Aus der Klinik und Poliklinik für Anästhesiologie und Intensivmedizin (Geschäftsführender Direktor: Univ.-Prof. Dr. med. Michael Wendt)

der Medizinischen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald

Atem-Muskulatur, glatte Muskulatur und lokale Zirkulation bei Trauma und Inflammation

(Respiratory skeletal muscle, smooth muscle and local circulation in trauma and inflammation)

Habilitationsschrift

zur

Erlangung des akademischen Grades

doctor medicinae habilitatus

(Dr. med. habil.)

an der Medizinischen Fakultät

der

Ernst-Moritz-Arndt-Universität Greifswald

vorgelegt von

Dragan Pavlovic

geboren am 4. 8. 1949

in Vranje (Jugoslawien)

Greifswald, 2006

2

Dekan: ..................................................................................................... 1. Gutachter: .............................................................................................. 2. Gutachter: .............................................................................................. 3. Gutachter: .............................................................................................. Tag der Habilitation: ......................................................................................

3

Respiratory skeletal muscle, smooth muscle and local circulation in trauma and inflammation Foreword

The text that follows is one of possible ways to summarise more then 20 years of research. It

started following the Exercise physiology course that I took in London and was inspired by

meeting number of famous physiologists including some of the British members of the Mount

Everest expedition from the fifties. I moved then to Paris to the laboratory that focussed on the

respiratory physiology and respiratory muscles including smooth muscle of the airways. My

interests were concentrated mainly on the muscle pathophysiology, skeletal and some years

later, smooth muscle, with some ramifications including respiratory mechanics, lung

ventilation, asthma research and related intensive care aspects of ventilation. Certainly, my

anaesthesiology background served as a link between physiology and clinics. After moving to

the anaesthesiology department in Greifswald, the anaesthesiology interests prevailed, although

interests in muscle physiology component remained. The focus was slightly moved towards

vascular smooth muscle and microcirculation, but the general frame remained the same. Some

other aspects of the anaesthesiology, obviously, had to be covered since the department of the

experimental anaesthesiology which I have been directing, had to accommodate its research to

the relatively large spectrum of interests that anaesthesiology and intensive care medicine

incorporate. Therefore, the present short report treats primarily (skeletal and smooth) muscle

research. However, it includes also related aspects of global microcirculation and the issues like

some aspects of pathophysiology of respiration or even pain research. In order to give more

realistic picture of may be one longer research career, the anaesthesiology related issues

belonging to the medical ethics have also been included.

4

Contents

Zusamenfasung (Deutsch) 6

Summary (English) 11

I. Respiratory responses trauma and inflammation 16 a. Modulation of Airway Tone 16

b. Diaphragm contractility and lung mechanics 22

II. Vascular response to inflammation and trauma 29

III. Other related aspects 38

a. Pain and alternative methods in medicine 38

b. Medical ethics 41

IV. References and chronological list of publications 44

V. Papers: Principle theme original research articles, 1st, second, or last author (not paginated) 1. Pavlovic, Dragan, Evelin Brione, Michel Fournier, and Michel Aubier: Partial inhibition by epithelium of tracheal smooth muscle relaxation induced by potassium channel activator BRL 38227. Br. J. Pharmacol., 110: 139-144, 1993. 2. Pavlovic, Dragan. , Naima Viires, Christine Zedda, Michel Fournier, and Michel Aubier: Effects of corticosteroids on epithelial structure and smooth muscle function in rat trachea. Eur. Respir. J. 11: 575-582, 1998 3. Helge Frieling, Matthias Gründling, Kai-Steffen Lauer, Michael Wendt, Thomas Hachenberg, Christian Lehmann, and Dragan Pavlovic Intraperitoneal instillation of polihexanide produces hypotension and vasodilation: in vivo and in vitro study in rats Int J Colorectal Dis. 21: 373-380, 2006. 4. Dragan Pavlovic, MD, Helge Frieling, MD, Kai-Stephan Lauer, MD, Vo Hoai Bac, MSc, Jorn Richter, Michael Wendt, MD, Christian Lehmann, MD, Taras Usicenko, MD, Konrad Meissner, MD, and Matthias Gruendling, MD. Thermostatic tissue platform for intravital microscopy: “the hanging drop” model Journal of Microscopy-Oxford (in press: 2006.) 5. T.I. Usichenko, S. Foellner, M. Gruendling, F. Feyerherd, Ch. Lehmann, M. Wendt, D. Pavlovic Akrinor®-induced relaxation of pig coronary artery in vitro is transformed into α 1-receptor-mediated contraction by the pre-treatment with propranolol J Cardiovasc Pharmacol, 47: 450-455, 2006.

5

VI. Erklerung 60

VII. Lebenslauf 61

6

Zusammenfassung

a. Die Modulation des Atemwegstonus Wir schildern im ersten Abschnitt aus historischer

Perspektive praktische und theoretische Probleme, im Zusammenhang mit unserer Forschung.

Diese führten zu einer Entwicklung eines neuen in vitro Modells der glatten

Trachealmuskulatur. Im zweiten Abschnitt werden Ergebnisse einiger Experimente dargestellt,

welche mit klassischen Modellen für in vitro Untersuchungen an Bronchialgewebe und

beatmeten Ratten gewonnen wurden. Das neue Modell ermöglicht es, die glatte

Trachealmuskulatur selektiv innen oder außen pharmakologisch zu stimulieren, um die

Beteiligung des trachealen Epithels bei der Regulation der Kontraktion der glatten Muskulatur

zu klären. Die bisherige Forschung weist auf eine aktive Rolle des Epithels der Atemwege bei

der Regulation des bronchomotorischen Tonus. Um dieses Phänomen weitergehend zu

untersuchen, entwickelten wir ein in vitro Modell, welches uns es erlaubt, die äußeren oder

inneren Gewebsschichten der Atemwege, getrennt mit Krebslösung zu perfundieren. So ist es

uns möglich diese Schichten selektiv pharmakologisch zu stimulieren. Zu diesem Zweck

exidierten wir eine Rattentrachea, brachten eine Kanüle in das Lumen ein und perfundierten

innere und äußere Gewebsschichten unabhängig von einander. Anschließend wurde der

Gewebsring mit zwei gegenüberliegenden Haken befestigt. Der untere Haken ist fixiert,

während der obere mit einem Transducer verbunden ist. Eine isometrische Kontraktion wurde

durch Perfusion mit Carbachol in einzelnen und kumulativen Konzentrationen hervorgerufen.

In der Hälfte der Experimente wurde das Epithel mechanisch entfernt. In dem ersten Teil

unserer Arbeit stellten wir fest, dass die Tonuserhöhung mit längerer Latenz zustande kam,

wenn wir es auf die innere Gewebsschicht applizierten. Dieser Effekt wurde durch Entfernung

des Epithels aufgehoben.

7

Zusätzlich stellte sich heraus, dass bei entferntem Epithel 1) die maximal Spannung

unabhängig von der Carbacholkonzentration zu nahm und 2) auch die Sensibilität sich

gegenüber Carbachol verstärkte. Zusätzlich war es uns möglich zu demonstrieren, dass bei

Epithelentfernung die Sensibilität gegenüber dem Relaxans BRL 38227 (Kalium-Kanal-

Aktivator) zunahm, so dass die Wirkung die von Aminophyllin übertroffen wurde. Gleichzeitig

beobachteten wir, dass Triamcinolon appliziert auf Trachealringe der Ratte, die Anzahl der

Epithelzellen und der „Cross-section-areas“ reduzierten, ebenso wie die 50% Tmax von

Carbachol (In) verringert wurde. Als Ursache dieser Effekte vermuten wir Atrophie als Folge

von Mangelerbnährung der glatten Trachealmuskulatur und des Trachealepithels durch

Kortikosteroide. Diese Ergebnisse sind die Vorbereitung für die Verbesserung des

Experimentalmodells. Basierend darauf untersuchten wir die die Wirkung von Carbocystein an

einem Versuchsaufbau bei dem zwischen dem alten Model und dem neuen gewechselt werden

konnte. Es stellte sich heraus, dass Carbocystein einen geringen inhibitorischen Effekt auf die

Sensibilitätssteigerung von Carbachol an glatter Trachealmuskulatur der Ratte ohne Epithel

bewirkt. Es wurde bereits eine ganze Reihe von standardisierten in vitro Experimenten

etabliert, einschließlich isoliert-perfundierter Trachea und Ringpräparationen um Kontraktilität

der glatten Muskulatur und den Einfluss des Epithels oder verschiedener Pharmaka auf den

Muskeltonus zu untersuchen. Molekular-biologische Techniken werden verwandt um

Signalwege, im Zusammenhang der allergischen Reaktion des bronchialen Gewebes und

unterschiedlicher potenter Therapeutika (DHEA- Dehydroepiandrosteron), nach zu verfolgen.

Des Weiteren umfasst diese Forschung nicotinerge und muskarinerge Arzneien, welche in der

Anästhesie angewandt werden. DHEA relaxiert bronchiale Muskulatur der Ratte und des

Menschen. Somit kann es als potenter Bronchodilatator bezeichnet werden. Relaxantien der

Skelettmuskulatur wirken mit unterschiedlicher Stärke auch auf glatte Muskulatur. Eine genau

Kenntnis dieser Eigenschaft, würde Vorteile für die gezielte Anwendung bringen. An einem zu

diesem Zweck entwickelten in vitro Model der Rattentrachea zeigten wir, dass das Epithel

8

Einfluss auf den Tonus der glatten Muskulatur hat. Aber das Epithel besitzt als

Diffusionsbarriere eine wichtige Funktion. Daher wird sich das oben beschriebene Modell als

wertvoll, bei der weiteren Erforschung der Rolle des Epithels bei Kontraktion glatter

Muskulatur, erweisen.

b. Zwerchfellkontraktion und Atemmechanik Das Zwerchfell ist als Quergestreifter Muskel

für die Atmung verantwortlich. Es kontrahierte sich ähnlich wie der Herzmuskel über die

gesamte Lebensdauer. An Hand verschiedener in vitro und in vivo Versuche mit

unterschiedlichen Tieren haben wir die Kontraktilität und die Wirkung verschiedener

pathophysiologischer Mechanismen sowie von Medikamenten untersucht. Opioidanalgetika

blockieren Aktionspotentiale in der Skelettmuskulatur durch nicht- spezifische und spezifische

Opioidrezeptoren.

Wir verwendeten Zwerchfellmuskelfasern der Ratte um zu untersuchen, ob diese Wirkstoffe

die Kontraktilität dieses Muskels beeinflussen. Eine isometrische Kraft erzeugten wir mit

elektrischer Stimulation ( 8/min über 0,3 ms), welche mit einem hoch empfindlichen

photoelektrischen Transducer gemessen wurde. Nach einer Kontrollmessung, wurde der

Präparation in Krebslösung Pentazocine hinzugefügt. Die elektrische Stimulation wurde

fortgesetzt und registriert. Durch die Steigerung der Konzentration von Pentazocine ermittelten

wir eine Dosis-Wirkungskurve. Dieses Experiment wiederholten wir mit Morphium,

Phenoderidin und Naloxon. Wir fanden heraus, dass Opioide die Spannung der isolierten

Zwerchfellmuskulatur der Ratte über längere Zeit verringern. Wahrscheinlich beruht dies auf

direkter Hemmung der kontraktilen Mechanismen in der Muskelzelle.

Kortikosteroide werden oft bei Patienten eingesetzt, denen ein akutes respiratorischen Versagen

droht (COPD, Asthma bronchiale). Ziel der Studie war es, die Wirkung einer

Kortikosteroidanwendung über acht Tage hinsichtlich der Muskelatrophie und kontraktilen

9

Eigenschaften zu untersuchen. In identischen in vivo und in vitro Experimentieranordnungen

wurde direkt der Nervus phrenicus stimuliert und der transdiaphragmatische Druck (Pdi).

gemessen. In beiden Versuchsanordnungen blieben „low-frequncy“ und „twitch“ unbeeinflusst.

