFR2 and p<0.005 in the case of Bcl-2, EGFR, MMP-9, PTEN, Rb (p110) expression.

CO N C L U S I O N S: Identification of differential proteinexpression gives a morpho-functional view of tissue com-position and physiological processes (metabolism, signa-lling etc...) taking place in it.

P-118 DISRUPTION OF PRECISION GRIP PAT-TERN IN ON/OFF PA R K I N S O N I A NPATIENTS USING AN A N T H R O P O-MORPHIC DEVICE.

Nombela, C. 1, Pedreño, J.L.2, Molina, J.2, RosBernal, F.1, Gomez, M.1, Herrero, M.T.1, Lopez-Coronado, J.2

1 Universidad de Murcia. 2 Universidad Politécnicade Cartagena.

Several parameters of grip and load force have beenstudied in Parkinson´s Disease (PD) by means of preci-sion grasping tasks. The aim of this work was to carry outan accurate study of reach-load-grip-hold-place-releasetasks in 27 patients (17 women, 10 men, 62.70 ±10.4years), in ON and OFF state, and 27 aged matched controlsubjects (16 women, 11 men, 61.26±20.74) using a senso-rized anthropomorphic device (5DT data Globe); to mea-sure the grip force (GF) it was used a piezorresistive sen-sor located over the data glove in the position of the indexfingertip and an accelerometer was placed on the wristand over the data glove in order to measure the accelera-tions of the hand. This apparatus can measure preciselyseveral parameters related to abnormalities in the move-ments performanced by PD patients. The experimentaltask consisted of gripping four experimental objects ofdifferent textures and weights, one by one, using indexand thumb to carry out the reaching, loading, holding, pla-cing and releasing tasks.

RESULTS: PD patients (ON and OFF states) needed sig-nificantly more time than the control group to completethe task; load velocity, acceleration, grip force and velo-city in the application of the grip force during executionof the tasks were lower in PD patients (independent oftheir state) than in the controls. Coordination between thetwo forces involved was impaired and down movementmade by PD patients was delayed with respect to the con-trol group, and to a greater extent in OFF than in ONpatients.

CONCLUSION: these delays could be a kind of freezingphenomenon occurring at the beginning of the differentsubtasks of the movement. This phenomenon would haveits origin in the basal ganglia, which has lost the capacityto generate discrete control actions at specific time land-marks during the execution of the task.

P-119 PROTECTIVE EFFECTS OF MEMANTI-NE A G A I N S T N E U R O D E G E N E R AT I O NO F THE BASALOCORT I C A L C H O L I-NERGIC SYSTEM.

Ros, F.1, Nombela, C.1, Gomez, A.1, Gomez,M.1, Antunez, C.2, Herrero, M.T.1

1 Universidad de Murcia. 2 Hospital UniversitarioVirgen de la Arrixaca.

Memantine (1-amino-3,5-dimethyladamantane) is anuncompetitive N-methyl-D-aspartate (NMDA) glutamatereceptor antagonist. We aimed to investigate the therapeu-tic efficacy of memantine in different behavioural outco-mes in a rat model of unilateral and bilateral corticaldevascularization (which induces a loss of the corticalcholinergic terminal network and a retrograde degenera-tion of the cholinergic projections that originate in thenucleus basalis de Meynert). A total of 28 Sprague-Daw-



ley rats were ramdomly divided in 7 groups: control, threegroups of animals with unilateral lesion (without treat-ment/saline, treated from 3 days before the lesion, and tre-ated after the lesion), and three groups of animals withbilateral lesion (without treatment/saline, treated from 3days before the lesion, and treated after the lesion). Thedose of memantine was 20 mg/kg/day was administeredvia Alzet minipumps implanted subcutaneously in theposterior thoracic region. Control and treated rats weretrained in an elevated T-maze and in the water maze befo-re starting the experiment and the behavioural effectswere assessed following a postsurgical delay of one week,and then repeated once a week after the lesion, until the28th day. All memantine-treated rats: i) recover spatialabilities lost after cortical devascularization (both unilate-ral and bilateral, and with treatment and ii) were signifi-cantly able to make more efficient use of their capacitiesto master the T- and the water maze tasks. These resultssuggest a neuroprotector action of systemically adminis-tered memantine, both starting before and after corticaldevascularization, and mitigates cholinergic degenerationin vivo.

