ARTICLE IN PRESS

J. Comp. Path. 2008,Vol.138, 23^31

0021-9975/$ - see fdoi:10.1016/j.jcpa.

Correspondence

www.elsevier.com/locate/jcpa

Distribution and Chemical Coding of IntramuralNeurons in the Porcine Ileum During

Proliferative Enteropathy

Z. Pidsudko, J. Kaleczyc, K. Wasowicz, W. Sienkiewicz, M. Majewski*,W. Zajacy and M. Łakomy

Division of Animal Anatomy, *Division of Clinical Physiology, Department of FunctionalMorphology, Faculty ofVeterinary

Medicine, University ofWarmia andMazury in Olsztyn, Oczapowskiego 13 Street, 10-719 Olsztyn, and yWetprom,Veterinary Clinic,

Zwyciestwa133 Street, 75-672 Koszalin, Poland

Summary

Enteric neurons are highly adaptive in their response to various pathological processes including in£ammation, sothe aim of this study was to describe the chemical coding of neurons in the ileal intramural ganglia in porcineproliferative enteropathy (PPE). Accordingly, juvenile LargeWhite Polish pigs with clinically diagnosed Lawsoniaintracellularis infection (PPE; n ¼ 3) and a group of uninfected controls (C; n ¼ 3) were studied. Ileal tissue fromeach animal was processed for dual-labelling immuno£uorescence using antiserum speci¢c for protein gene pro-duct 9.5 (PGP 9.5) in combination with antiserum to one of: vasoactive intestinal polypeptide (VIP), substance P(SP), calcitonin gene-related peptide (CGRP), somatostatin (SOM), neuropeptideY (NPY) or galanin (GAL). Ininfected pigs, enteric neurons were found in ganglia located within three intramural plexuses: inner submucosal(ISP), outer submucosal (OSP) and myenteric (MP). Immuno£uorescence labelling revealed increases in thenumber of neurons containing GAL, SOM,VIP and CGRP in pigs with PPE. Neuropeptides may therefore havean important role in the function of porcine enteric local nerve circuits under pathological conditions, when thenervous system is stressed, challenged or a¥icted by disease such as PPE. However, further studies are required todetermine the exact physiological relevance of the observed adaptive changes.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: ileum; porcine proliferative enteropathy; neuropeptide; enteric neuron

Introduction

Proliferative enteropathy (PPE) is a disease of pigscaused by infection with the bacterium Lawsonia intra-

cellularis (Lawson et al., 1993; Lawson and Gebhart,2000). Contrary to other in£ammatory diseases of theporcine alimentary tract in which atrophic or necroticchanges occur (i.e. swine dysentery), L. intracellularis-induced in£ammation results in proliferative changes.These proliferative changes may occur at di¡erent lo-cations in the intestinal tract, and may be modi¢ed bysecondary changes to primary lesions that alter thegross pathological appearance (Lawson and Gebhart,

ront matter2007.09.003

to: Zenon Pidsudko (e-mail: zenekp@uwm.edu.pl)

2000).This porcine disease thus presents many speci¢clesions, con¢ned to the intestinal tract, that most fre-quently involve the lower ileum and less commonlythe large intestine. Generally, a¡ected areas of intestinemay show irregular patchy sub-serosal oedema, withsmall £ecks of necrotic material on the surface of thethickened mucosa. In mild cases, the mucosal thicken-ing takes the form of small, raised, opaque islands with-in the normal epithelium. With increasing severitythese lesions become con£uent and show an irregularnodular or folded surface (Lawson and Gebhart, 2000).In£ammatory processes such as those that occur in

PPE may a¡ect all components of intestinal tissue. En-teric neurons are known to show a high degree of plas-ticity in response to in£ammation (Sharkey and

r 2007 Elsevier Ltd. All rights reserved.

ARTICLE IN PRESS

Z. Pidsudko et al.24

Kroese, 2001; Ekblad and Bauer, 2004).These adaptivechanges include both up- and down-regulation of ex-pression of neurotransmitters and the induction ofnew genes in enteric neurons. Such changes developedto help enteric neurons survive under pathological con-ditions and also to help the in£amed intestine to re-cover from pathological challenge.The enteric nervous system (ENS) is organized into

three layers called plexuses. The inner submucosalplexus (ISP) is located close to the muscularis mucosa,the outer submucosal plexus (OSP) is located near theexternal circular muscle layer (Balemba et al., 1999;Timmermans et al., 2001) and the myenteric plexus(MP) is situated between the longitudinal and circularmuscle layers. The ENS receives extrinsic innervationfrom both the prevertebral and dorsal root ganglia(Timmermans et al.,1994, 2001; Furness, 2000; Sharkeyand Kroese, 2001). Each of these plexuses contains aheterogeneous population of neurons with di¡erentfunctions and targets.The observation that a single en-teric neuron may contain more than one transmitter orneuron-speci¢cmarker has led to the concept of chemi-cal coding of enteric neurons that is related to theirfunction and segmental position (Costa et al., 1984;Costa and Furness, 1984; Ekblad et al., 1984; Furnesset al., 1984; Keast et al., 1984; Scheuermann et al., 1987;Barbiers et al.,1994;Timmermans et al.,1994).Most data on the plasticity of the ENS in in£amma-

tion have been derived from experimental models(Holzer, 1998; Evangelista, 2001; Feher et al., 2001;Sharkey and Kroese, 2001; Abad et al., 2003). By con-trast, information concerning the adaptive changes ofenteric neurons in the course of natural in£ammationassociated with diseases a¡ecting the gastrointestinaltract is very limited (Romanska et al., 1993; Balembaet al., 2001). For this reason, the aim of the presentstudy was to investigate alterations in the ENSduring the course of PPE, a spontaneously arising in-£ammatory disease that is widespread in the pig popu-lation and characterized by proliferative changes withthe intestine.