Aber die standardisierter tetanische Kraft war in der ST-Gruppe signifikant verringert,

verglichen mit der PF-Gruppe (p<0.001). Musculus soleus und EDL-Muskel wurden ebenfalls

in vitro untersucht. Steroide zeigten keine Wirkung. EDL bewirkte eine leichte Verringerung

(11%) bei standardisierter tetanischer Kraft. Kalziumionen spielen eine bedeutende Rolle für

alle Arten der Muskulatur. Besonders der Herzmuskel ist hoch sensitiv gegenüber der

extrazellulären Kalziumkonzentration. Die Dosis-Wirkungs-Kurve von BAY K 8644 und

Nifedipin wurde in vitro erstellt (3). Bei beiden Wirkstoffen beobachteten wir eine

Veränderung der „twitch“. In Medium mit geringer Kalziumkonzentration (0,5 mM) wurde die

„twitch“ Potenzierung durch beide Wirkstoffe verstärkt (Steigerung der Konzentration auf

60% von 10(-5) M Bay K 8644 oder Nifedipin). Dies bestätigt das Zwerchfellmuskulatur sich

in Bezug auf den Kalziumtransport vom Skelettmuskel unterscheidet. Die Wirkung einer

dreitägigen Pneumokokkeninfektion auf die Kraft und Ausdauer des Diaphragmas wurden an

einem Rattenmodel untersucht. Wir folgern aus den Ergebnissen, dass ein vier tägiger Mangel

eine Reduktion des Zwerchfellgewichtes hervorruft, in Kombination mit verringerter Kraft und

Ausdauer. Im Zusammenhang mit Sepsis (Endotoxinmodell) und Hypoxie wurden die Effekte

noch deutlicher. Ob maschinelle Beatmung die Ursache von Muskelschwäche, welche zu einer

verlängerten Entwöhnungsdauer führt, konnte nicht demonstriert werden. Wir untersuchten die

Folgen 48-stündiger Beatmung auf Gewicht, kontraktile Eigenschaften und enzymatisches

Profile des Diaphragmas, des Musculus soleus und des Musculus extensor digitorum longus

(EDL). Wir denken das eine Studie basierend auf Muskeln nach der Rehabilitation von

Skelettmuskeln nach Langzeitimmobilisierung der Extremitäten nach einem Trauma oder

Operation weitere Erkenntnisse bringen könnte, ebenso Untersuchungen an Diaphragmata bei

10

Quadriplegie, nach elektrischer oder magnetischer Stimulation während andauernder

mechanischer Beatmung.

Mit vorhergehender Studie an Hunden zeigten wir, dass denervierte Diaphragmata resistent

gegenüber elektrischer Stimulation wurden. Selbst bei Platzierung mehrer Elektroden

benötigten wir starke Spannungen, um messbare Kontraktionen zu verursachen. Deshalb

schlagen wir vor, dass in einer anderen Studie die funktionelle Kapazität des denervierten

Diaphragmas durch Reinervierung mit dem Nervus vagus untersucht wird.

Unsere jetzige Arbeit fand in Verwendung einer Mehrkanalbeatmungskanüle statt. Diese

Kanüle sicherte einen ausreichenden Gasaustausch bei geringerer Flussrate und geringem

Risiko durch Druckschädigung der Lunge. Die beschriebene Kanüle hat trotz dieser

vorteilhaften Eigenschaften noch keinen Platz im klinischen Alltag von Patienten mit Risiko

der Druckschädigung durch maschinelle Beatmung. Es stellte sich bei Erwachsenen aufgrund

des großen exspiratorischen Volumens und dem Verlust an „tidal volumen“ als schwierig

heraus. Wie auch immer, die Idee des konstanten minimalen Flusses bei der maschinellen

Beatmung kann andere bedeutende Vorteile haben. Wir führten zwei Studien durch, mit einer

Kanüle, welche als Luftpumpe verwendet werden kann und eine aktive Inspiration und

Exspiration, ermöglicht. Beide Studien zeigten das, dass selbe Gasvolumen zugeführt und

abgesaugt werden konnte. Wenn ein adäquater I:E-Quotient und eine genügende Flussrate

gewährleistet werden, könnten eine akute maschinelle Beatmung physiologischer und

pathologischer Lungen mit einer 16G Kanüle beherrscht werden. Dazu genügt ein einfaches

Beatmungsgerät, welches mit Sauerstoff versorgt wird. Diese Ergebnisse wurden bereits

veröffentlicht.

11

Summary

a. Modulation of Airway Tone. We describe, in the first section, with an historical

perspective, theoretical and practical problems related to our research works that were linked to

the development of a new model of tracheal smooth muscle in vitro. In the second section we

give some recent experimental results produced in the classical in vitro model of bronchial ring

and in the cannulated rat trachea. The model presented permits to stimulate pharmacologically

selectively inside (in) or outside (out) of the tracheal smooth muscle and to obtain some

information of the involvement of the tracheal epithelium in the regulation of the smooth

muscle tone.

There is evidence implying an active role of airway epithelium in the modulation of

bronchomotor tone. To study this phenomenon, we designed an in vitro system allowing

pharmacological stimulation of either the inside or outside of the airway lumen. Rat tracheas

were excised, cannulated, and their inside and outside perfused independently with Krebs

solution. Two hooks were inserted through opposite sides of the tracheal wall; the lower one

was attached to a fixed point, while the upper one was connected to a force transducer.

Isometric contractions of the tracheal muscle were elicited by carbachol solution perfused in

single and cumulative concentrations. In one-half of the preparations the epithelium was

mechanically removed. In our first works we found that the time course of tension development

was longer when carbachol was administered inside the trachea: an effect that was abolished

when the epithelium was removed. In addition, removal of the epithelium was found 1) to

increase the maximal tension irrespective of the route of carbachol perfusion and 2) to increase

the sensitivity of the preparation to carbachol stimulation. In addition, we were able to

demonstrate that epithelium removal increased the sensitivity to the relaxant effect of BRL

12

38227 (potassium channel activator) that had a more potent relaxant effect than aminophylline.

Equally, we examined the effects of triamcinolone administration on the rat trachea and found

that that agent reduced the number of epithelial cells and the tracheal smooth muscle cross-

sectional area, induced a decrease in maximal tension and reduced the time required to reach

50% Tmax in carbachol (In) preparations. We conclude that the observed changes resulted

from atrophy of tracheal smooth muscle induced by undernutrition and atrophy of tracheal

smooth muscle and tracheal epithelium induced by corticosteroid treatment. Mentioned works

were a prerequisite to the recent improvements of the model. We examined the effects of

carbocisteine in a model that now could be used, interchangeably for ring and cannulated

preparation. We conclude that carbocisteine has small inhibitory effects on the sensitivity to

carbachol of the rat tracheal smooth muscle denuded of epithelium. A number of unique

methods were employed in in vitro experiments, including the isolated, perfused trachea model

and airway ring preparations for measurement of both contractile activity of the smooth muscle

and influence of either epithelium or various pharmacological agents on the smooth muscle

tension. Molecular biology techniques were used to explore in detail the transduction pathways

involved in allergic responses of bronchial tissue and pharmacological effects of some potential

therapeutic agents (dehydroepiandrosterone, DHEA) or to estimate some accompanying effects

of nicotinic and muscarinic agents that are extensively used in anaesthesia. DHEA was found to

relax rat and human bronchial muscle and is considered as potential bronchodilator. Skeletal

muscle relaxant agents were found to relax smooth muscle to a various degrees. Knowledge of

their potential smooth muscle relaxing action is valuable for their proper use as muscle relaxing

agents. We demonstrated that in one for that purpose conceived rat tracheal model in vitro,

airway epithelium modulates smooth muscle tone. It has nevertheless an important function as

a diffusion barrier. Described model is valuable for examining the effects of bronchial

epithelium on bronchial smooth muscle contraction.

13

b. Diaphragm Contractility and Lung mechanics. Diaphragm is a skeletal muscle that

provides for ventilation of the lungs. It contracts permanently during the whole life span

similarly to the heart. We examined in vitro end in vivo its functioning in various animal

models its contractility and the effect of various pathohysiological circumstances, including

drug effects.

Opioid analgesics block action potential production in skeletal muscles by a non-specific and

opiate drug receptor mediated blockade. To investigate whether those drugs also affect skeletal

muscle fibre tension generation we obtained costal diaphragmatic fibres from rats. Isometric

force generated in response to supramaximal electrical stimulation (8 times/min, duration of

stimulation 0.3ms) was registered with a highly sensitive photoelectric transducer. After a

period of control measurements, preparation was perfused with Krebs solution which contained

pentazocine, while the fibres were continuously stimulated and twitches recorded. Incremental

concentrations of pentazocine were tried and cumulative dose-response curve was constructed.

In separate experiments preparations were perfused with different concentrations of

pentazocine, morphine, phenoperidine and naloxone. We found that opiods have long lasting

depressant effect on tension generation of rat isolated diaphragmatic fibres which is probably

result of their direct inhibitory action on contractile mechanisms of muscle cells. Similarly,

corticosteroids are the agents frequently used in patients that potentially may develop

respiratory failure (COPD, asthma patients). The study was undertaken to determine the effects

of 8 days of corticosteroid administration on diaphragmatic atrophy and contractile properties.

In identical in vitro setting and in in vivo experiment where phrenic nerve was directly

stimulated and transdiaphragmatic pressure measured (Pdi). In both preparations, twitch and

low-frequency force were unaffected, whereas normalised tetanic force, in the ST group was

markedly reduced compared with that in the PF group (p less than 0.001). Soleus and EDL

14

muscles were also studied in vitro. Although steroid treatment had no effect on the soleus, in

the EDL, a slight (11%) decrease in normalised tetanic tension was observed. Calcium ions

play important role for both muscle, although the hearth muscle is highly sensitive to the

calcium extracellular concentration. The dose-response effects of BAY K 8644 and nifedipine

on diaphragmatic contractility were assessed in vitro (3). We found that both BAY K 8644 and

nifedipine altered twitch characteristics. In low-calcium media (0.5 mM) twitch potentiation

produced by the two drugs was further enhanced (increasing 60% for 10(-5) M BAY K 8644 or

nifedipine). This confirmed that diaphragm could be, in respect of Ca-transport, different from

skeletal muscles. The effects of a 3-day pneumococcal infection on diaphragmatic strength and

endurance capacity were studied in in vivo rat model. We conclude that a 4-d total fast

produces a reduction in diaphragmatic weight, which is associated with a decreased

diaphragmatic strength and reduced endurance capacity. If combined with sepsis (endotoxine

model) or with hypoxia we demonstrated that the deleterious effects were even more

pronounced.

Whether mechanical ventilation is the cause of muscle fatigue that results in prolonged

weaning was not conclusively demonstrated. We measured in rats the effects of 48 h of

mechanical ventilation on the weight, contractile properties, and enzymatic profile of the

diaphragm, the soleus and the extensor digitorium longus (EDL) muscles. We proposed, in

another work, that based on experience with the rehabilitation of skeletal muscles during long-

term immobilisation of the extremities following trauma or surgery and on experience with

diaphragm pacing in quadriplegics, intermittent electrical or magnetic stimulation of the

diaphragm while the lungs are still being mechanically ventilated may be beneficial.

In a preliminary study in dogs we were able to demonstrate that denervated diaphragm rapidly

becomes resistant to the electrical stimulation and even placement of multiple electrodes

15

requires very high currents to produce almost undetectable muscle contractions. Therefore we

proposed , in another work, that restoration of functional capacity of denervated diaphragm

may be achieved by reinervating it with vagus nerve.

Our recent works were related to the multiple channel jet ventilation tube (McETV). That tube

ensured adequate gas exchanges with lower insufflation pressures and could diminish positive

pressure ventilation-induced injury. The described endotracheal tube although it was found to

be quite suitable, has not yet its place in every-day ventilation of the patients that are at risk

from high-pressure ventilation. It proved to be difficult to use in adults, because of high

backward flow through larger endotracheal tubes and loss of tidal volume. However the idea of

constant minimal flow ventilation could have other important advantages. We conceived a

venturi valve that could be used as an air pump, providing active inflation and deflation of the

lungs and performed two studies that are submitted for publication. In both studies we

demonstrated that the same amount of gas could be insufflated and suctioned with our venturi

pump. If an adequate I:E ratio and appropriate flow are used, satisfactory emergency

ventilation of normal and pathological lungs could probably be managed with a 16G cannula

and a simple venturi pump supplied with pressurized oxygen.

Keywords: Smooth muscle models; tracheal epithelium; contraction, relaxation,

hyperreactivity, diaphragm contractility, weaning, jet ventilation

16

Respiratory skeletal muscle, smooth muscle and local

circulation in trauma and inflammation

Following my Diploma in Sports Medicine (1), obtained in London, I continued research

activity in Paris and was involved mainly with skeletal and smooth muscle pathophysiology.

The research of the department (INSRM 226 and 408) was centred on respiratory physiology

and my work was, thereby, respiratory oriented. Later works, particularly after moving to the

anaesthesiology department in Greifswald, concerned larger spectrum of anaesthesia linked

field.

1. Respiratory responses to inflammation and trauma

Bronchial hyperreactivity (BHR) is the main feature of asthma. There are many factors that

have been identified as to be linked to BHR, but none of them has been proved so far to be the

ultimate cause of the condition. In this section we will be mainly concerned with the role of

bronchial epithelium in the modulation of the airway smooth muscle tone with the scope to get

further insight into the mechanisms of BHR.

a. Modulation of Airway Tone.

We describe, in the first section, our works, with an historical perspective, and the theoretical

and practical problems related research works that were linked to the development of a new

model of tracheal smooth muscle in vitro. In the second section we give some recent

experimental results produced in the classical in vitro model of bronchial ring and also in the

cannulated rat trachea. The model that is presented (5, 11, 14), permits to stimulate

pharmacologically selectively inside (IN) or outside (OUT) of the tracheal smooth muscle and

17

to obtain some information of the involvement of the tracheal epithelium in the regulation of

the smooth muscle tone.

From the ref. 16.