Supported by Lundbeck-Spain.

P-120 TIME COURSE OF THE MORPHOLOGI-CALEXPRESSION C-FOS AND HSP-90 INTHE CNS AFTER ACUTE EXPOSURE TO900 MHz GSM RADIATION OFPICROTO-XIN MODEL OF EPILEPTIC RATS.

López-Martín, E., Jorge-Mora, T., Misa, M.J.,Suarez, J., Quintans, M., García Caballero-Parada, T., Jorge-Barreiro, F.J.

Universidad de Santiago de Compostela.

Electromagnetic fields (EMFs), with no thermalstress, are suspected to have several biological effects andinduction of genes. In previous studies we have foundneuronal activation markers (c–fos) in the brain of ratspre–treated with subconvulsive doses of picrotoxin, after2hs exposure to a 900 MHz GSM (Global System forMobile communications) radiation, at intensities similarto those emitted by mobile phones. In this study we analy-zed –by means of immunochemical testing of relevantanatomical areas– the relationship between the inductionof c–fos and heat shock proteins HSP90 markers in acuteexperimentally models of epilepsy (induced by picroto-xin), to establish the effects on the CNS (Central NervousSystem) of 50 adult male Sprague-Dawley rats after 60minutes, 24 hours and 3 days of exposure to EMF radia-tion. After exposure, the animals were sacrificed and theirbrains were cut into sections, which were processed for c-Fos and HSP90 immunodetections. C-Fos positives cellswere counted in cortical and hippocampal areas, using amicroscope connected to a morphometric software,having expressed counts per field as means ± s.e.m. Thesignificance of between–group differences was estimatedthrough two–way analysis of variance Tukey or Krus-kal–Wallis tests. 60 minutes after the radiation (AR), thec-Fos positive cells were double in all cortical areas,except in entorhinal cortex, where there were smaller dif-ferences; 24 hours AR there were differences in paleocor-tex and none in neocortex; after 3 days the counts weresimilar in the same areas of the exposed–and–treated andnon–exposed–and–treated animals. In hippocampal areas

the c-Fos expression showed differences after 60 minutesand 24 hours in CA1 and CA3 areas, but did not in Denta-te Gyrus. After 3 days, none of them showed differences.The HSP90 expression showed differences in DentateGyrus, CA1 and CA60 minutes AR, while did not showafter 24 hours in all areas; after 3 days in there were dif-ferences only in CA1.

The time course of the effects after the GSM radiationon the CNS showed to be recovered after 3 days.

P-121 C-FOS DISTRIBUTION-PATTERN INBRAIN OF MICE CHRONICALLY EXPO-SED TO POWER-FREQUENCY E L E C-TROMAGNETIC FIELDS AT ENVIRON-MENTAL POLLUTION DENSITIES.

Terol, F.F., Sánchez, M., Poveda, A., Correa, J.,Almenar, V., Sánchez, F.

Dpto. de Histología y Anatomía, División de Anato -mía y Embriología Humana, Universidad MiguelHernández, Campus de San Juan, Alicante.

Last years some epidemiological studies related seve-ral pollution levels of power-frequency electromagneticfields (50 Hz) with different pathologies, particularly chil-dren’s leukaemia. These people were chronically exposedto high levels (upper 0.2 microTesla) of these physic-polluted environmental as those living near power-lines.However these results have been refuted by other studiesand today these health-effects are still confused. In orderto explore effects into laboratory, we have developed anexperimental system that produces electromagnetic fieldssimilar to the above mentioned. Inside a large tube-sole-noid ten mice were introduced in individual dielectric-boxes where animals were chronically exposed at veryhomogeneous electromagnetic fields (10.0 microTeslaand 50.0 Hz) during a very long period (9 month) and asham-system with ten mice were used as a control. Imme-diate early genes were used as a model to explore diffe-rent distribution-patterns in cortical and sub-cortical brainareas compared to exposed and sham groups. We havefound some differences in both exposed/sham brains anda statistical significant reduction of c-Fos expression atboth habenular and paraventricular thalamic nucleus andsome evidences to a global reduction of immuno-reactiveexpression in the brains exposed.

P-122 MORPHOMETRIC EVA L U ATION FORTHE USE OF MAGNETIC RESONANCEIMAGING TO CHARACTERIZE NEURO-DEGENERATION IN MOUSE MODELS.