Materials and Methods

The study was performed on 6 juvenile pigs of theLargeWhite Polish breed (10 kg body weight, aged 6weeks) obtained from a commercial fattening farm inŁomianki, Poland.The animals were housed and trea-ted in accordance with rules approved by the localEthical Commission (conforming to the Principles ofLaboratoryAnimal Care, NIH publication No. 86-23,revised 1985). The pigs were divided into a controlgroup (C) consisting of 3 normal, clinically healthy an-imals, and a group consisting of 3 pigs with laboratorydiagnosed L. intracellularis infection (PPE).The diagno-

sis of PPEwas con¢rmedwith apolymerase chain reac-tion (PCR)-based test performed at a StateVeterinaryResearch Institute in Pulawy, Poland.The animals were killed with an overdose of sodium

pentobarbital (Thiopental, Sandoz, Kundl-RakuŁ sko,Austria; 20mg/kg body weight given intravenously)and perfused intracardially with 4% bu¡ered parafor-maldehyde (pH 7.4). Samples of the ileum were col-lected, post-¢xed by immersion in the same ¢xative forseveral hours and, ¢nally, stored in 18% sucrose untilsectioned. Cryostat sections (10 mm) were processedfor double-labelling immuno£uorescence as describedpreviously (Pidsudko et al., 2001). In order to studythe distribution of intramural nerve structuresone of the two antibodies used to label each sectionwas that against protein gene product 9.5 (PGP-9.5).In order to determine the chemical coding of thesestructures, the second of the two antibodies used hadspeci¢city for one of the following molecules: vasoac-tive intestinal polypeptide (VIP), substance P (SP), cal-citonin gene-related peptide (CGRP), somatostatin(SOM), neuropeptide Y (NPY) and galanin (GAL)(Table1).Negative controls employed in the immuno£uores-

cence procedure included pre-absorption of the neuro-peptide antisera with 1mg of appropriate antigen per1ml of corresponding antibody at working dilution.Antigens used for this purpose were purchased fromPeninsula (St Helens, Merseyside, UK), Sigma (StLouis, USA) or Dianova (Hamburg, Germany). Addi-tional negative controls involved the omission and re-placement of all primary antiseraby non-immune sera.To determine the chemical coding of particular neu-

ronal populations, at least 500 PGP-9.5 labelled neu-rons in a particular ileal plexus (MP, ISP and OSP)were examined for expression of each of the biologi-cally active substances. Sections labelled for the samecombination of the antigens and assigned to quantita-tive analysis were separated by at least 100 mm to avoiddouble-counting of individual neurons. Only neuronscontaining distinct nuclei were counted. The labelledsections were studied and photographed with a ZeissAxiophot £uorescence microscope equipped with epi-illumination and an appropriate ¢lter set for £uores-cein isothiocyanate (FITC) orTexas Red.The relation-ship between expression of the two £uorescent labelswas examined by interchanging the ¢lters. Sectionswere also examined with a laser confocal microscope(Bio-RadMicroradianceMR2).Data from each plexus in each animal were pooled.

All results are expressed as means 7 standard devia-tions. Statistical analysis was performed with the Stu-dent’s t-test (GRAPHPAD PRISM v.2.0, GraphPadSoftware Inc., San Diego, CA). Di¡erences were con-sidered as being statistically signi¢cant when Pp0.05.

ARTICLE IN PRESS

Table 1List of primary and secondary antisera used in the study

Antigen Species of origin Code Dilution Supplier

Primary antiseraPGP-9.5 Rabbit 13C4 1in 2000 Biogenesis, UKCGRP Rabbit RPN1842 1in1600 Amersham, UKGAL Rabbit 4600-5004 1in1600 Biogenesis, UKSOM Rat 8330-0009 1 in 30 Biogenesis, UKSP Rat 8450-0505 1in 250 Biogenesis, UKVIP Rabbit 20077 1in 200 Incstar, MO, USANPY Rat NZ1115 1in 200 A⁄niti, UK

Secondary antiseraFITC-conjugated goat anti-rabbit IgG 1in 400 Jackson Immunoresearch, USAFITC-conjugated goat anti-mouse IgG 1in 400 Jackson Immunoresearch, USAFITC-conjugated goat anti-rat IgG 1in 400 Jackson Immunoresearch, USABiotinylated goat anti-rabbit IgG 1in 400 Dako, DKBiotinylated goat anti-rat IgG 1in 400 Amersham, UKBiotinylated goat anti-mouse IgG 1in 400 Amersham, UKStreptavidin-conjugated CY 3 1in 4000 Dianova, Hamburg, GER

Fig. 1. Confocal laser scanning microscopic (CLSM) image show-ing three distinct plexuses in the wall of the porcine ileum.The inner submucosal plexus (ISP) lies between the muscu-laris mucosa and the lamina propria.The outer submucosalplexus (OSP) lies within the submucosa and the myentericplexus (MP) is locatedbetween the longitudinal andcircularlayers of the muscularis. Bar, 200 mm.