There is evidence implying an active role of airway epithelium in the modulation of

bronchomotor tone. To study this phenomenon, we designed the mentioned in vitro system

allowing pharmacological stimulation of either the inside (trans-epithelial) or outside (trans-

serosal) of the airway lumen. Rat tracheas were excised, cannulated, and their inside and

outside perfused independently with Krebs solution. Two hooks were inserted through opposite

sides of the tracheal wall; the lower one was attached to a fixed point, while the upper one was

connected to a force transducer. Isometric contractions of the tracheal muscle were elicited by

carbachol solution perfused in single and cumulative concentrations. In one-half of the

18

preparations the epithelium was mechanically removed. In our first works we found that the

time course of tension development was longer when carbachol was administered inside the

trachea: an effect that was abolished when the epithelium was removed.

From the ref. 16.

In addition, the removal of the epithelium was found 1) to increase the maximal tension

irrespective of the route of carbachol perfusion and 2) to increase the sensitivity of the

preparation to carbachol stimulation. In addition, we were able to demonstrate that epithelium

removal increased the sensitivity to the relaxant effect of BRL 38227 (potassium channel

activator) that had a more potent relaxant effect than aminophylline (14, 24). Equally, we

examined the effects of triamcinolone administration on the rat trachea and found that that

agent reduced the number of epithelial cells and the tracheal smooth muscle cross-sectional

area, induced a decrease in maximal tension and reduced the time required to reach 50% Tmax

in carbachol (IN) preparations (epithelial stimulation) (25, 44). We conclude that the observed

changes resulted from atrophy of tracheal smooth muscle induced by undernutrition and

atrophy of tracheal smooth muscle and tracheal epithelium induced by corticosteroid treatment.

However, liberation of an relaxing factor could not be demonstrated in a model of simultaneous

perfusion of epithelial and serosal side of rat trachea (30). On the other hand, it was found that

19

chronic hypoxia, from birth to 3 months of age, produced diminished sensitivity of rat tracheal

rings to carbachol (31).

Effects of BRL 38227 (From ref. 17)

Mentioned works were a prerequisite to the recent improvements of the model. We examined

the effects of carbocisteine in a model that now could be used, interchangeably for ring and

cannulated preparation (26, 58, 96). We concludeed that carbocisteine has small inhibitory

effects on the sensitivity to carbachol of the rat tracheal smooth muscle denuded of epithelium.

20

It is possible to explain our results by the existence of some epithelium derived contracting

factor (EpDRF, figure). Nevertheless, more experiments are to be done to verify this

hypothesis.

A number of unique methods were employed in in vitro experiments, including the isolated,

perfused trachea model and airway ring preparations for measurement of both contractile

activity of the smooth muscle and influence of either epithelium or various pharmacological

agents on the smooth muscle tension or tracheal smooth muscle mass (25, 44). Molecular

biology techniques were used to explore in detail the transduction pathways involved in allergic

responses of bronchial tissue (32, 36, 38, 39, 46, 47, 98).

In vitro: normal sensitivity (excitatory / inhibitory balance)

EpDCF?

After

CCS

treatme

nt

- Ep - Ep

+ capsaicin

B. In vitro: normal C. In vitro: inhibition D. In vitro: normal

Sensitive nerve Inhibitory mediators Smooth muscle Tracheal epithelium

Excitatory mediators

21

In the mentioned works the pharmacological effects of some potential therapeutic agents were

examined. Similarly, it was tried to estimate some accompanying effects of nicotinic and

muscarinic agents that are extensively used in anaesthesia. DHEA was found to relax rat and

human bronchial muscle and is considered as potential bronchodilator (48, 60, 65) although its

mechanism of action remains to be elucidated. In addition, in a classical rat tracheal ring

preparation, the skeletal muscle relaxant agents were found to relax smooth muscle to a various

degrees, a muscarinic receptor effects which was epithelium independent (51, 53, 76, 97).

Knowledge of their potential smooth muscle relaxing action and possible selective action on

muscarinic receptors subtypes is valuable for their proper use as muscle relaxing agents and for

the design of new muscle relaxing agents.

22

Effects on pilocaropine induced tone

-50

0

50

100

45678910

SMR concentration, -log M

%Tm

ax (

pilo

carp

ine 5

x1

0-6

M) mivacurium

cisatracurium

pancuronium

succinylcholine

rocuronium

Conclusion. We demonstrated that, in one for that purpose conceived rat tracheal model in

vitro, airway epithelium modulates smooth muscle tone and probably number of factors

contribute to that epithelial function. Bronchial epithelium has, nevertheless, an important

function as a diffusion barrier also. Described model is valuable for examining the effects of

bronchial epithelium on bronchial smooth muscle contraction. Number of other laboratories

continue to use our model what proves again its importance.

b. Diaphragm Contractility and Lung mechanics.

Diaphragm is a skeletal muscle that provides for ventilation of the lungs. It contracts

permanently during the whole life span similarly to the heart. Whether the function has

changed its structure and has influenced mechanisms of contraction is not clear. Customary the

research that has been done on diaphragm compared it to the other skeletal muscles or, less

often, to the heart that is of course very much different type of muscle. The other difference is

in the fact that its complete paralysis in otherwise healthy humans does not compromises life,

23

since it is responsible for only about 75% of the air that is pumped in the lungs, the rest being

done by the action of the intercostal muscles. And quite on the contrary, if the ventilation and

gas exchange are already seriously compromised by a disease (COPD for example) slight

diaphragm weakness may be incompatible with life. Its weakness and fatigue often accompany

other lung disease and knowledge of its physiology and pathophysiology are essential for an

effective therapy that may be, in these circumstances, indispensable.

When a reliable model of the mechanism of muscle contraction was described in the early

fifties muscle research started to flourish. Diaphragm research followed and was particularly

intensive in 70s and 80s. This was stimulated by the finding that respiratory muscle

insufficiency often followed chronic pulmonary diseases (COPD and asthma, which were

found to be linked to extremely high mortality. Great interest in sport performance stimulated

muscle research also and a conviction that muscle work can be influenced pharmacologically

led to new research efforts and naturally, a lot of disappointment. Number of agents were

described that could affect negatively respiratory muscle contractility. However a wonder agent

that would help compromised muscle to recover from fatigue or to increase its capacity to

contract has not been found. This was another difference from the heart for which relatively

efficient therapy was long available.

We examined in vitro end in vivo its functioning in various animal models, its contractility and

the effect of various pathohysiological circumstances, including drug effects (2, 3, 4, 12, 28,

29). Opioid analgesics block action potential production in skeletal muscles by a non-specific

and opiate drug receptor mediated blockade. To investigate whether those drugs also affect

skeletal muscle fibre tension generation we obtained costal diaphragmatic fibres from rats.

Isometric force generated in response to supra maximal electrical stimulation (8 times/min,

24

duration of stimulation 0.3ms) was registered with a highly sensitive photoelectric transducer

(7).

Train of repeated electrical stimulation produce stabile twitch tension that is rapidly depressed

by pentasozine.

After a period of control measurements, preparation was perfused with Krebs solution which

contained pentazocine, while the fibres were continuously stimulated and twitches recorded.

Incremental concentrations of pentazocine were tried and cumulative dose-response curve was

constructed. In separate experiments preparations were perfused with different concentrations

of pentazocine, morphine, phenoperidine and naloxone. We found that opiods have long lasting

depressant effect on tension generation of rat isolated diaphragmatic fibres which is probably

result of their direct inhibitory action on contractile mechanisms of muscle cells. Similarly,

corticosteroids are the agents frequently used in patients that potentially may develop

respiratory failure (COPD or asthma patients). The study was undertaken to determine the

effects of 8 days of corticosteroid administration on diaphragmatic atrophy and contractile

properties (15). In experimental protocols similar to those done in vitro setting, in vivo

experiment where performed where phrenic nerve was directly stimulated and

transdiaphragmatic pressure measured (Pdi). In both preparations, twitch and low-frequency

Pentazocine 10-5M

100mg/10min

Pavlovic et al., Jugoslav. Physiol. Pharmacol. Acta,1988.

25

force were unaffected, whereas normalised tetanic force, in the starvation (ST) group, was

markedly reduced as compared with that in the pair feed (PF) group (p less than 0.001). Soleus

and EDL muscles were also studied in vitro.

Set up for measuring transdiaphragmatic pressure in anaesthetised rat.

Although steroid treatment had no effect on the soleus, in the EDL, a slight (11%) decrease in

normalised tetanic tension was observed. A similarity of heart and diaphragm was observed in

respect to Ca++ importance. Calcium ions seam to play important role for both muscle,

although the hearth muscle is highly sensitive to the calcium extracellular concentration while

diaphragm seemed to be less sensitive, still more sensitive than skeletal muscle. To further

investigate these mechanisms, the dose-response effects of BAY K 8644 and nifedipine on

diaphragmatic contractility were assessed in vitro (16). We found that both BAY K 8644 and

nifedipine altered twitch characteristics. In low-calcium media (0.5 mM) twitch potentiation

produced by the two drugs was further enhanced (increasing 60% for 10(-5) M BAY K 8644 or

26

nifedipine). This further confirmed that diaphragm could be, in respect of Ca++-transport,

different from skeletal muscles.

Typical twitch tension after BAY K8644, nifedipine, and both. (from Ref. 3)

Undernutrition and particularly sepsis are often linked to compromised respiration. The effects

of a 3-day pneumococcal infection on diaphragmatic strength and endurance capacity were

studied in in vivo rat model (8, 9, 13). We conclude that a 4-d total fast produces a reduction in

diaphragmatic weight, which is associated with a decreased diaphragmatic strength and reduced

endurance capacity. If combined with sepsis (endotoxine model) or with hypoxia we

demonstrated that the deleterious effects were even more pronounced. These findings may be

clinically relevant since septic condition is frequently linked to both conditions. The diaphragm

fatigue in these patients may be further complicated by disuse atrophy.

Whether mechanical ventilation is the cause of muscle fatigue that results in prolonged

weaning was not conclusively demonstrated (10). We measured in rats the effects of 48 h of

mechanical ventilation on the weight, contractile properties, and enzymatic profile of the

diaphragm, the soleus and the extensor digitorium longus (EDL) muscles (27). The obtained

results made us propose, in another work (66), that based also on experience with the

27

rehabilitation of skeletal muscles during long-term immobilisation of the extremities following

trauma or surgery and on experience with diaphragm pacing in quadriplegics, intermittent

electrical or magnetic stimulation of the diaphragm while the lungs are still being mechanically

ventilated may be beneficial. The experiments have been planed to be done in the near future.

A combination of magnetic phrenic stimulation and biofeedback or each of these techniques

alone, may be, at least theoretically beneficial. (90).

Certainly disuse atrophy may be combined with denervation and rehabilitation of such muscle

is particular chalendge. In a preliminary study in dogs (10) we were able to demonstrate that

denervated diaphragm rapidly becomes resistant to the electrical stimulation and even

placement of multiple electrodes requires very high currents to produce almost undetectable

muscle contractions. Therefore we proposed , in another work, that restoration of functional

capacity of denervated diaphragm may be achieved by reinervating it with vagus nerve (67).

This hypothesis remains to be proved.

High pressure and high volume mechanical ventilation may be deleterious and various modes

of ventilation have been tested to diminish risks of ventilator induced lung injury (VILI). Our

recent works were related to the multiple channel jet ventilation tube (McETV). That tube

ensured adequate gas exchanges with lower insufflation pressures and could diminish positive

pressure ventilation-induced injury. The described endotracheal tube, although it was found to

be quite suitable, has not yet its place in every-day ventilation of the patients that are at risk

from high-pressure ventilation. It proved to be difficult to use in adults, because of high

backward flow through larger endotracheal tubes and loss of tidal volume (17-23, 34, 35, 37).

28

Bousignac tube (From ref. 13.)

Whether it will find it place out of emergency conditions, is not clear. However the idea of

constant minimal flow ventilation could have other important advantages, like during

percutaneous tracheaotomy which seem to be quite secure procedure (81, 88, 89). Our solution

for some improvement of the technique has yet to be tested (100). Emergency lung ventilation

would require, in some exceptional “can’t intubate, can’t ventilate” situations some more

reliable method to these available, at least temporarily. The main problem of available devices

for transtracheal emergency lung ventilation is possible air retention and build up of dangerous

intrinsic PEEP and subsequent overinflaton or hypoventilation. We conceived a venturi valve

that could be used as an air pump, providing active inflation and deflation of the lungs and

performed two studies that are submitted for publication (55, 56, 59). In both studies we

demonstrated that the same amount of gas could be insufflated and suctioned with our venturi

pump. If an adequate I:E ratio and appropriate flow are used, satisfactory emergency

ventilation of normal and pathological lungs could probably be managed with a 16G cannula

and a simple venturi pump supplied with pressurized oxygen.

29

Venturi pump

2. Vascular responses to inflammation and trauma

Vascular smooth muscle in vitro

Our interest in airway smooth muscle has been extended, following increased interests for the

pathophysiology of conditions linked to anesthesia and intensive care patients, to the smooth

muscle in general and in particular, arterial smooth muscle.