Carretero, J.1, Carretero, M.4, Blanco, E.1,Rubio, M.1, Sierra, E.1, Herrero, J.J.3, Prieto,A.1,2, Burks, D.J.1,2

1 Department of Human Anatomy and Histology,L a b o r a t o ry of Neuroendocrinology of INCyL.Faculty of Medicine, University of Salamanca, Spain.2 Institute of Cellular Biology, Valencia Spain. 3

Department of Surgery, Faculty of Medicine, Univer -sity of Salamanca, Spain. 4 Area of Physics and Mat -hematics, Department of Educational Sciences,School of Educational Sciences, University Pontificiaof Salamanca, Spain.



Common anatomical manifestations of neurodegene-rative diseases such as Alzheimer include loss of neurons,formation of plaques and tangles, and inflammation.However, detection and analysis of these pathologicalchanges by histological methods can only be performedpostmortem, thus complicating an accurate diagnosis inhumans or limiting aging studies and therapeutic trials inthe case of animal models. Therefore, approaches allo-wing the direct, in vivo visualization of brain pathophy-siology would greatly facilitate assessment of disease sta-tus in humans and would enable researchers to fullyexploit mouse models of neurodegenerative disorders.Here we present the use of magnetic reasonance imaging(MRI) as an analytical tool in the characterization of neu-rodegeneration the IRS-2 knockout model.

Insulin and IGF-I exert their various physiologicale ffects by phosphorylating and activating insulin receptorsubstrate (IRS) proteins. Deletion of IRS-2 in mice drama-tically reduces brain size due to impaired neuronal prolife-ration during development. The absence of IRS-2 in adultbrain produces an additional pathology: hyperphosphory-lation and accumulation of the microtubule-associated pro-tein tau. Young IRS-2 knockouts display defects in lear-ning and memory as assessed by tests such as Morris watermaze. We reasoned that IRS-2 deficiency might representa reasonable mouse model of neurodegeneration. T h e r e f o-re, we allowed these animals to age and subjected them toMRI at 18 months of age (Bruker Pharmascan system, Ins-tituto Biomedicas, Madrid). This analysis revealed a verystriking pathological difference in cerebral ventricles ofaged KOs as compared to W T controls. We used theseimages to measure area, diameters and perimeter of lateral,medial and fourth ventricles and thus, have detected signi-ficant increases of these structures in the brains of IRS-2KOs. Interestingly, enlarged ventricles have been reportedfor several neuronal disorders including schizophrenia andAlzheimer~s. Subsequent histological analysis of thesesame brains revealed other abnormalities including neuro-nal loss and amyloid deposits joint to decreases of the thikof cortical areas and basal ganglions. Thus, our initialobservations suggest that MRI provides a valuable tool forin vivo detection of pathological changes in neurodegene-rated brains of mouse models.

S u p p o rted by I+D+I program: Project (CICYT)B M C 2 0 0 2 - 0 0 8 7 2

P-123 M O R P H O L O G I C A L D ATA S U P P O RTINDUCED SYNCHRONIZING A C T I V I T YUNDER APPLIED MAGNETIC FIELDSIN ELEMENTA L TWO-NEURON NET-WORKS OF MOLLUSCS NERVOUS S Y S T E M .

Lahoz, M.1,2, Aísa, J.1,2, Junquera, C.1,2, Pérez-Castejón, M.C. 1,2, Azanza, M.J.1,2, Raso, M.1,2,Pes, N.1,2, Martínez Ciriano, C.1,2, Pérez Bru-zón, R.N.(1,2), Maestu, C.(2), Vera-Gil, A.(1,2),del Moral, A.2,3

1 Laboratorio de Magnetobiología.Departamento deAnatomía e Histología Humanas. Facultad de Medi -cina. Universidad de Zaragoza. 50009 Zaragoza,Spain. 2 Fundación Humanismo y Ciencia (Madrid,Spain). 3 Laboratorio de Magnetismo. Departamentode Física de Materia Condensada. Facultad de Cien -cias e ICMA. Universidad de Zaragoza-CSIC, 50009Zaragoza, Spain.