Intramural Neurons in Porcine Proliferative Enteropathy 25

Results

Immune labelling of PGP-9.5 revealed three distinctplexuses in the wall of the porcine ileum (Fig. 1).Theseincluded an ISP between the muscularis mucosa andlaminapropria (Fig.2), anOSP found in the submucosa(Figs. 3, 5), and a myenteric plexus (MP) located be-tween the longitudinal and circular muscle layers ofthe ileal muscle coat (Fig. 4).Immunolabelling revealed an increase in the num-

ber of neurons containingGAL, SOM,VIPandCGRPin all three plexuses within ileal tissue from pigs withPPE (Table 2). Immunolabelling for SP did not reveala consistent increase in expression of this neuropeptide,and in fact a reduction in the number of neurons posi-tively labelled for this molecule occurred withinthe OSP ganglia of pigs with PPE. No neuron in anyof the intramural ganglia of both animal groupslabelled for NPY.Nerve ¢bres expressing CGRP, SOM, GAL,VIP and

SPwere found in all layers of the ilealwall, including themucosa andmuscle layer as well as in themyenteric andsubmucosal plexuses. In general, those immunolabellednerve ¢bres observed in the muscle layer and myentericplexus outnumbered those found in the remaininglayers. A small number of SOM-immunoreactive nerveterminals was encountered in the muscle layer andmyenteric plexus only. In contrast, few CGRP-positivenerve ¢bres were observed and these were only found inthe mucosa and submucosal plexuses.

Discussion

The results of the present study have shown elevation inexpression of the neuropeptides GAL, SOM,VIP and

CGRP by neurons located in the intramural ganglia ofthe porcine ileum during PPE. By contrast, there was aless consistent change in the neuronal expression of SP,and neurons expressing this molecule were fewer in theOSPof animals with PPE.It is now recognized that SP plays a role as amediator

of neurogenic in£ammation (HolzerandHolzer-Petsche,1997;Maggi,1997). SPandCGRParealso regardedas the

ARTICLE IN PRESS

Fig. 2. The ileal inner submucosal plexus (ISP) in a control pig (2a) and a pig with proliferative enteropathy (2b). CLSM image showing thedistribution of neurons expressing PGP-9.5 (green £uorescence) and/or GAL (red £uorescence). Red and green channels are digitallysuperimposed. Dual-labelled (PGP-9.5 andGAL-positive) neurons are yellow. Note that in the pig with PPE almost all of the neuronsexpress GAL, whilst in the control animal many neurons do not label for this peptide. Bar, 50 mm.

Fig.3. The ileal outer submucosal plexus (OSP) in a control pig (3a) and apig with proliferative enteropathy (3b). CLSM image showing thedistribution of neurons expressing PGP-9.5 (green £uorescence) and/or CGRP (red £uorescence). Red and green channels are digi-tally superimposed. Dual-labelled (PGP-9.5 and CGRP-positive) neurons are yellow. Note that in the pig with PPE almost one half ofthe neurons express CGRP, whilst in the control animal many neurons do not label for this peptide. Bar, 50 mm.

Fig. 4. The ileal myenteric plexus (MP) in a control pig (4a) and a pig with proliferative enteropathy (4b). CLSM image showing the dis-tribution of neurons expressing PGP-9.5 (green £uorescence) and/or SOM (red £uorescence). Red and green channels are digitallysuperimposed. Dual-labelled (PGP-9.5 and SOM-positive) neurons are yellow. Bar, 50 mm.

Z. Pidsudko et al.26

major pro-in£ammatory neuropeptides.There is mount-ing evidence that SP plays a role in the derangement ofgastrointestinal motor activity inducedby intestinal ana-phylaxis, infections, in£ammation, trauma or stress(Holzer, 1998). In addition, this peptide may play a rolein mediating pathological changes in gastrointestinal£uid and electrolyte secretions which accompany infec-tions, in£ammation and tissue damage.There is increas-ing evidence that SP participates in a variety ofhypersecretory and in£ammatory reactions in the gut,where this tachykinin may take part in reactions asso-

ciated with anaphylaxis, delayed-type hypersensitivityandTrichinella spiralis infections, but not in the diarrhoeacaused by cholera toxin (Pothoulakis et al.,1994). Finally,there is also a hypothesis which suggests that SP andCGRPact asmessengers at the interfacebetweenthener-vous and immune system, and that intestinal mast cells,lymphocytes, granulocytes and macrophages are underthe in£uence of peptidergic neurons (Maggi,1997).The interrelationship between neuropeptides and

the immune system is of a reciprocal nature as exempli-¢ed by the potential role of tachykinins in the secretory

ARTICLE IN PRESS

Table 2Percentage of ileal plexus neurons expressing speci¢c neuropeptides in control pigs and pigswith proliferative enteropathy (PPE)

Myenteric plexus

(mean7 standard deviation)

Outer submucosal plexus

(mean7 standard deviation)