Effects of Akrinor in pig coronary arteries (62, 63, 64, 125)

Akrinor (AKR, original solution: ORIG ), a mixture of theodrenaline (TDR) and cafedrine

(CDR), is a sympatomimetic agent used to counter transitory hypotension. Although some

cases of vascular complications associated with AKR have been reported there is no

O2

To patient

30

experimental data about its direct effects on coronary arteries. The effects of AKR, TDR, CDR

and its most frequently used alternative ephedrine (EDR) on the isometric contraction of the

ring preparations of pig coronary arteries in vitro which were pre-contracted with 20x10-3

mol/L KCl were studied. Influence of the endothelium removal, or preincubation with

nonselective β-adrenoreceptor antagonist propranolol (PROP), α1-adrenoreceptor antagonist

prazosin (PRAZ), dopamine receptor antagonist SCH-23390, or adenosine receptor antagonist

CGS-15943 were also tested. AKR, TDR and CDR produced relaxation of the preparations

(EC50: 2.96, 0.34, ORIG; 4.09, 0.36; and 3.79, 0.34; -log, mol; mean, SEM) while preparations

without endothelium were more sensitive to the AKR relaxing effects (4.58, 0.34, ORIG). EDR

alone produce increase in the tension of the pig coronary artery (143.3, 49.7; % over basic

tension; mean, SEM). In the preparations pretreated with PROP, the lower concentrations of

AKR, TDR and EDR produced contraction (163.6, 9; 180.12, 14.1: 4, 1; %, mean; SEM) while

higher concentrations of the AKR only produced full relaxation (EC50: 2.16, 0.11. ORIG;

mean, SEM). The AKR induced contractions could be prevented by the pretreatment with

PRAZ. Adenosine and dopamine receptor antagonists did not influence relaxing effects of

AKR. AKR and its constituents induce relaxation of pig coronary artery preparations pre-

contracted with KCl. In the preparations pre-treated with PROP, lower concentrations of AKR,

TDR or EDR produced contraction while higher concentrations of AKR and TDR, but not of

EDR, induced relaxation of the coronary artery. The observed contractions were probably due

to stimulation of unmasked α1-adrenoreceptors.

Dopexamine has been proposed as a selective peripheral vasodilator in the treatment of sepsis

and septic shock. As a catecholamine it may have large spectrum of effects, including the

effects on coronary arteries, which has not been examined.

31

Dopexamine elicited concentration dependent contractions in the KCl pre-contracted

preparations (given as EC50, means ±SEM) (4.38 ±0.23; n=7); whereas dopamine produced a

relaxation (6.61 ±0.26; n=6). Dopexamine induced contractions were endothelium (ENDO)

dependent (-ENDO vs. +ENDO: 3.78 ±0.38; n=5, vs. 4.38; ±0.23; n=7; P=0.0002). Pre-

incubation with adrenergic alpha-receptor antagonist prazosin (10-5M), beta-receptor antagonist

propranolol (10-6M), or dopamine DA1 receptor antagonist SCH23390 (10-7M) displaced dose-

response-curves of dopexamine to the right, as compared to the controls. (respectively: 4.07

±0.40, n=7; 4.10 ±0.27, n=7; and 3.95±0.19, n=8; all P<0.001). Pre-incubation with verapamil

only partially inhibited dopexamine elicited contractions (verapamil vs. controls: 2.54 ±1.68;

vs. 4.38 ±0.23; n=7, P <0.001) while in Ca2+ free solution (+ EDTA, 5x10-4M) dopexamine

elicited contractions of coronary arteries that were weakly pre-contracted with KCl (control vs.

Ca2+ free solution: 4.30 ±0.36 vs. 3.96 ±0.30; n=7, P<0.001). The incubation with the cGMP-

inhibitor ODQ (10-4M) or phosphatase inhibitor cantharidin (10-6M) both partially inhibited

contractions caused by dopexamine (respectively: 3.87 ±0.18; n=6; P=0.004; and 3.65 ±0.28;

n=6; P=0.028). It was found that dopexamine contracted coronary artery of the pig, probably as

a result of adrenergic and dopaminergic (DA1) receptors stimulation, but also by some

unidentified extra cellular Ca2+ independent mechanism.

Hypotension is an often seen side effect of antidepressive treatment. It still remains

controversial whether this is a central or peripheral effect. We tested some antidepressant

agents for their vasoactive properties in vitro. (84, 102) In isometric ring preparations of the rat

thoracic aorta, with and without endothelium (+ and – ENDO) the relaxing effects of

amitriptyline (AMI), fluoxetine (FLUO), tranylcypromin (TRAN) and venlafaxine (VENLA)

were tested by examining the effects of cumulative concentrations of the agents (10nM-

100µM) in the preparations pre contracted with 20mM KCl. The effects of pre-treatment with

antidepressant agents (pre incubation for 20 min, with 500µM of each) on KCl (5 – 100mM)

32

contractions were also examined. In the KCl pre contracted aortal preparations AMI, FLUO

and VENLA produced complete relaxation. The preparations +End were more sensitive to

FLUO (EC50: 5.57 ±0.04 vs. 4.96 ±0.03; + vs. -End; - mean ± SEM; n=4; P<0.01) and VENLA.

The AMI relaxant effects were not ENDO dependent, while TRAN produced only small

relaxation in preparations –End. Incubation with antidepressants had no significant effect on

the sensitivity to KCl although FLUO showed tendency to increase while AMI and VENLA to

reduce maximum tension. We found that AMI, FLUO and VENLA had direct relaxing effects

on vascular smooth muscle tone. The relaxing effects of FLUO and VENLA were partially

endothelium-dependent. These results indicate that hypotension due to antidepressant treatment

may partially be caused by peripheral, vascular effects of the drugs.

Vanadate

Endotoxemia is characterised by vascular hyposensitivity (HS) that is partially caused by the

excess of nitric oxide (NO) production. We induced vascular HS in rat aorta with endotoxin

(lipopolysaccharide escherichie coli, LPS 10-3g/l, 6h incubation) in vitro and tried to reverse it

by the preincubation (10min) with K+ channel blockers glibenclamide, 4-amynopyrridine,

charibdotoxin or tetraethylammoniumchloride (TEA), or with Ca++ transport blockers sodium-

orthovanadate (OV, PMCa++ATP-ase and tyrosine phosphatase inhibitor), sodium azide

(mitochondrial Ca++ transport inhibitor) or thapsigargin (sarcoplasmatic Ca++ ATP-ase

inhibitor). Aortal rings with or without endothelium (±ENDO) were incubated in MEM

medium (INC; 37°C), in presence or absence of LPS (12 groups, n=7 each) and isometric

contractions elicited by phenylephrine (PE), were examined. LPS INC and INC alone

diminished sensitivity to PE vs. controls (CONT) (EC50: 6.13 ±0.07; 6.64 ±0.12; 7.1 ±0.06;

respectively). Maximal tension (Tmax) was also depressed by LPS incubation (+ENDO LPS

INC: 0.65 ±0.22 vs. CONT +ENDO: 1.31 ±0.2; g). These effects were partially endothelium

dependent. Preincubation only with TEA (4x10-4 M) reversed aortal HS (LPS -ENDO TEA vs.

33

LPS -ENDO; EC50: 6.96 ±0.26 vs. 5.93 ±0.19) and increased Tmax (Tmax: 1.05 ±0.15).

Incubation with OV 10-7 and 10-6M increased Tmax as compared to INC alone. (Tmax: 0.85

±0.23; 0.78 ±0.1; 0.44 ±0.12, respectively). We found that LPS induced in vitro aortal HS may

be caused by an overproduction of NO and activation of TEA sensitive potassium channels.

This effect could be reversed by an inhibition of calcium removal and/or inhibition of tyrosine

phosphatase (72, 80).

Microcirculation

Gut mucosal hypoperfusion plays a major role in the pathogenesis of ongoing sepsis and

multiple organ dysfunction syndrome (MODS). Traditionally, therapy included increasing

systemic flow, thus secondarily augmenting blood flow to the gut. Direct manipulation of the

gut mucosal microcirculation avoiding systemic effects, i.e., selective gut microcirculatory

control, has not been consistently tested.. The pathogenesis of the syndrome might be multi-

causal (69, 85, 87). An impaired intestinal barrier function is associated with a decreased

separation of intraluminal bacteria and toxins and systemic circulation, which may induce

sepsis and multiple organ failure, i.e. systemic inflammatory response syndrome (SIRS).

Hypoperfusion during shock, reperfusion injury of the splanchnic mucosa, immunologic and

hormonal disturbances are important underlying pathophysiological mechanisms. Alternative

therapeutic concepts have been proposed such as nutritive and metabolic treatment by means of

immunomodulating nutrients, parenteral substitution of glutamine, early onset of enteral

nutrition, normalisation of gut motility and selective decontamination of the gut. However the

controversy persist. An aggressive treatment of shock and avoidance of microcirculatory

disturbances are of principal importance for prophylaxis of multiple organ failure. (1)

34

Model/Method

Intravital microscopy (IVM) imposes the particular problem of the combined control of the

body temperature of the animal and control of the local temperature of the observed organ or

tissues. We constructed and tested, in the preparation of the rat ileum microcirculation, a new

tissue support platform (93, 127). The platform consisted of the organ bath filled with

physiological solution, and contained the suction tube, superfusion tube, an intestine support

hand which was attached to a micromanipulator, and the thermometer probe. To cover the

intestine we used a cover glass plate with the plastic ring which was glued on its upper surface.

After a routine procedure (anaesthesia, monitoring and surgery) the intestine segment (2-3cm

long) was gently exteriorised and placed on the “hand” of the tissue support. A small part of the

intestine formed a little “island” in the bath that was filled with physiological solution. The

cover glass was secured in place. The physiological solution from the superfusion tube which

was pointed to the lower surface of the cover glass formed a “hanging drop”. Then, the

objective of the microscope was immersed into distilled water that was formed by the cover

glass plastic ring. The “hanging drop” technique prevented any tissue quenching, assured

undisturbed microcirculation, provided for stable temperature and humidity, and permitted a

clear visual field. The model has been approved in the recent experimental studes (128, 129).

35

Treatment of peritonitis may include abdominal lavage with local disinfectants. We examined

an disinfectant agent polyhexanide (POLY), available as 0.04% solution (“Lavasept 0.2%®”,

POLY-LS), for eventual use in abdominal lavage (54, 74, 75, 78, 103). We examined, in

healthy rats, the effects of peritoneal installation of POLY-LS or NaCl 0.9% (10ml each, during

10min, n=5) on mean arterial pressure (MAP) and measured, in 7 other animals, using intravital

microscopy (IVM), diameter of terminal ileum submucosal blood vessels before and after local

superfusion with POLY-LS. Furthermore, in an in vitro isometric preparation of rat thoracic

aortal rings, with and without endothelium (+ and – End), we tested POLY-LS and POLY for

their vasoactive properties. It was found that POLY-LS peritoneal instillation produced

decrease of MAP while superfusion with POLY-LS caused local vasodilatation of intestinal

wall blood vessels. In vitro, POLY-LS and/or POLY produced endothelium dependent

relaxation in the preparations precontracted with phenylephrine (PE) (EC50= POLY-LS: 2.53 ±

0.16, vs. 1.36 ± 0.16, n=4, P<0.05; POLY: 4.02 ± 0.12 vs. 3.21 ± 0.10, n=12, P<0.001; + vs. –

36

End, respectively; -log g%;) which (in aortas +End) could be attenuated by either N(G)-nitro-L-

arginine methyl ester (L-NAME) - nitric oxide (NO) generation inhibitor, or 1H-

(1,2,4)Oxodiazolo-(4,3-a)quinoxalin-1-one (ODQ) - an inhibitor of guanylyl cyclase. The

relaxing effect of POLY (aortas –End) were not affected by K+-channel blocking agents

charibdotoxin, tetraethylammoniumchloride, glibenclamide, or 4-aminopyrridine, while POLY

had no effects on 40mM KCl contractions. This implies that polyhexanide may promote nitric

oxide liberation, potassium channel activation and vasodilatation that may result in

hypotension.

37

Taurolidine

We further examined the effects of peritoneal instillation (PInst) of taurolidine (Taurolin® 2%,

TL) or polyhexanide (chlorhexidine polimer Lavasept® 0.2%; poly-L) on intestinal

microcirculation using intravital microscopy in experimental endotoxemia (15 mg/kg

lipopolysaccharide - LPS, i.v.) in the rat (n=40) ((43, 79, 129).