Synchronizing activity in pairs of pacemaker neuronsfrom the central nervous system (CNS) of molluscs (Helixaspersa) has been observed to be induced under appliedsinusoidal magnetic fields (AMF) of extremely low fre-quency (50 Hz) and low intensity (1-15 mT) (1, 2). Theaim of this work has been to find out a morphological sup-port for the synchronizing activity. It has been widelyaccepted that in mammals the synchronization of neuronsbioelectric activity is promoted through gap junctions,being astrocytes the main cells responsible for the trans-mission of excitability between neuron distant networks.We have found by electron microscopy (EM) and immu-nocytochemistry (IC) methods morphological data whichsupport the described synchronization in molluscan neu-rons induced by applied AMF. EM shows neuron-neuron,neuron-glia and glia-glia gap-like junctions. We havemade the characterization of Helix aspersa nervous cellswith connexin 26 (Chemicon), connexin 43 (Chemicon),enolase (Chemicon) and glio fibrillary acidic protein(GFAP, DAKO) antisera. Positive and negative controlshave been made on Wistar rat brain samples. We haveconcluded that in molluscs CNS: i) there are two kind ofglia cells distributing respectively among neurons somaand neuron fibres in the neuropile; ii) neurons and gliacells express connexin 26; iii) neither enolase nor conne-xin 43 and GFAP are good markers for molluscan CNScells. Such a reaction differences could be the consequen-ce of either non-specificity for mammals antisera or phy-logenetic differences in the expression of the proteins tes-ted in molluscs with respect to mammals nervous cells.We have shown that applied 50 Hz MF mimic the effectof glutamate on neurons (3). MF stimulation is mediatedby calcium ions which are free liberated due to the inte-raction of MF with neuron membrane accordingly withour proposed model (2). We have come to the conclusionthat applied 50 Hz-MF induces simultaneous stimulationof neuron-glutamaergic and glutamate-glia-dependentpathways, as a result neurons synchronization is facilita-ted by applied sinusoidal MF.

(1) M.J. Azanza, A.C. Calvo, A. del Moral. J Magn MagnMat 226-230, 2101-2103 (2001).

(2) M.J. Azanza, A.C. Calvo, A. del Moral. ElectromagnBiol Med 209-220 (2002).

(3) A.C. Calvo, M.J.Azanza. Comp Biochem Physiol C 124,99-107 (1999).

P-124 CORTICAL AXONAL PROJECTIONS TOSOMATO-SENSORY AREAS IN RABBITCORTEX.

Fuentes, T., Alejo, A., Mendizabal-Zubiaga, J.L.,Pró-Sistiaga, P., Reblet, C., Bueno-López, J.L.

Departamento de Neurociencias, Facultad de Medi -cina y Odontología, Universidad del PaísVasco/EHU.

The axonal connections presented here had been scar-cely studied in rabbits to date. In doing this, we have addi-tionally estimated the occurrence of inverted pyramidal(IP) cells out of the examined projection cells. Youngadult rabbits received 1% CTb (0,2-0,4 ml) injections eit-her in primary somatosensory cortex (SI), the rim betwe-en somatosensory and visual areas (S-V), or in precentralareas, both medial (PrCM) and lateral (PrCL).

• Injections in PrCM caused retrograde labelling thatwas important in PrCL, SI, SII and anterior limbic cortex



and scarce in the posterior limbic cortex and prelimbiccortex.

• Injections in PrCL yielded important regrogradelabelling in SI. They also labelled SII, PrCM, prelimbicareas and anterior limbic areas.

• Injections in posterior SI produced retrograde labe-lling in SII, PrCL and S-V. In turn, injections in medial SIdid so in PrCM and several polimodal associative areassuch as area 25 (which is prefrontal), anterior cingulateareas (especially the rostralmost one) and perirhinal cortex.

• Injections in posterior S-V revealed major retrogradelabelling in SI and VI, minor in SII and scarce in VII,PrCL y PrCM.

In the insular claustrum, there was either minor retro-grade cell labelling following the injections in SI, S-V, ormajor following those in PrCM. The latter injections alsolabelled projection cells in anterior endopiriform claus-trum and basolateral amigdala.

There was usually many retrogradely labelled IP cellsamong labelled infragranular neurons. Out of the lattercells, IP-cell percentage was higher for projections fromassociative areas (S-V, VII, SII) to SI, PrCL to S-V andVI-VII to PrCM, in said order.