Inner submucosal plexus

(mean7 standard deviation)

Neuropeptide Control pigs PPE pigs Control pigs PPE pigs Control pigs PPE pigs

GAL 18.6171.33 28.0771.51 22.1070.80 35.6070.88 23.5070.62 67.2071.13SOM 7.7070.87 20.8070.86 3.170.79 36.3070.73 17.4071.25 46.6071.00VIP 4.1270.81 8.6970.87 5.1770.86 13.5871.32 36.5471.30 41.3071.27CGRP 15.6071.14 25.7071.22 17.1070.97 41.6071.15 7.3071.16 53.8071.31SP 2.8871.00 9.2071.10 25.9671.34 10.6770.94 23.1771.20 40.8471.25NPY No expression No expression No expression No expression No expression No expression

Fig.5. The ileal outer submucosal plexus (OSP) in a control pig (5a) andapig with proliferative enteropathy (5b). CLSM image showing thedistribution of neurons expressing PGP-9.5 (green £uorescence) and/or SP (red £uorescence). Red and green channels are digitallysuperimposed.Dual-labelled (PGP-9.5 and SP-positive) neurons are yellow. Note that in the pig with PPE only a fewof the neurons areSP-positive. Bar, 50 mm.

Intramural Neurons in Porcine Proliferative Enteropathy 27

response of the rat colon to interleukin1b (Eutamene etal.,1995). It is also noteworthy that immune cells are notonly a target uponwhich SPacts to modify immune re-sponses, but under pathological conditions cells of theimmune system can also synthesize and release tachy-kinins and other neuropeptides. Additionally, it is nowrecognized that SPand CGRPare important factors inin£ammatory processes which are regulated by nervegrowth factor (NGF) (Lindsay and Harmar, 1989).NGF has the ability to increase the level of these pep-tide neurotransmitters in mature neurons. In in£am-matory bowel disease (IBD), NGFand its high-a⁄nityreceptor (trkA) are greatly over-expressed in the in-£amed tissue (di Mola et al., 2000), and it is clear thatNGF controls in£ammatory processes by regulatingrelease of peptidergic neurotransmitters (Reinshagenet al.,1998).Many studies have shown that neuropeptides such as

SP and CGRP are protective in acute (Reinshagenet al.,1994) and chronic (Reinshagen et al.,1996) modelsof experimental colitis. The protective e¡ect of neuro-tropic factors can partly be explained by a speci¢cmodulation of neuropeptide expression during in£am-mation. Therefore, in experimental in£ammation ofthe rat gut, NGFand neurotropin-3 seem to functionin a protective manner. Because the in£uence of neuro-tropic factors on the function and development of theENS is highly complex, the role of the ENS and neuro-

tropic factors in chronic in£ammatory states, such asoccurs in PPE, is not yet fully understood.VIP is widely accepted as an anti-in£ammatory fac-

tor (Delgado et al., 2001; Delgado and Ganea, 2001;Abad et al., 2003). In the present study, this neuropep-tide was found to be expressed by more neurons in allganglia studied in the PPE group of animals than incontrols.This observation accords well with results ob-tained for trinitrobenzene sulphonic acid (TNBS)-treated rats during the chronic phase of in£ammation(Miampamba and Sharkey, 1998). Kishimoto et al.(1992) also observed an increased number of VIP-ex-pressing neurons and nerve ¢bres in both plexuses ofthe colon in dextran sulphate-induced colitis in the rat.By contrast, during the colitis associated with experi-mental Schistosoma japonicum infection there was dimin-ished expression of VIP in the ISP and OSP, and in themost severely damaged regions of the intestine therewas total absence of expression of this neuropeptide(Balemba et al.,1998). Similarly, inTrichinella spiralis-in-fested ferrets, immunoreactivity toVIP was found tobelower than in control animals (Palmer and Green-wood,1993). ReducedVIP expression has also been re-ported in the human colon during the course ofulcerative colitis (Renzi et al.,1998). It is not clear whydi¡erences between these studies were found, but re-gional variation, disease state and sampling techniquemay have all contributed.

ARTICLE IN PRESS

Z. Pidsudko et al.28

Another neuropeptide considered to have anti-in-£ammatory and anti-nociceptive actions is SOM. Inan immunohistochemical study performed on intestinefrom pigs with swine dysentery (Sienkiewicz et al.,2004), aswell as in our present study, an increased num-ber of SOM-positive neurons was observed in the wallof the ileum and colon in the MP and OSP ganglia.SOMmay be actively involved in the pathophysiologyof intestinal in£ammation, and possibly of other organs(Reubi et al., 1994). Exogenously administered SOM isknown to mediate systemic anti-in£ammatory andanti-nociceptive e¡ects (Abd El-Aleem et al., 2005;Pinter et al., 2006). It has been also recognized thatSOM acts as a modulator of gastrointestinal activityandcanbe found in the gastrointestinalmucosa (Evansand Potten, 1988; Satoh et al., 1988), enteric neurons(Costa et al., 1980; Pompolo and Furness, 1998) andgut-associated lymphoid tissue (Reubi et al., 1992).SOM is expressed by a speci¢c subgroup of choliner-gic-descending interneurons in the myenteric plexus ofthe small intestine in the guinea-pig (Portbury et al.,1995; Pompolo and Furness, 1998) and pig (Timmer-mans et al., 2001), where it is reported to exert an e¡ecton both the contractile response and acetylcholinerelease. A previous study (Vizi et al., 1984) on guinea-pig ileum provided evidence that SOM causes releaseof endogenous substances responsible for its presynap-tic inhibitory e¡ect. It hasbeen reported that SOMalsocauses the release of noradrenaline in the guinea-pigileal wall. Noradrenaline then acts via a2 adrenorecep-tors which results in the release of transmitters whichinhibit contraction. Furthermore, SOM plays an im-portant role in the reduction of in£ammatory nocicep-tion, nociceptor excitation and sensitization (Hasleret al.,1993; Plourde et al.,1993).Apart from its role in neurotransmission, SOM also