38

One hour after LPS injection peritoneal instillation (5 mL solution, PInst) was performed and

30 min after, the microcirculation of terminal ileum was examined. LPS produced hypotension

(98.8 ±9.5; mmHg vs. 130.4 ±10.5 mmHg; MAP, mean ±SD) which was further pronounced

after PInst with TL (78.8 ±10.8) [p<0.005] or with poly-L (78.1 ±6.0) [p<0.001]. The number

of temporary adhering leukocytes in the V1 and V3 venules in LPS rats was reduced after PInst

with TL, poly-L or NaCl (controls) while the number of firmly adhering leukocytes in V1 and

V3 venules in LPS rats was increased. In the V3 venules of the TL group the number of firmly

adhering leukocytes did not significantly increase as much as in the poly-L group. The

functional capillary density (FCD, cm/cm2) in the mucosa of the LPS rats diminished after

PInst with Poly-L (259.7 ±54.1) as compared to NaCl (337.1 ±35.5). The FCD in the

longitudinal muscular layer in LPS rats diminished after PInst with TL (119.8 ±14.8), as

compared to the Poly-L (148.9 ±18.8), NaCl (159.8 ±13.2), or to the NaCl (no-LPS) (147.7

±23.4). In vitro, both agents had vasodilating properties, which were partially endothelium

dependent. We found that, in experimental endotoxemia, contrary to the PInst with NaCl, PInst

with poly-L or TL could produce hypotension and intestinal microcirculatory disturbances and

that TL may prevent some inflammatory responses.

Some of these effects could have been direct results of vasodilating properties of these agents.

Indeed, volume replacement is of paramount importance not only in sepsis, but also in typical

emergencies. Therefore we designed an intravenous cannula that has a property to change its

diameter with temperature change (94). The model has yet to be examined in vivo.

39

3. Other related aspects

a. Pain and Alternative Methods in Medicine

Reducing venipuncture pain using “cough-trick”: a volunteer study

Existing methods to reduce venipuncture (VP) pain are not free from drawbacks like additional

costs, efforts of the staff and pain on application of local anaesthetics. In Traditional Chinese

Medicine insertion of the acupuncture needles simultaneously with coughing on command

(cough-trick, CT) leads to almost painless procedure. To investigate whether CT can reduce

pain on peripheral VP and influence heart rate, blood pressure and blood sugar we performed a

one-centre single blind study in which each participant served as his own control (56, 61, 86). It

was found that the pain intensity according to VAS on venipuncture with CT procedure was

lower, than without CT (median: 31 vs 46; p<0.05). Pain intensity on catheter insertion was

also lower during procedure with CT (median: 14 vs 21; p<0.05). Heart rate and blood pressure

decreased through the course of the study insignificantly and as the other outcome parameters

revealed no correlation with pain intensity changes.

Conclusion Cough-trick revealed to be effective in VP pain reduction, although the mechanism

remains unclear. Nevertheless it might be a simple and cheap method to reduce pain on VP in

clinical practice.

0

20

40

60

80

100

1 2

With CT Without CT

VA

S-1

00

40

Detection of Ear Acupuncture Points

The aim of the next study (68) was the detection of ear acupuncture points (EAP) by measuring

the electrical skin resistance under general anesthesia. EAP with lower skin resistance were

examined in 25 patients scheduled for elective orthopedic surgery on the day before, during the

operative procedure and a few hours after it. EAP, detected in more than 25% of patients were

further analyzed using a logistic regression model and compared to those in 15 healthy

volunteers. The following EAP were identified in more than 50% of patients: Clavicle, Lung,

Shenmen and points corresponding to the site of surgery. Point Clavicle was found in 16

patients (64%) throughout the study period. Shenmen was detected in 15 patients (60%) before

surgery, whereas during and after surgery it was represented in 5 (20%) of them. EAP

corresponding to the site of surgery were detected in 20 patients (80%) during the operative

procedure. These EAP were detected more frequently in patients in comparison with healthy

volunteers. The side of examination showed no significant differences throughout the study

The frequently found patterns of EAP with lower skin resistance in patients during orthopedic

surgery can be useful for treatment of preoperative anxiety and postoperative pain relief.

11

11

4

11

11

2 1

1

1

41

Auricular acupuncture for pain relief

Auricular acupuncture (AA) is known to be effective in treatment of various pain conditions,

but still there are no randomized controlled studies of AA for treatment of acute postoperative

pain. Therefore we tested whether AA of specific points is superior to sham acupuncture for

complementary analgesia after total hip arthroplasty in a patient-anesthesiologist-evaluator-

analyst blinded study (95). Fifty-four patients (29 AA and 25 controls) completed the study.

Piritramide requirement during 36 h after surgery in AA group was lower than in control: 37 ±

18 vs. 54 ± 21 mg; mean ± SD; P=0.004. Pain intensity on VAS-100 and incidence of

analgesia-related side effects were similar in both groups. The differences of patients’ opinion

concerning credibility of blinding between the groups were not significant. Findings from our

study suggest that auricular acupuncture shows promise for the postoperative pain relief.

b. Medical Ethics

Ethics of Concerns and Life Cessation Decisions: When Emotions are All What Remains

(106, 120)

Background. It is generally claimed that, in some exceptional circumstances, the taking of

human life may be justified on moral grounds. Rare but possible or largely theoretical life

situations are often presented to describe either a dilemma within which all outcomes involve

loss of human life, or a dilemma which offers at least two alternatives, whereby to act in a way

to not maintain human life could be morally preferable. In practical situations, in some rare

occasions when such inevitable decisions (to terminate or not human life) have to be taken -

when no alternative exists, the decision could not be left to be accidental but would have to be

based on same reasons. We argue that the taking of human life could not be morally justified,

42

and we propose here a solution based on emotional motives. Aim of the essay is, therefore, to

examine moral reasons for or against maintenance of human life in medically uncontroversial

circumstances in the patients whose mental life is estimated to be absent.

Ethics of concerns. The problem of the justification of the taking of human life is not only a

moral but, above all, an ontological (existential) question which, consequently, could not be

morally justified and requires other conceptual frame of reference. In medical practice, the

currently accepted criteria for the termination of life support are morally acceptable reasons

including patients’ desire, together with medical reasons, like absence of mental life, for

example the case of Terri Schiavo, March 2005), as well as the certainty that sensible life could

not be prolonged. A decision which should be taken has to accommodate to all concerns that

are involved. However, our concerns are closely linked to our emotions and may relate not only

to persons but also to physical objects. If our intentional life were understood to be based on

our concerns and if our acting would be understood to be predominantly, also, biased by our

concerns, the emotional attitude of the actor would have to be largely influencing almost all

hers/his decisions. These would include decisions that not necessarily have to have a direct link

with emotions, like various rational decisions, and would certainly include moral decisions.

Applying the morality of concerns. The reasons for the maintainance or cessation of the life

support may be based on direct or indirect patient’s interests and/or Interests of others. If there

are no patient’s interests, when rational choice is missing, and when moral arguments fail,

decisions could be based on reasons based on the emotional concerns, which are only morally

justifiable reasons that remain, while reasons stemming from material motives could not be

accepted.

Conclusion. The emotional approach may be of particular value in the above described

circumstances, and in critically ill patients where a dilemma exists as to whether to maintain

life support or not, when all other approaches would fail, and should be estimated as necessary

and sufficient for decision making. Those persons that invested the most of emotions (the

43

closest relatives of the patient) would be the persons whose opinion should significantly

influence a decision about life cessation in patients that otherwise would not qualify for life

support. Then, intensive communication with the persons emotionally concerned would be an

appropriate method that may lead to the decision which could satisfy moral and ontological

criteria.

For an indeterminist ethics. The emptiness of the rule in dubio pro vita and life cessation

decisions (77) It is generally claimed that there exist exceptional circumstances when taking human life may be

approved and when such actions may be justified on moral grounds. Precise guidelines in the medical

field for making such decisions concerning patients who are terminally ill or have irreparable injuries

incompatible with a bearable life, are difficult to establish. Recommendations that take the particular

logical form of a rule, such as “in dubio pro vita”, have been suggested and in some countries

incorporated into legal texts. We claim here that such a rule is of no value since it is open-ended and

always allows for doubt, and a decision to employ measures that would support human life could always

be argued to be a valid choice. Preservation of this rule could be encouraged, but giving it the force of

law may put physicians at risk, as they may be challenged for choosing to terminate life in otherwise

ethically and medically uncontroversial circumstances.

* * *

Keywords: Smooth muscle models; epithelium, endothelium; contraction, relaxation,

hyperreactivity, diaphragm contractility, weaning, jet ventilation, microcirculation, alternative

medicine, acupuncture, medical ethics

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References (Given as a chronological list of publications, year and full reference, with impact factor 2003 or Richtlinien - Leistungsorientierte Vergabe von investiven Mitteln, Greifswald 2002.) ____________________________________________________________________________ 1985 Articles 1. Pavlovic, Dragan Anaesthesia and analgesia in sports injuries. Thesis, The London Hospital Medical School, London, 1985. (0.4) 1986 Abstracts 2. Dureuil, B. N. Viires, D. Pavlovic, R. Pariente, and M. Aubier. Alteration in nerve conduction and diaphragmatic function after phrenic nerve hypothermia. Proceedings, VII Congres Europeen DíAnesthesiology, Vienne, 1986. 3. Dureuil, B. , N. Viires, D. Pavlovic, R. Pariente, M. Aubier, and J. M. Desmonts. Effects of phrenic nerve cooling on nerve conduction and diaphragmatic function in dogs. Poceedings, International Symposium on Anaesthesia for Cardiac Patients, Munchen, 1986. 1987 Abstracts 4. Dureuil, B. , N. Viires, B. Weber, D. Pavlovic, M. Aubier, R. Pariente, and J. M. Desmonts. Effects of acute starvation on diaphragmatic function. Am. Rev. Resp. Dis. 4: A329 (suppl), 1987. 1988 Abstracts 5. Pavlovic, D. , M. Fournier, M. Aubier, and R. Pariente. Modulation of the tracheal smooth muscle response to carbachol stimulation by the epithelium in the rats. Am. Rev. Resp. Dis. 137 (suppl): 243, 1988. 6. Jorga, V. I. , I. K. Jorga, D. V. Pavlovic, Olivera Bosnic, Snezana Vukovic, M. Aubier, and V. M. Mujovic. Prostaglandins in top karate sportsmen during specific work load test. Jugoslav. Physiol. Pharmacol. Acta. 24 (Suppl 6): 161), 1988.

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Articles 7. D. Pavlovic, Jorga, I. K., V. Mujovic, M. Aubier and R. Pariente. Opioids have depressant effect on diaphragmatic fiber tension generation. Jugoslav. Physiol. Pharmacol. Acta. 24 (Suppl 6): 159-160) 1988. (0.2) 8. Boczkowski, J. , B. Duireil, N. Viires, D. Pavlovic, D. Murciano, R. Pariente, and M. Aubier. Effects of sepsis on diaphragmatic function in rats. Am. Rev. Resp. Dis. 138: 260-265, 1988. (5.443) 1989 Abstracts 9. Boczkowski, J. , B. Dureuil, D. Pavlovic, R. Pariente, M. Aubier. Preventive effect of indomethacin on contractile diaphragmatic alteration in septic rats. Am. Rev. Resp. Dis. 4: A163 (suppl), 1989. 10. Nussaume, O. , Murciano, G. , Viires, N., and Pavlovic, D. Stimulation musculaire - travail preliminaire, Proceedings, Socite de Chirurgie Thoracique et Cardio-Vasculare de Langue Franáaise, Versailles, 1989. Articles 11. Pavlovic, D. , M. Fournier, M. Aubier, and R. Pariente. Epithelial versus serosal stimulation of tracheal muscle: role of epithelium. J. Appl. Physiol. 67: 2522-2526, 1989. (2.297) 12. Dureuil, B. , N. Viires, B. Veber, D. Pavlovic, R. Pariente, J. M. Desmonts, and M. Aubier. Acute diaphragmatic changes induced by starvation in rats. Am. J. Clin. Nutr. 49: 738-744, 1989. (5.012) 1990 Abstracts 13. Pingleton, S. K. , J. Boczkowski, N. Viires, D. Pavlovic, R. Pariente, and M. Aubier. Effect of combined starvation and sepsis upon diaphragmatic function. Am. Rev. Resp. Dis. 1990 (suppl). 14. Pavlovic Dragan, Evelin Brione, Michel Fournier, et Michel Aubier. L’epithelium inhibe la relaxation du muscle lisse tracheal de rat induit par un activateur des canaux potassiques (BRL 38227). Proceedings of Journee de la Recherche (UFR Xavier Bichat), Paris, Sept. 1990.