The results of the present study show that the motorprecentral cortex can be further subdivided in the rabbitcortex. Because of its axon connections, in particular withthe amigdala, PrCM would correspond with the motorsuplementary area of other mammals.

This work was granted by MEC-BSA2001-11 7 9 ,9/UPV00212.327-15837/2004 and Fundación Gangoiti.

P-125 ANALGESIC-INDUCED A N T I N O C I C E P-TION MAY BE MEDIATED BY THE A7NORADRENERGIC NEURONS IN T H ERAT.

Valverde Navarro, A . A., Teruel Martí, V., Cer-vera Ferri, A., Cabanes Vila, J., Hernández Gil deTejada, T., Ruiz To r n e r, A., Martínez Soriano, F.

Departamento de Anatomía y Embriología Humanas.Universidad de Valencia.

The endogenous antinociceptive circuitry, originallydescribed by Basbaum and Fields, involves the mesen-cephalic periaqueductal gray (PAG) and the rostral ven-tromedial medulla (RVM). It has been clearly establishedthat spinally-projecting neurons located in the RVM,which includes the nucleus raphe magnus and the nucleusreticularis gigantocellularis pars a, constitute an importantcomponent of this descending system. However, modula-tion of nociceptive transmission induced by activation ofRVM neurons is mediated in part by noradrenergic neu-rons that project to the spinal cord. Anatomical tract tra-cing studies have demonstrated that a significant popula-tion of neurons in the RVM project to the pontine A7catecholamine cell group that provide the major noradre-nergic innervation of the spinal dorsal horn. The aim of

this work was to provide more evidence that activation ofneurons in the RVM produces antinociception by activa-ting noradrenergic neurons in the A7 cell group.

We studied the expression of Fos-like immunoreacti-vity into the tyrosine hydroxylase-positive A7 neurons ofthe rat after intraperitoneal injection of two analgesics:morphine and metamizol.

Fos-like immunoreactive neurons were localized nearthe A7 cell group both in the morphine treated and in themetamizol-treated animals. However, only a few Fos-positive cells were also tyrosine-hydroxylase-immunore-active A7 neurons.

Our results are consistent with the possibility thatsome of the analgesic effects of morphine and metamizolmay be attributable to activation of the A7 noradrenergicneurons.

P-126 ANALGESIC-INDUCED FOS-LIKEI M M U N O R E A C T I V I T Y IN THE RATROSTRAL VENTROMEDIALMEDULLA.

Hernández Gil de Tejada, T., Valverde Nava-rro, A.A., Teruel Martí, V., Cervera Ferri, A.,Ruiz Torner, A., Martínez Soriano, F.

Departamento de Anatomía y Embriología Humanas.Universidad de Valencia.

The rostral ventromedial medulla (RVM) is a criticalrelay from the mesencephalic periaqueductal gray (PAG)to the spinal cord dorsal horn. The RVM includes thenucleus raphe magnus and the nucleus reticularis giganto-cellularis pars a. All these structures were originally des-cribed by several authors as a part of an endogenous anti-nociceptive circuitry. The goal of the experiment was todetermine if the intraperitoneal injection of a mild analge-sic such as the metamizol or an opioid analgesic such asthe morphine were able to induce expression of c-fos pro-toncogene into the nuclei of the RVM.

Experiment was carried out in adult male Sprague-Dawley rats. We used Fos immunocytochemistry todemonstrate the activated neurons. A systemically admi-nistration of metamizol was chosen as antinociceptivenon-opiate stimulus while morphine was the narcoticanalgesic. Rats were subdivided into three groups: a meta-mizol-treated group (500 mg/kg, i.p.), a morphine-treatedgroup (10 mg/kg, i.p.) and a control group (saline, i.p.).Animals were sacrificed 2 hours and half an hour after theanalgesic (or saline) stimulation.

The results showed Fos-like immunoreactivity in thenucleus raphe magnus and nucleus gigantocellularis parsa of both metamizol-treated and morphine-treated rats incontrast with the control rats where Fos-like immunoreac-tive elements were absent.

These results support that the RVM is an importantcomponent of endogenous antinociceptive circuitry.The results are also consistent with a role of descendingprojections from RVM in both opiate and non-opiatea n t i n o c i c e p t i o n .



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