has an active role in the immune response to in£amma-tion.There is convincing evidence that SOMacts as animmunoregulatory and anti-in£ammatory peptidethat down-regulates pro-in£ammatory cytokine ex-pression and release (Blum et al., 1992; Chowers et al.,2000), lymphocyte proliferation (Stanisz et al., 1986)and immunoglobulin production (Blum et al., 1993;Ten Bokum et al., 2000). It is therefore possible that ana-logous functions are played by SOM in the wall of theporcine ileum during PPE, but this hypothesis requiresfurther investigation.GAL is present in nerves supplying the ileum in var-

ious mammalian species (Bishop et al., 1986; Yau et al.,1986; Botella et al.,1992; Akehira et al.,1995) but the roleof GAL in bowel in£ammation is still not fully recog-nized (Mantyh et al., 1989; Kuraishi et al., 1991; Benyaet al.,1998; Majewski et al., 2002).A ¢nal important aspect of the intestinal in£amma-

tory reaction is the altered expression of NGF. NGF is

crucial for development and survival of sympatheticneurons; however, a subclass of primary sensory neu-rons is also strongly NGF-dependent and requires thismolecule for survival. Depriving sympathetic neuronsof NGF leads to a set of well-described ‘‘post-axotomy’’changes including the induction of the expression ofGAL,VIPand sometimes SP, and the down-regulationof expression of catecholamine-synthesizing enzymesand NPY. However, NGF expression is strongly up-regulated in enteritis (di Mola et al., 2000) so it is di⁄-cult to attribute the rise in GAL expression in intestinalin£ammation to alterations in NGF synthesis. In thisrespect, the naturally occurring NGF antagonist,FIZZ1 (also called resistin-like molecule a) is knownto be synthesized in the ileum and colon. Expression ofFIZZI is elevated in enteritis and this molecule may in-teract with NGF-regulated processes (Sharkey andMawe, 2002). Both previous and present results suggestthat the action of GAL is, at least in part, mediated bySP, and that GAL may act on primary a¡erent term-inals to increase release of SP evoked by in£ammation(Kuraishi et al.,1991).In summary, the present results show that neuropep-

tides may have an important role in the function ofporcine enteric nerve pathways not only under physio-logical, but also pathological conditions such as in PPE.However, the exact relevance of these adaptive changesremains to be elucidated in detail.

Acknowledgments

The authors wish to thank M. Marczak, G. Greniukand A. Penkowski for excellent technical assistance.This study was supported by funding from the PolishState Committee of Scienti¢c Research, Grant 6P06K 012 20.

References

Abad, C.,Martinez, C., Juarranz,M.G., Arranz, A., Leceta,J., Delgado, M. and Gomariz, R. P. (2003). Therapeutice¡ects of vasoactive intestinal peptide in the trinitroben-zene sulfonic acidmicemodel of Crohn’s disease.Gastroen-terology, 124, 961^971.

Abd El-Aleem, S. A., Morales-Aza, B. M., McQueen,D. S. andDonaldson, L. F. (2005). In£ammation alters so-matostatin mRNA expression in sensory neurons in therat. EuropeanJournal of Neuroscience, 21,135^141.

Akehira, K., Nakane,Y., Hioki, K. andTaniyama, K. (1995).Site of action of galanin in the cholinergic transmission ofguineapig small intestine.EuropeanJournal of Pharmacology,284,149^155.

Balemba, O. B., Grondahl, M. L., Mbassa, G. K., Semugur-uka,W. D., Hay-Smith, A., Skadhauge, E. and Dantzer,V. (1998). The organisation of the enteric nervous systemin the submucous andmucous layers of the small intestine

ARTICLE IN PRESS

Intramural Neurons in Porcine Proliferative Enteropathy 29

of the pig studied byVIP and neuro¢lament protein im-munohistochemistry. Journal of Anatomy, 192, 257^267.

Balemba, O. B., Mbassa, G. K., Semuguruka,W. D., Assey,R. J., Kahwa, C. K., Hay-Schmidt, A. and Dantzer,V. (1999). The topography, architecture and structure ofthe enteric nervous system in the jejunum and ileum ofcattle. Journal of Anatomy, 195,1^9.

Balemba, O. B., Semuguruka, W. D., Hay-Schmidt, A.,Johansen, M.V. and Dantzer,V. (2001).Vasoactive intest-inal peptide and substance P-like immunoreactivitiesin the enteric nervous system of the pig correlate withthe severity of pathological changes induced by Schistoso-

ma japonicum. International Journal for Parasitology, 31,1503^1514.