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Articles 15. Viires, N. , D. Pavlovic, R. Pariente, and M. Aubier. Effects of steroids on diaphragmatic function in rats. Am. Rev. Resp. Dis. 124: 34-38, 1990. (5.443) 1991 Articles 16. Viires, N. , D. Pavlovic, and M. Aubier. Multiple effects of BAY K 8644 and nifedipine on isolated diaphragmatic fibres in vitro. J. Appl. Physiol. 71: 841-846, 1991. (2.297) 1992 Abstracts 17. Pinquier, D. , G. Boussignac, D. Pavlovic, and F. Beaufils. Sonde d'intubation multi-canaux: Une voie pour limiter le barotraumatisme pendant la ventilation mÈcanique. Proceedings of Journee de la Recherche (UFR Xavier-Bichat), Paris: 105, 1992. 18. Pinquier, D. , G. Boussignac, D. Pavlovic, and F. Beaufils. Sonde d'intubation multi-canaux: Interet dans le sevrage de la ventilation mecanique. Proceedungs of Journe de la Recherche (UFR Xavier-Bichat, Paris): 106, 1992. 19. Pinquier, Didier, Georges Boussignac, Dragan Pavlovic, Michel Aubier, and F. Beaufils. Multichannel endotracheal tube: a way to limit barotrauma. Intensive Care Med. 18 (S2), 164, (A 186), 1992. 20. Pinquier, Didier, Georges Boussignac, Dragan Pavlovic, Michel Aubier, and F. Beaufils. Multichannel endotracheal tube: a way to improve weaning from mechanical ventilation. Intensive Care Med. 18 (S2), 164 (A 185), 1992. 1993 Abstracts 21. Pinquier, Didier, Georges Boussignac, Dragan Pavlovic, Michel Aubier, and F. Beaufils. Ventilation intratracheale multichanalaire. Rev. mal. Resp. , 10 (S1), 18 (A 32), 1993 22. Gacic, Z. , M. Grgurevic, D. Pavlovic, and V. Nedeljkov. The effect of penicillin on retzius nerve cells of the leech. Proceedings of III Congress of Yugoslav Neurologists, 210, 1993. 23. Grgurevic, M. , S. Malobabic, Z. Gacic, D. Pavlovic, and V. Nedeljkov.

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The dendroarchitectural organization of the neurons in the substantia gelatinosa of the spinal cord. Proceedings of III Congress of Yugoslav Neurologists, 405, 1993. Articles 24. Pavlovic, Dragan. , Evelin Brione, Michel Fournier, and Michel Aubier: Epithelium partialy inhibits tracheal smooth muscle relaxation induced by potassium channel activator BRL 38227. Br. J. Pharmacol. 110: 139-144, 1993. (3.689) # 1994 Abstracts 25. Pavlovic, Dragan. , Naima Viires, Christine Zedda, Michel Fournier, and Michel Aubier: Effects of corticosteroids on epithelial structure and smooth muscle function of rat trachea Am. J. Respir. Crit. Care Med. , 4: A905, 1994. 26. Pavlovic, D. , Cheik-Zeinedinne, T. , Fournier, M. , and Fournier, M. Carbocisteine diminishes rat tracheal smooth muscle reactivity in epithelium denuded preparations. Eur. Resp. J. Vol 7, suppl 18: 76s, 1994. Articles 27. Genevieve Le Bourdelles, Naima Viires, Jorge Boczkowski, Natalie Seta, Dragan Pavlovic, and Michel Aubier. Effects of mechanical ventilation on diaphragmatic contractile properties in rats. Am. J. Respir. Crit. Care Med. , Vol. 149: 1539-1544, 1994. (5.443) 1995 Abstracts 28. Viires, N. , Pavlovic, D. , Moutaouakkil, S. , and Aubier, M. Effects of chronic hypoxia on diaphragmatic contractility, capillarity and fibre type distribution Proceedings of XXIV European Muscle Conference, Firence, Italy, 13-16 Sept., 1995. 29. Viires, N. , Pavlovic, D. , Zedda, C. , and Aubier, M. Effects of chronic hypoxia on diaphragmatic contractility, capillarity and fibre type distribution Eur. Resp. J. Vol 7, suppl 18: 88s, 1995. 30. Pavlovic, D. , Moldovan, F. , and Aubier, M. Selective perfusion of rat tracheal preparation: no evidence for EpDRF secretion

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Eur. Resp. J. Vol 8, suppl 19: 43s, 1995 1996 Abstracts 31. D. Pavlovic, N. Viires, F. Roux, and M. Aubier Chronic hypoxia diminishes sensitivity of rat tracheal smooth muscle to carbachol applied epithelialy Am. J. Resp. Crit. Care. Med. , Vol. 153, N∞ 4 (suppl. , part 2): A840, 1996. 32. Ouksel, H. , D. Pavlovic, N. Viires, Y. Amrani, N. Seta, and M. Aubier Modifications in MLCK expression induced by repeated bronchoconstriction Proceedings, EMC, XXV European Muscle Congress, Montpellier, France, 14 - 17 Sept., 1996. 33. Nedeljkov, V. Cemerikic, D. , Grgurevic, M. , Dragovic, S. , and Pavlovic, D. The effect of Ni2+ on Retzius nerve cells of the leech. XV Yugoslav. Symp. of Epileptology, 1996. Articles 34. Didier Panquier, Dragan Pavlovic, Georges Boussignac, Michel Aubier, and FranÁois Beaufils Benefits of the low pressure multichannel endotracheal ventilation Am. J. Resp. Crit. Care. Med. 154: 82 - 90, 1996. (5.443) 1997 Abstracts 35. Didier Panquier, Dragan Pavlovic, G. Fromont - Hankard, L. Ferkdadji, G. Boussignac, M. Peuchmaur, M. Aubier, and F. Beaufils Effects of tracheal gas insuflation during CMV on pulmonary pressures and airway histology Am. J. Resp. Crit. Care. Med. vol. 155, N∞ 4, (suppl. , part 2): A773, 1997. 36. Ouksel, H. , N. Viires, D. Pavlovic, C. Peiffer, N. Seta, Y. Amrani, and M. Aubier Modifications in MLCK Expression in guinea-pig airway smooth muscle: Role of repeated bronchoconstrictions and role of inflamation Am. J. Resp. Crit. Care. Med. Vol 155, N∞ 4 (suppl, part 2): A370, 1997. 37. Didier Panquier, Dragan Pavlovic, G. Fromont - Hankard, L. Ferkdadji, G. Boussignac, M. Peuchmaur, M. Aubier, and F. Beaufils Effects of TGI during CMV on pulmonary pressures and airway histology Eur. Resp. J. vol 10, 80s (suppl), 1997. 39. Ouksel, H. , N. Viires, D. Pavlovic, C. Peiffer, C. Zedda, and M. Aubier MLCK and SERKA expression in tracheal smooth muscle from sensitized guinea-pigs Eur. Resp. J. vol 10, 291s (suppl), 1997.

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1998 Abstracts 39. Ouksel, H. , N. Viires, D. Pavlovic, C. Peiffer, C. Zedda, M. Pretolani, C. RuffiÈ, and M. Aubier Effects of Inflamation on myosin light chain kinase (MLCK) expression in guinea-oig model of bronchial hyperreactivity Am. J. Crit. Care Med. , Vol 157, 3 (Suppl): A519, 1998 40. Grgurevic-Nedeljkov, Marica, N. Puskas, L. Puskas, M. Malis, S. Malobabic, D. Pavlovic, and V. Nedeljkov The distribution of neuropeptide Y (NPY) in the rat spinal cord Folia Anatomica, vol 26 (Suppl. 2): 109, 1998. 41. Grgurevic-Nedeljkov, Marica, N. Puskas, L. Puskas, M. Malis, S. Malobabic, D. Pavlovic, and V. Nedeljkov Calcitonin gene-related peptide (CGRP) in the rat spinal cord: a light microscopic analysis Folia Anatomica, vol 26 (Suppl. 2): 110, 1998. 42. Grgurevic -Nedeljkov, M. , N. Puskas, M. Malis, S. Malobabic, D. Pavlovic, and V. Nedeljkov Immunohistochemical localization of substance P in cat spinal dorsal horn The Eleventh European Anatomical Congress, Timisoara, Procedings, p. 102, Sept 10-13, 1998. 43. Grgurevic -Nedeljkov, M. , N. Puskas, L. Puskas, M. Malis, S. Malobabic, D. Pavlovic, and V. Nedeljkov The cat spinal cord: neuropeptide Y (NPY) immunoreactivity The Eleventh European Anatomical Congress, Timisoara, Procedings, p. 103, Sept 10-13, 1998. Articles 44. Pavlovic, Dragan. , Naima Viires, Christine Zedda, Michel Fournier, and Michel Aubier: Effects of a high dose corticosteroids on smooth muscle function in rat trachea. Eur. Respir. J., 11: 575-582, 1998. (2.590) 1999 Articles 45. Grguvevic Marica, Malobabic Slobodan, Pavlovic Dragan and Nedeljkov Vladimir Large multipolar neurons in the substantia gelatinosa (Rolandi) of the cat spinal cord Folia Anatomica, 1: 51-55, 1999. (0.2)

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2000 Abstracts 46. H. Ouksel, N. Viires, D. Pavlovic, C. Ruffie, C. Zedda, C. Peiffer, C. Vizzuzaine, M. Pretolani, M. Aubier Allergic bronchial hyperreactivity in the guinea-pig is associated with an inflammation-dependent increase in the expression of MLCK in the tracheal smooth muscle Am. J. Crit. Care Med, 161: A840, 2000. 47. A. Samb, C. Lisdero, J. Callebert, D. Pavlovic, M. Pretolani, M. Aubier, J. Boczkowski Decreased lung expression and activity of type I nitric oxide synthase (nNOS) after ovalbumin immunisation and aerosol challenge in guinea pigs. Am. J. Crit. Care Med. 161: A919, 2000. 2001 Abstracts 48. D. Pavlovic, F. Zimmerman, T. Hachenberg, M. Wendt Dehydroepiandrosterone induced relaxation of rat trachea and aorta is not affected by potassium channel blocking agents Am. J. Resp. Crit. Care Med. , Vol. 163, No 5, A: 976, 2001. 49. Grgurevic, M. , Pavlovic, D. , Ristanovic, D. , Wendt, M. , and Nedeljkov, V. A Golgi study of the neurons of the lamina VI of the rat spinal cord Proceedings, 83eme Congres de l'Association des Morphologistes, and 12th European Anatomical Congress, p. LIII, LYON, FRANCE (23-27 MAY), 2001. 50. Pavlovic, D., Grgurevic, M. , Puskas, N. , Ristanovic, D. , Wendt, M. , and Nedeljkov, V. The morphology of Golgi-stained neurons in lamina IV of the rat spinal cord Proceedings, 83eme Congres de l'Association des Morphologistes, and 12th European Anatomical Congress, p. LII, LYON, FRANCE (23-27 MAY), 2001. 2002 Abstracts 51. Meissner, Konrad, Michael Wendt, and Dragan Pavlovic Steroid muscle relaxants inhibit carbachol induced tracheal smooth muscle tone in the rat Europ J Anaesthesiol, 19 (Suppl 24), 2002. 52. S. NJ. Lopicic, D. Cemerikic, D. Pavlovic, M. Wendt, M. Grgurevic, D. Ristanovic, V. Nedeljkov The excitatory effects of β-N-methylamino-L-aspartate on Retzius nerve cells of the leech Haemopis sanguisuga European Journal of Neurology 9 (Suppl 2): 139, 2002.

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53. Milchert, M, Meissner, K., Usichenko, T., Lehmann C., Wendt, M. und Pavlovic, D. Muskelrelaxanzien hemmen Kontraktion der glatten Tracheamuskulatur der Ratte NAT (Norddeutsche Anästhesie-Tage), Hamburg,.5-6. 10. 2002. 54. Helge Frieling, Mathias Gruendling, Christoph Strang, Thomas Hachenberg, Michael Wendt, and Pavlovic, Dragan Lavasept relaxing effects on rat aorta are partially nitric oxide mediated Intensiv Medizin und Notfallmedizin 39 (Suppl 1): 37, 2002. 55. D. Pavlovic, K. Meissner, H.-E. Wagner, U. Bartels, and M. Wendt Bi-directional venturi pump for emergency transtracheal lung ventilation Intensiv Medizin und Notfallmedizin 39 (Suppl 1): 49, 2002. 56. Usichenko T. I., S. Foelner, D. Pavlovic, M. Wendt Reducing venipuncture pain using ”cough-trick”: a volunteer study Der Schmertz, 16 8 (Suppl 1): 590, 2002. Articles 57. Pavlovic, Dragan and Wendt, Michael A device for manual venturi-jet ventilation (German Patent Office N° 202.13.420.2, August 29, 2002.) (0.8) 58. Dragan Pavlovic Modulation des Tonus der Atemwege durch das Epithel: Effekte von Carbocistein Medizinischen Fakultät Der Ernst – Moritz - Arndt Universität, Greifswald German Doctoral Thesis, 2002 (0.8) 2003 Abstracts 59. K. Meissner, T. I. Usichenko, Ch. Lehmann, M. Wendt and D. Pavlovic Bi-directional venturi pump for short term emergency transtracheal lung ventilation ASA 2003. 60. Dragan Pavlovic, Juliane Maskow, Taras Usichenko, and Michael Wendt Dehydroepiandrosterone induces relaxation of human and rat bronchial smooth muscle and rat aorta Eur Resp J, Vol. 22 Suppl- 45: 105s, 2003. 61. Usichenko TI, Pavlovic D, Foellner S, Wendt M. Reducing venipuncture pain by a “cough-trick”: a randomized cross-over study. Eur J Anaesth. 20(Suppl.30):180, 2003. 62. Foellner S, Usichenko TI, Lehmann Ch, Wendt M, Pavlovic D. Akrinor produces transitory contraction of pig coronary artery pre-treated with propranolol. Eur J Anaesth. 20(Suppl.30):134. 2003.