Barbiers, M., Timmermans, J. P., Scheuermann, D. W.,Adriaensen, D., Mayer, B. and Groodt-Lasseel, M. H.(1994). Nitric oxide synthase-containing neurons in thepig large intestine: topography, morphology, and viscero-fugal projections. Microscopy Research and Technique, 29,72^78.

Benya, R.V.,Matkowskyj, K. A., Danilkovich, A. andHecht,G. (1998). Galanin causes Cl-secretion in the human co-lon. Potential signi¢cance of in£ammation-associatedNF-kappa B activation on galanin-1 receptor expressionand function. Annals of the NewYork Academy of Science, 863,64^77.

Bishop, A. E., Polak, J. M., Bauer, F. E., Christo¢des, N. D.,Carlei, F. and Bloom, S. R. (1986). Occurrence and distri-bution of a newly discovered peptide, galanin, in themammalian enteric nervous system. Gut, 27, 849^857.

Blum, A. M., Metwali, A., Cook, G., Mathew, R. C., Elliott,D. andWeinstock, J.V. (1993). Substance Pmodulates anti-gen-induced, IFN-gamma production in murine schisto-somiasis mansoni. Journal of Immunology, 151, 225^233.

Blum, A. M., Metwali, A., Mathew, R. C., Cook, G., Elliott,D. andWeinstock, J.V. (1992). GranulomaT lymphocytesin murine schistosomiasis mansoni have somatostatin re-ceptors and respond to somatostatin with decreased IFN-gamma secretion. Journal of Immunology, 149, 3621^3626.

Botella, A., Delvaux, M., Bueno, L. and Frexinos, J. (1992).Intracellular pathways triggered by galanin to inducecontraction of pig ileum smooth muscle cells. Journal ofPhysiology, 458, 475^486.

Chowers,Y., Cahalon, L., Lahav,M., Schor, H.,Tal, R., Bar-Meir, S. and Levite, M. (2000). Somatostatin through itsspeci¢c receptor inhibits spontaneous and TNF-alpha-and bacteria-induced IL-8 and IL-1 beta secretion fromintestinal epithelial cells. Journal of Immunology, 165,2955^2961.

Costa,M. and Furness, J. B. (1984). Somatostatin is present ina subpopulation of noradrenergic nerve ¢bres supplyingthe intestine.Neuroscience, 13, 911^919.

Costa, M., Furness, J. B., Smith, I. J., Davies, B. and Oliver,J. (1980). An immunohistochemical study of the projec-tions of somatostatin-containing neurons in the guinea-pig intestine.Neuroscience, 5, 841^852.

Costa, M., Furness, J. B., Yanaihara, N., Yanaihara, C. andMoody,T.W. (1984). Distribution and projections of neu-rons with immunoreactivity for both gastrin-releasing

peptide and bombesin in the guinea-pig small intestine.Cell andTissue Research, 235, 285^293.

Delgado, M., Abad, C., Martinez, C., Leceta, J. andGomariz, R. P. (2001). Vasoactive intestinal peptide pre-vents experimental arthritis by downregulating bothautoimmune and in£ammatory components of the dis-ease.NatureMedicine, 7, 563^568.

Delgado, M. and Ganea, D. (2001). Inhibition of endotoxin-induced macrophage chemokine production by vasoac-tive intestinal peptide and pituitary adenylate cyclase-activating polypeptide in vitro and in vivo. Journal of Im-munology, 167, 966^975.

diMola, F. F., Friess, H., Zhu, Z.W., Koliopanos, A., Bley,T.,di Sebastiano, P., Innocenti, P., Zimmermann, A. andBuchler, M.W. (2000). Nerve growth factor andTrk higha⁄nity receptor (TrkA) gene expression in in£ammatorybowel disease. Gut, 46, 670^679.

Ekblad, E. and Bauer, A. J. (2004). Role of vasoactive intest-inal peptide and in£ammatory mediators in entericneuronal plasticity. Neurogastroenterology and Motility, 16,S123^S128.

Ekblad, E., Hakanson, R. and Sundler, F. (1984). VIP andPHI coexist with an NPY-like peptide in intramural neu-rones of the small intestine. Regulatory Peptides, 10, 47^55.

Eutamene, H., Theodorou, V., Fioramonti, J. and Bueno,L. (1995). Implication of NK1 and NK2 receptors in ratcolonic hypersecretion induced by interleukin1beta: roleof nitric oxide. Gastroenterology, 109, 483^489.

Evangelista, S. (2001). Involvement of tachykinins in intest-inal in£ammation. Current Pharmaceutical Design, 7,19^30.

Evans, G. S. and Potten, C. S. (1988).The distribution of en-docrine cells along themouse intestine: a quantitative im-munocytochemical study.Virchows Archive B: Cell PathologyIncludingMolecular Pathology, 56,191^199.

Feher, E., Altdorfer, K., Bagameri, G. and Feher, J. (2001).Neuroimmune interactions in experimental colitis. Animmunoelectron microscopic study. Neuroimmunomodula-tion, 9, 247^255.

Furness, J. B. (2000).Types of neurons in the enteric nervoussystem. Journal of theAutonomic Nervous System, 81, 87^96.

Furness, J. B., Costa,M. andKeast, J. R. (1984). Choline acet-yltransferase- and peptide-immunoreactivity of submu-cous neurons in the small intestine of the guinea-pig. CellandTissue Research, 237, 329^336.

Hasler,W. L., Soudah, H. C. andOwyang, C. (1993). A soma-tostatin analogue inhibits a¡erent pathways mediatingperception of rectal distention. Gastroenterology, 104,1390^1397.

Holzer, P. (1998). Implications of tachykinins and calcitoningene-related peptide in in£ammatory bowel disease.Digestion, 59, 269^283.

Holzer, P. and Holzer-Petsche, U. (1997). Tachykinins in thegut. Part II. Roles in neural excitation, secretion and in-£ammation. Pharmacology andTherapeutics, 73, 219^263.

Keast, J. R., Furness, J. B. and Costa, M. (1984). Origins ofpeptide and norepinephrine nerves in the mucosa of theguinea pig small intestine. Gastroenterology, 86, 637^644.

Kishimoto, S., Kobayashi, H., Shimizu, S., Haruma, K.,Ta-maru,T., Kajiyama, G. and Miyoshi, A. (1992). Changes

ARTICLE IN PRESS

Z. Pidsudko et al.30

of colonic vasoactive intestinal peptide and cholinergicactivity in rats with chemical colitis. Digestive Diseases and

Sciences, 37,1729^1737.Kuraishi, Y., Kawabata, S., Matsumoto, T., Nakamura, A.,

Fujita, H. and Satoh, M. (1991). Involvement of substanceP in hyperalgesia induced by intrathecal galanin. Journalof Neuroscience Research, 11, 276^285.

Lawson, G. H. and Gebhart, C. J. (2000). Proliferative en-teropathy. Journal of Comparative Pathology, 122,77^100.

Lawson, G. H., McOrist, S., Jasni, S. and Mackie, R. A.(1993). Intracellular bacteria of porcine proliferative en-teropathy: cultivation and maintenance in vitro. Journalof ClinicalMicrobiology, 31,1136^1142.

Lindsay, R. M. and Harmar, A. J. (1989). Nerve growth fac-tor regulates expression of neuropeptide genes in adultsensory neurons.Nature, 337, 362^364.

Maggi, C. A. (1997).The e¡ects of tachykinins on in£amma-tory and immune cells. Regulatory Peptides, 70,75^90.

Majewski, M., Bossowska, A., Gonkowski, S., Wojtkiewicz,J., Brouns, I., Scheuermann, D.W., Adriaensen, D. andTimmermans, J. P. (2002). Neither axotomy nor target-tis-sue in£ammation changes the NOS- orVIP-synthesis ratein distal bowel-projecting neurons of the porcine inferiormesenteric ganglion (IMG). Folia Histochemica et Cytobiolo-

gica, 40,151^152.Mantyh, P.W., Catton,M.D., Boehmer, C. G.,Welton,M. L.,

Passaro, E. P. J., Maggio, J. E. andVigna, S. R. (1989). Re-ceptors for sensory neuropeptides in human in£amma-tory diseases: implications for the e¡ector role of sensoryneurons. Peptides, 10, 627^645.

Miampamba, M. and Sharkey, K. A. (1998). Distributionof calcitonin gene-related peptide, somatostatin, sub-stance P and vasoactive intestinal polypeptide in experi-mental colitis in rats. Neurogastroenterology and Motility, 10,315^329.

Palmer, J. M. and Greenwood, B. (1993). Regional content ofenteric substance P and vasoactive intestinal peptide dur-ing intestinal in£ammation in the parasitized ferret. Neu-ropeptides, 25, 95^103.

Pidsudko, Z., Kaleczyc, J., Majewski, M., Lakomy, M.,Scheuermann, D.W. andTimmermans, J. P. (2001). Di¡er-ences in the distribution and chemical coding betweenneurons in the inferior mesenteric ganglion supplying thecolon and rectum in the pig. Cell andTissue Research, 303,147^158.

Pinter, E., Helyes, Z. and Szolcsanyi, J. (2006). Inhibitorye¡ect of somatostatin on in£ammation and nociception.Pharmacology&Therapeutics, 112, 440^456.

Plourde,V., Lembo,T., Shui, Z., Parker, J., Mertz, H.,Tache,Y., Sytnik, B. andMayer, E. (1993). E¡ects of the somatos-tatin analogue octreotide on rectal a¡erent nerves in hu-mans. AmericanJournal of Physiology, 265, G742^G751.

Pompolo, S. and Furness, J. B. (1998). Quantitative analysis ofinputs to somatostatin-immunoreactive descending inter-neurons in the myenteric plexus of the guinea-pig smallintestine. Cell andTissue Research, 294, 219^226.

Portbury, A. L., Pompolo, S., Furness, J. B., Stebbing, M. J.,Kunze,W. A., Bornstein, J. C. andHughes, S. (1995). Cho-linergic, somatostatin-immunoreactive interneurons in

the guineapig intestine: morphology, ultrastructure, con-nections and projections. Journal of Anatomy, 187, 303^321.