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63. Sebastian Foellner, Taras.I.Usichenko, Christian. Lehmann, Michael Wendt, and Dragan Pavlovic Akrinor induced relaxation of pig coronary artery is transformed into transitory contraction by propranolol HAI 2003. 64. Sebastian Foellner, Taras.I.Usichenko, Christian. Lehmann, Michael Wendt, and Dragan Pavlovic In the presence of propranolol akrinor induces transitory contraction of the pig coronary artery Proceedings of Pan-Hellenic Cardiology Congress, p 41, Rhodes, Greece, 2003. 65. Juliane Maskow, Matthias Gründling, Frank Feyerherd, Albrecht Stier, Michael Wendt, and Dragan Pavlovic Dehydroepiandrosterone induces relaxation of human and rat bronchial smooth muscle and rat aorta HAI 2003- Articles 66. Dragan Pavlovic and Michael Wendt Hypothesis. Diaphragm pacing during prolonged mechanical ventilation of the lungs could prevent from respiratory muscle fatigue Medical Hypotheses, 3: 398-403, 2003. (0.76) 67. Dragan Pavlovic and Michael Wendt Hypothesis that Vagal Reinervation of Diaphragm Could Sensitise it to Electrical Stimulation Medical Hypotheses, 3: 404-407, 2003. (0.76) 68. Usichenko TI, Lysenyuk VP, Groth MH, Pavlovic D. Detection of ear acupuncture points by measuring the electrical skin resistance in patients before, during and after orthopedic surgery performed under general anesthesia. Acupunct Electrother Res, 28 (3-4):167-73, 2003. (1.4) 69. Birnbaum J, Lehmann C, Stauss HM, Weber M, Georgiew A, Lorenz B, Pulletz S, Grundling M, Pavlovic D, Wendt M, Kox WJ. Sympathetic modulation of intestinal microvascular blood flow oscillations in experimental endotoxemia. Clin Hemorheol Microcirc, 28 (4):209-20, 2003. (0.623) 70. T. I. Usichenko and D. Pavlovic Suggesting the optimal control procedure for acupressure studies Anesth Analg, Oct; 97(4): 1196-7, 2003. (2.2 i.e. 0.44) 71. Usichenko TI, Pavlovic D, Groth M. The effect of auricular acupuncture on anaesthesia: a search for optimal design. Anaesthesia: 58 (9): 928-9, 2003. (2.3) i.e. 0.46

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2004 Abstracts 72. Henning Erber, Christian Lehmann, Matthias Gruendling, Thomas Hachenberg, Michael Wendt and Dragan Pavlovic Lipopolysaccharide induced hyporeactivity of rat aorta in vitro is reversed by potassium channels blocking agents and orthovanadate Paris WCA 2004 73. Kai-Steffen Lauer, Helge Frieling, Theoni Kanellopoulou, Dragan Pavlovic, Christian Lehmann, and Matthias Gründling Peritonal lavage with taurolidin and polyhexanid modulates intestinal microcirculation in experimental endotoxemia Paris WCA 2004 74. Helge Frieling, Matthias Grundling, Kai-Steffen Lauer, Christian Lehmann, Michael Wendt, and Dragan Pavlovic Polyhexanide relaxing effects in rat aorta are partially mediated by nitric oxide and cGMP Paris WCA 2004 75. Helge Frieling, Matthias Gruendling, Kai-Steffen Lauer, Michael Wendt, Christian Lehmann, and Dragan Pavlovic Lavasept® induced hypotension during peritoneal lavage is due to its vasodilating properties: in vivo and in vitro study in the rat Paris WCA 2004 76. Marcin Milchert, Konrad Meissner, Taras Usichenko, Matthias Grundling, Christian Lehmann, Michael Wendt, and Dragan Pavlovic Skeletal muscle relaxants inhibit tracheal smooth muscle tone in the rat (Paris WCA 2004) 77. Pavlovic D. (1), Wendt M. Moral and ontological grounds of the life cessation decisions and emptiness of the rule "in dubio pro vita" Paris WCA 2004, CD072. 78. Helge Frieling, Matthias Grundling, Kai-Steffen Lauer, Christian Lehmann, Michael Wendt, und Dragan Pavlovic Mechanismen der Lavasept® (Polyhexanid) - bedingten Vasodilatation während Peritoneallavage HAI, Berlin, 2004 79. Kai-Steffen Lauer, Helge Frieling, Theoni Kanellopoulou, Dragan Pavlovic, Christian Lehmann und Matthias Gründling Auswirkungen einer Peritoneal-Lavage mit Taurolidin bzw. Polyhexanid auf die intestinale Mikrozirkulation im septischen Rattenmodell HAI, Berlin, 2004 80. Henning Erber, Christian Lehmann, Matthias Gründling, Michael Wendt, Dragan Pavlovic

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In-vitro-Untersuchungen zur Beeinflussbarkeit der LPS-induzierten vaskulären Hyporeaktivität HAI. Berlin, 2004. 81. Kuhn, S., Friesecke, S., Abel, P., Feyerherd, F., Pavlovic, D., Gründling, M. Comparison of the percutwist and ciaglia blue rhino tracheostomy techniques: first results. ESCIM, Berlin 200 82. Lopicic, SNj., Nedeljkov, V., Pavlovic, D., Cemerikic, D., Ristanovic, D. Bicarbonate augments the excitatory effect of beta-N-methylamino-L-alanine on Retzius nerve cells of the leech (Haemopis sanguisuga) Proceedings of the 22nd International symposium on biophysics, St Stephan-Belgrade, Sebia-Montenegro. S3: 14, 2004. 83. Nedeljkov, V., Lopicic, S.Nj., Pavlovic, D., Cemerikic, D., Ristanovic, D. Effects of beta-N-methylamino-L-alanine on input membrane resistance of directly polarised membrane of the Retzius nerve cells of the leech (Haemopis sanguisuga) Proceedings of the 22nd International symposium on biophysics, St Stephan-Belgrade, Sebia-Montenegro, S3: 16, 2004. 84. Helge Frieling, Dragan Pavlovic, Johannes Kornhuber, Christian Lehmann Effekte verschiedener Antidepressiva auf den Gefäßtonus der thorakalen Aorta der Ratte, DGPPN KONGRESS, Berlin, 2004. Articles 85. Lehmann C, Birnbaum J, Luhrs C, Ruckbeil O, Spies C, Ziemer S, Grundling M, Pavlovic D, Usichenko T, Wendt M, Kox WJ. Effects of C1 esterase inhibitor administration on intestinal functional capillary density, leukocyte adherence and mesenteric plasma extravasation during experimental endotoxemia. Intensive Care Med. 30 (2):309-14. 2004. (2.314) 86. Taras I. Usichenko, Dragan Pavlovic, Sebastian Foellner, Michael Wendt Reducing venipuncture pain by A ”Cough-Trick”: a randomized cross-over, volunteer study Anaesth-Analg, 98:343-5, 2004 (2.2) 87. Lehmann C, Pavlovic D, Usichenko TI. Sphingomyelinase pathway activation in septic myocardial depression--another brick in the wall? Crit Care Med. 32 (2):609-10. 2004- (3.486) 88. Grundling M, Pavlovic D, Kuhn SO, Feyerherd F. Capnography vs. bronchoscopy for percutaneous tracheostomy placement. Anaesthesia. 59 (3):301-2. 2004. (2.3 i.e. 0.46) 89. Grundling M, Kuhn SO, Nees J, Westphal K, Pavlovic D, Wendt M, Feyerherd F. PercuTwist dilational tracheostomy: prospective evaluation of 54 consecutive patients

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Anaesthesist. 53 (5):434-40, 2004 (0.2) 90. Hannich, H. J., U. Hartmann, Ch. Lehmann, M. Grueundling, D. Pavlovic, F. Reinhardt Biofeedback as a supportive method in weaning long-term ventilated critically ill patients Med Hypotheses, 63 (1):21-5. 2004 (0.6) 91. Kumar Sinha P, Manikandan S, Usichenko TI, Pavlovic D. Reducing venipuncture pain by cough trick Anesth Analg. 99 (3):952-3. 2004. (2.2) 92. Pavlovic D, Usichenko T. B-Aware: recall of intraoperative events Lancet. 364 (9437): 841. 2004 (18.316 i.e. 3.66) 93. Dragan Pavlovic, Matthias Gruendling, Helge Frieling, Kai-Stephal Lauer, Vo Hoai Bac, Michael Wendt, and Christian Lehmann Thermostatic tissue platform for intravital microscopy: “the hanging drop” model DIVI, Berlin, 2004 94. Dragan Pavlovic Intravenous cannula with changeable diameter: a new shock therapy device German Patent Office, 2005. (0.8) 95. Usichenko T.I., Dinse M., Hermsen M., Witstruck T., Pavlovic D., Lehmann Ch. Auricular acupuncture for pain relief after total hip arthroplasty – a randomized controlled study Pain. 114 (3):320-7, 2005 (4.556) 2005 Abstracts 96. Christian Lehman, Vo Haoai Bac, Dragan Pavlovic, Martina Lustig, Claus-Dieter Heidecke, Michael Wendt, and Matthias Gründling Metronidasole has antiinflamatory effects and could improve microcirculation in rat model of sepsis International Congress on microcirculation, Hawaii 2005. 97. Andreas Knöck, Stephan Diedrich, Dragan Pavlovic, Michael Wendt, Christian Lehmann Activated protein C improves intestinal microcirculation in experimental endotoxemia in the rat, ESCIM, Brussels Crit Care 9 (Suppl 1), S31-32, 2005. 98. Marc Fogliata, Thomas Feyerherd, Dragan Pavlovic, Matthias Gruendling, Michael Wendt, and Christian Lehmann Ketamine and propofol do not affect intestinal microcirculationin in the rat edotoxemia: experiments in a new model for intravital microscopy Crit Care 9 (Suppl 1), S31-32, 2005.

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99. Henning Erber, Christian Lehmann, Matthias Gruendling, Thomas Wenzel, Albrecht Stier, Michael Wendt and Dragan Pavlovic Reversal of lipopolysaccharide induced hyporeactivity in rat aorta and human mesenteric artery in vitro by orthovanadate First Prise for Poster, ESCIM, Brussels 2005. Critical Care 9 (Suppl 1): S61-62, 2005. 100. T.I. Usichenko, K. Bolz, M. Dinse, M. Wendt, Ch. Lehmann, F. Feyerherd, D. Pavlovic Naloxone suppresses the pain relief effect of “cough-trick” during venipuncture: a crossover volunteer study Eur J Anaesth 22, suppl 34: 187 (A-727), 2005. 101. T.I. Usichenko, M. Hermsen, T. Witstruck, M. Dinse, D. Pavlovic, C. Lehmann, F Feyerherd. Auricular acupuncture reduces intraoperative fentanyl requirement during total hip arthroplasty – a randomised controlled study Eur J Anaesth 22, suppl 34: 189 (A-734), 2005. 102. T.I. Usichenko, S. Foellner, Ch. Lehmann, M. Wendt, D. Pavlovic Theodrenalin but neither cafedrine nor ephedrine produces transitory contraction of pig coronary artery pre-treated with propranolol Eur J Anaesth 22, suppl 34: 135 (A-515), 2005. 103. Konrad Meissner, Thomas Iber, Jan-Patrick Roesner, Christian Mutz, Christina Layher, Christian Lehmann, Michael Wendt, and Dragan Pavlovic In-vivo tests of a bi-directional venturi pump for emergency transtracheal lung ventilation Eur J Anaesth 22, suppl 34: 181 (A-701), 2005. 104. Marc Fogliata, Thomas Feyerherd, Dragan Pavlovic, Matthias Gruendling, Michael Wendt, and Christian Lehmann Ketamine and propofol do not affect intestinal microcirculationin in the rat edotoxemia J Anästhesie Intensivbehandlung 2: S. 162, 2005. 105. Dragan Pavlovic, Konrad Meissner, Thomas Iber, Jan Roesner, Christian Mutz, Christina Layher, Christian Lehmann, and Michael Wendt Bi-directional venturi pump can provide satisfactory transtracheal lung ventilation during more then 1 hour in 40kg pigs J Anästhesie Intensivbehandlung 2: S. 178, 2005. 106. Dragan Pavlovic and Snezana Divac: Ethics of concerns and life cessation decisions: when emotions are all what remains, Infection 33 (Suppl. 1): 15, 2005. 107. Andreas Knöck, Stephan Diedrich, Dragan Pavlovic, Michael Wendt, Christian Lehmann Activated protein C improved intestinal microcirculation without affecting the cytokine release in experimental endotoxemia in the rat Infection 33 (Suppl. 1): 36, 2005. 108. Christian Lehman, Vo Haoai Bac, Dragan Pavlovic, Martina Lustig, Claus-Dieter Heidecke, Michael Wendt, and Matthias Gründling