Pothoulakis, C., Castagliuolo, I., Lamont, J. T., Ja¡er, A.,O’Keane, J. C., Snider, R. M. and Leeman, S. E. (1994).CP-96,345, a substance Pantagonist, inhibits rat intestinalresponses to Clostridium di⁄cile toxin Abut not cholera tox-in. Proceedings of the National Academy of Sciences of the USA,91, 947^951.

Reinshagen, M., Flamig, G., Ernst, S., Geerling, I., Wong,H., Walsh, J. H., Eysselein, V. E. and Adler, G. (1998).Calcitonin gene-related peptide mediates the protectivee¡ect of sensory nerves in a model of colonic injury. Jour-nal of Pharmacology and Experimental Therapeutics, 286,657^661.

Reinshagen, M., Patel, A., Sottili, M., French, S., Sternini,C. and Eysselein,V. E. (1996). Action of sensory neuronsin an experimental rat colitis model of injury and repair.AmericanJournal of Physiology, 270, G79^G86.

Reinshagen, M., Patel, A., Sottili, M., Nast, C., Davis, W.,Mueller, K. and Eysselein, V. (1994). Protective functionof extrinsic sensory neurons in acute rabbit experimentalcolitis. Gastroenterology, 106,1208^1214.

Renzi, D., Mantellini, P., Calabro, A., Panerai, C., Amorosi,A., Paladini, I., Salvadori, G., Garcea,M. R. and Surren-ti, C. (1998). Substance P and vasoactive intestinalpolypeptide but not calcitonin gene-related peptide con-centrations are reduced in patients with moderate and se-vere ulcerative colitis. ItalianJournal of Gastroenterology andHepatology, 30, 62^70.

Reubi, J. C., Horisberger, U.,Waser, B., Gebbers, J. O. andLaissue, J. (1992). Preferential location of somatostatin re-ceptors in germinal centers of human gut lymphoid tissue.Gastroenterology, 103,1207^1214.

Reubi, J. C., Mazzucchelli, L. and Laissue, J. A. (1994).Intestinal vessels express a high density of somatostatinreceptors in human in£ammatory bowel disease.Gastroen-terology, 106, 951^959.

Romanska, H. M., Bishop, A. E., Brereton, R. J., Spitz,L. and Polak, J.M. (1993). Immunocytochemistry for neu-ronal markers shows de¢ciencies in conventional histol-ogy in the treatment of Hirschsprung’s disease. Journal ofPediatric Surgery, 28,1059^1062.

Satoh, Y., Oomori, Y., Ishikawa, K., Satoh, T. and Ono,K. (1988). Application of immunohistochemistry to theisolated mucosa of the mouse gastrointestinal tract, withspecial reference to somatostatin cells. Acta Anatomica

(Basel), 133, 229^233.Scheuermann, D.W., Stach,W. andTimmermans, J. P. (1987).

Topography, architecture and structure of the plexus sub-mucosus internus (Meissner) of the porcine small intes-tine in scanning electron microscopy. Acta Anatomica

(Basel), 129, 96^104.Sharkey, K. A. and Kroese, A. B. (2001). Consequences of

intestinal in£ammation on the enteric nervous system:neuronal activation induced by in£ammatory mediators.Anatomical Record, 262,79^90.

Sharkey, K. A. and Mawe, G. M. (2002). Neuroimmune andepithelial interactions in intestinal in£ammation. CurrentOpinion in Pharmacology, 2, 669^677.

ARTICLE IN PRESS

Intramural Neurons in Porcine Proliferative Enteropathy 31

Sienkiewicz,W., Kaleczyc, J. and Lakomy, M. (2004). Soma-tostatin-immunoreactive nerve structures in the ileumand large intestine of pigs undergoing dysentery. PolishJournal ofVeterinary Sciences, 7,115^118.

Stanisz, A.M., Befus,D. andBienenstock, J. (1986).Di¡erentiale¡ects of vasoactive intestinal peptide, substance P, andsomatostatin on immunoglobulin synthesis and prolifera-tions by lymphocytes from Peyer’s patches, mesentericlymph nodes, and spleen. Journal of Immunology,136,152^156.

Ten Bokum, A. M., Ho£and, L. J. and Van Hagen, P. M.(2000). Somatostatin and somatostatin receptors in theimmune system: a review. European Cytokine Network, 11,161^176.

Timmermans, J. P., Barbiers, M., Scheuermann, D. W.,Stach,W., Adriaensen, D., Mayer, B. and Groodt-Lasseel,M. H. (1994). Distribution pattern, neurochemical fea-

tures and projections of nitrergic neurons in the pig smallintestine. Annals of Anatomy, 176, 515^525.

Timmermans, J. P., Hens, J. andAdriaensen, D. (2001).Outersubmucous plexus: an intrinsic nerve network involved inboth secretory and motility processes in the intestine oflarge mammals and humans. Anatomical Record, 262,71^78.

Vizi, E. S., Horvath,T. and Somogyi, G.T. (1984). Evidencethat somatostatin releases endogenous substance(s)responsible for its presynaptic inhibitory e¡ect on rat vasdeferens and guinea pig ileum. Neuroendocrinology, 39,142^148.

Yau,W.M., Dorsett, J. A. andYouther,M. L. (1986). Evidencefor galanin as an inhibitory neuropeptide on myentericcholinergic neurons in the guinea-pig small intestine.Neuroscience Letters, 72, 305^308.