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Metronidasole has antiinflamatory effects and could improve microcirculation in rat model of sepsis Proceeding of the 2nd International Symposium on Microcirculation and mitochondrial disfunction in Intensive Care Medicine, Amsterdam, 24th Sept, 2005. 109. Hackenberg I, Usichenko T I, Gruendling M, Meissner K, Rothe K F, Lehmann C, Wendt M, Pavlovic D Dopexamine induces contraction in pig coronary artery in vitro Intensive Care Medicine, Vol. 31 (Suppl. 1): S17 (051), 2005. 110. Vo Hoai Bac, Dragan Pavlovic, Martina Lustig, Claudia Spies, Claus-Dieter Heidecke, Michael Wendt, Matthias Gründling, and Christian Lehmann, Effects of imipenem on intestinal microcirculation and cytokine release in septic rats J Vasc Res 2006; 43:27-60 (First Prise for Poster, Rostock, Germany, 2005) 111. Lehmann Ch, Vo Hoai Bac, Dragan Pavlovic, Claus-Dieter Heidecke, Michael Wendt, Matthias Gründling. Metronidazol affect intestinal microcirlation in the rat DAK, Dresden, 2005. Articles 112. Dragan Pavlovic Intravenöse Kanüle, insbesondere für die schocktherapie (Intravenous cannula with changeable diameter: a new shock therapy device) German Patent Office, No: 10 2005 005 387.4; 04.02. 2005. (0.8) 113. Usichenko T.I., Dinse M., Hermsen M., Witstruck T., Pavlovic D., Lehmann Ch. Auricular acupuncture for pain relief after total hip arthroplasty – a randomized controlled study Pain. 114 (3):320-7, 2005. (4.556) 114. Christian Lehmann, Taras I. Usichenko, and Dragan Pavlovic Heparins in sepsis-induced disseminated intravascular coagulation: low weight – high impact? (Editorial) Crit Care Med, Jun; 33 (6):1455-7. 2005. (4.0) 115. Usichenko TI, Hermsen M, Witstruck T, Hofer A, Pavlovic D, Lehmann C, Feyerherd F. Auricular acupuncture for pain relief after ambulatory knee arthroscopy-a pilot study. Evid Based Complement Alternat Med. 2005 Jun; 2(2):185-189. (..) 116. Vladimir Nedeljkov, Srdjan Lopicić, Dragan Pavlović, and Dušan Cemerikić Electrophysiological effect of β-N-methylamino-L-alanine on Retzius nerve cells of the leech Haemopis sanguisuga, Ann. N.Y. Acad. Sci. (Series: Biophysics from Molecules to Brain: In Memory of Radoslav K. Andjus) 1048: 349–351, 2005. (1.890) 117. Helge Frieling, Matthias Gründling, Kai-Steffen Lauer, Michael Wendt, Thomas Hachenberg, Christian Lehmann, and Dragan Pavlovic Intraperitoneal instillation of polihexanide produces hypotension and vasodilation: in vivo and in vitro study in rats

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Int J Colorectal Dis. 21: 373-380, 2006. (1.848) 118. Christian Lehmann, Taras I. Usichenko, and Dragan Pavlovic From scurvy to sepsis: Vitamin C - a pill for all seasons? (Editorial) Crit Care Med, 33 (8): 1881-2. 2005. (4.0) 119. Grundling M, Pavlovic D, Kuhn SO, Feyerherd F. Is the method of modified percutaneous tracheostomy without bronchoscopic guidance really simple and safe? Chest. 128(5):3774-5. 2005. (3.264) 120. Dragan Pavlovic and Snezana Divac Ethics of concerns and life cessation decisions: when emotions are all what remains Eubios Journal of Asian and International Bioethics (EJAIB). 15: 178-182 (November) 2005. 2006 T.I. Usichenko, S. Foellner, M. Gruendling, F. Feyerherd, Ch. Lehmann, M. Wendt, D. Pavlovic Akrinor®-induced relaxation of pig coronary artery in vitro is transformed into α 1-receptor-mediated contraction by the pre-treatment with propranolol J Cardiovasc Pharmacol, 47: 450-455, 2006. (1.576) 122. Ch. Lehmann, V. H. Bac, D. Pavlovic, M. Lustig, S. Bohnenstengel, C.-D. Heidecke, M. Wendt, M. Gründling: Metronidazol verbessert die intestinale Mikrozirkulation bei septischen Ratten, in: Natscke K., Knaut, M, and Jung, F. (Ed.): Myocardial Microcirculation, PABST SCIENCE PUBLISHERS, 2005. 123. Jurgen Birnbaum, Ortrud Vargas Hein, Carsten Luhrs, Oskar Ruckbeil, Claudia D Spies, Sabine Ziemer, Matthias Grundling, Taras Usichenko, Konrad Meissner, Dragan Pavlovic, Wolfgang J Kox and Christian Lehmann Effects of coagulation factor XIII on intestinal functional capillary density, leukocyte adherence and mesenteric plasma extravasation in experimental endotoxemia Critical Care Manuscript: C50927P, in press. 124. Christian Lehmann MD, Frank Feyerherd MD, Thomas Feyerherd, Marc Fogliata, Matthias Gründling MD, Taras I. Usichenko MD, Konrad Meissner MD, Michael Wendt MD, and Dragan Pavlovic MD Intravenous anesthesia with pentobarbital and ketamine does not affect intestinal microcirculation in experimental endotoxemia Laboratory Animals (in press) (1.7) 125. Ch. Lehmann, V.H. Bac, D. Pavlovic, M. Lustig, S. Bohnenstengel, S. Maier, F. Feyerherd, T. I. Usichenko, K. Meissner, M. Wendt, C.-D. Heidecke, M. Gründling

Metronidazole improves intestinal microcirculation in septic rats independently of bacterial burden Clin Hemorheol Micro 34: 427-438, 2006. (0.63) 126. Dragan Pavlovic and matthias Gruendling

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Tracheotomy could have been performed by the Greeks in the fourth centudy B.C. Journal für Anästhesie und Intensivbehandlung, 3: 86-87, 2006. 127. Dragan Pavlovic, MD, Helge Frieling, MD, Kai-Stephan Lauer, MD, Vo Hoai Bac, MSc, Jorn Richter, Michael Wendt, MD, Christian Lehmann, MD, Taras Usicenko, MD, Konrad Meissner, MD, and Matthias Gruendling, MD. Thermostatic tissue platform for intravital microscopy: “the hanging drop” model Journal of Microscopy-Oxford, in press: 2006. (1.74) 128. Helge Frieling, MD*, Kai-Steffen Lauer, MD*, Matthias Gründling, MD, Taras Usichenko, MD, Konrad Meissner, MD, Theoni Kanellopoulou, Christian Lehmann, MD, Michael Wendt, MD, and Dragan Pavlovic, MD Peritoneal instillation of taurolidine or polihexanide modulates intestinal microcirculation in experimental endotoxemia Int J Colorectal Dis (in press) (1.848) 129. Martina Katja Lustig, Vo Hoai Bac, Dragan Pavlovic, Stefan Maier, Matthias Gründling, Olaf Grisk , Michael Wendt, Claus-Dieter Heidecke, Christian Lehmann CASP (Colon Ascendens Stent Peritonitis) – a model of sepsis adopted to the rat: physiological, microcirculatory and laboratory changes Shock (in press) (3.1)

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Erklärung

Hiermit erkläre ich, dass diese Arbeit bisher von mir weder der Medizinischen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald noch einer anderen wissenschaftlichen Einrichtung zum Zwecke der Habilitation eingereicht wurde. Ferner erkläre ich, dass ich diese Arbeit selbständig verfasst und keine anderen als die darin angegebenen Hilfsmittel benutzt habe. Ort, Datum Unterschrift des Habilitanden 15. 7. 2006

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LEBENSLAUF Dragan Pavlovic ______________________________________________________________________ Persönliches Geburtsdatum und-ort: 4. August 1949, Vranje, Jugoslawien Staatsangehörigkeit: Französisch Adresse: Dienstlich: Klinik und Poliklinik für Anästhesiologie und Intensivmedozin Ernst-Moritz-Arndt-Universität, 17475 Greifswald Friedrich-Loeffler-Strasse 23b Tel.: (49) 3834 865848 Portable: (49) 170242332 Fax: (49) 3834 - 86 5802 e-mail: pavlovic@mail.uni-greifswald.de Privat: Walter-Rathenau-Str. 46 (2.6) 17489 Greifswald Tel.: (49) 3834 - 86 2928 e-mail: dragan@hotmail.com

5, place de la Sorbonne, 75005 Paris, France, Tel. /Fax: (33 1) 56 24 90 99 ______________________________________________________________________ Akademische Grade Dr. med. Doktor der Medizin, Universität Belgrad, Jugoslawien (25. Juni 1976), Approbation : 25. 10. 1977 D.S.M. Diplom in Sportmedizin, The London Hospital Medical College, London, Groß Britannien. (Juni 1984) Dr. med. Doktor der Medizin, Ernst-Moritz-Arndt-Universität, Greifswald, Deutschland, November 2003. _______________________________________________________________________ Praktische Tätigkeiten 1976-1977 Ärztlicher Vorbereitungsdienst (AiP), Medizinische Fakultät der Universität Belgrad, Jugoslawien ( 4. 7. 1976 - 17. 7. 1977) und Sportmedizinische Praxis “Medicinar”, Belgrad, Jugoslawien 1977-1978 Allgemeinarzt, Ambulanz Porec und Koper, Jugoslawien. (18. 7. 1977 - 20. 8 1978)

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1978-1979 Assistenzarzt, Abteilung für Anästhesie und Intensivmedizin, Paracelsus Klinik, Marl, Deutschland. (1. 9. 1978 - 31. 7. 1979) 1979-1981 Sportmedizinische Praxis (Ärztlicher Vorbereitungsdienst Sportmedizin), ”Medicinar”, Karate Club, Belgrad, Jugoslawien. (1. 8. 1979 – 30. 7. 1981) 1981-1983 Assistenzarzt, Abteilung für Anästhesie und Intensivmedizin, Waiblingen, Deutschland. (15. 8. 1981 - 31. 5. 1983) 1983-1984 Sportmedizin, Fachausbildung, The London Hospital Medical College, London, Groß Britannien. (25 September 1983- 5 Juni 1984) 1985 - 2000 Wissenschaftlicher Mitarbeiter (seit 1987 Leiter) der Abteilung für glatte Muskulatur, Faculté Xavier - Bichat, INSERM U408 (226) und Beaujon-Hospital, Abteilung für Pneumologie und Intensivmedizin, Clichy, Paris, Frankreich. (6. 3. 1985 - 31.12. 1999) 2000 - ... Assistenzarzt und Wissenschaftlicher Mitarbeiter, Klinik und Poliklinik für Anästhesiologie und Intensivmedizin, Ernst-Moritz-Arndt Universität, Greifswald, B. R. Deutschland (seit 1. 1. 2000 - ... ) ______________________________________________________________________ Weitere Ausbildung 1979-81 Englische Sprache und Literatur Studium, Kilburn Polytechnics, London, Groß Britannien. 1983 Kursus “British Medical Practice” an der Postgraduate Medical School, University of Exeter, Groß Britannien (April 1983 - Juni 1983). 1985-86 Kursus in Physiologie und Pharmakologie des Atemweges, Universität Paris V., Frankreich. (September 1985-Juni 1986) Sommer 1989, Kursus “patch-clamp“, Faculté des Sciences (Prof. M. Lazdunski), Nizza, Frankreich, und Faculté de Pharmacie, Strassburg, Frankreich (Prof. Stockle). Sommer 1991, Kursus "Optical techniques of membrane potential recording and intracellular ion concentration recording", Faculté de Medicine, Marseille (Prof. Bongrand), Marseille, Frankreich. ___________________________________________________________________________ Forschung 1985 - 1999 Wissenschaftlicher Mitarbeiter, Unitè INSERM U408, Facultè Xavier - Bichat, Paris, Frankreich (6. 3. 1985-31. 12. 1999). 2000 - ... Wissenschaftlicher Mitarbeiter, Klinik und Poliklinik für Anästhesiologie und Intensivmedizin, Ernst-Moritz-Arndt Universität, 17487 Greifswald, Deutschland. (1. 1. 2000 -....) ______________________________________________________________________

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Positionen 1987 - 2000 Leitung: Abteilung für glatte Muskulatur, Unitè INSERM U408, Facultè Xavier - Bichat, Universität Paris 7, Paris, Frankreich. 2000- ... Wissenschaftlicher Mitarbeiter, Klinik und Poliklinik für Anästhesiologie und Intensivmedozin, Ernst-Moritz-Arndt-Universität, Greifswald, Deutschland (1. 1. 2000- ...). _______________________________________________________________________ Mitgliedschaften Societe de Pneumologie d’íIle-de-France. The Mind Association _______________________________________________________________________ Editorial Boards Redacteur associe / Associated Editor - Computer science advisor, Artificial Intelligence and Expert Systems, Paris, 1991 - 1999. _______________________________________________________________________ Fremdsprachenkenntnisse Serbokroatisch Französisch Englisch Deutsch ______________________________________________________________________ RADIO 1990 "Bioethique et legislation en France" (at Radio France Internationale, Paris). "Debat sur líeuthanasie se poursuit en France" (at Radio France Internationale, Paris). "Pollution et climat" (at Radio France Internationale, Paris). "Líinformatique a la croise des chemins" (at Radio France Internationale, Paris). ___________________________________________________________________________ Dragan Pavlovic