Vasoactive intestinal peptide rescues cultured rat myenteric neurons from lipopolysaccharide induced cell death

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Regulatory Peptides 146 (2008) 218 – 223 www.elsevier.com/locate/regpep

Vasoactive intestinal peptide rescues cultured rat myenteric neurons from lipopolysaccharide induced cell death Marcin B. Arciszewski a , Elin Sand b , Eva Ekblad b,⁎ a

b

Department of Animal Anatomy and Histology, Agricultural University, Lublin, Poland Department of Experimental Medical Science, Unit of Neurogastroenterology, BMC B11, Lund University, SE-22184 Lund, Sweden Received 9 May 2007; received in revised form 22 August 2007; accepted 6 September 2007 Available online 19 September 2007

Abstract The role of the enteric nervous system in intestinal inflammation is not fully understood and the plethora of cellular activities concurrently ongoing in vivo renders intelligible studies difficult. In order to explore possible effects of bacterial lipopolysaccharide (LPS) on enteric neurons we utilised cultured myenteric neurons from rat small intestine. Exposure to LPS caused markedly reduced neuronal survival and increased neuronal expression of vasoactive intestinal peptide (VIP), while the expression of Toll-like receptor 4 (TLR4) was unchanged. TLR4 was expressed in approximately 35% of all myenteric neurons irrespective of if they were cultured in the presence or absence of LPS. In neurons cultured in medium, without LPS, 50% of all TLR4-immunoreactive neurons contained also VIP. Addition of LPS to the neuronal cultures markedly increased the proportion of TLR4-immunoreactive neurons also expressing VIP, while the proportion of TLR4 neurons devoid of VIP decreased. Simultaneous addition of LPS and VIP to the neuronal cultures resulted in a neuronal survival comparable to controls. Conclusions: LPS recognition by myenteric neurons is mediated via TLR4 and causes neuronal cell death. Presence of VIP rescues the neurons from LPS-induced neurodegeneration. © 2007 Elsevier B.V. All rights reserved. Keywords: Myenteric neurons; Lipopolysaccharide; Vasoactive intestinal peptide; Enteric nervous system; Inflammatory bowel disease; Irritable bowel syndrome

1. Introduction Innate immunity is the immediate defence mechanism developed to fight invading microorganisms. The gut is constantly exposed to a number of bacteria, both gram positive and negative, and the primary function of the innate defence is to recognise and fight pathogens. Enteric bacterial endotoxins like lipopolysaccharide (LPS) activate an immune response by way of Toll like receptors (TLR) expressed on myeloid cells and epithelia (for recent reviews see [1,2]). The role of bacteria in the development of inflammatory bowel diseases (IBD) is probably important, but enigmatic since IBD is a chronic inflammation in the absence of any identified pathogen. The role of bacteria is also enigmatic in the large and diverse group of patients suffering from irritable bowel syndrome (IBS). A significant number of IBS cases are estimated to be postinfectious [3,4]. Several disease ⁎ Corresponding author. Tel.: +46 46 2220688; fax: +46 46 2224546. E-mail address: [email protected] (E. Ekblad). 0167-0115/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2007.09.021

symptoms e.g. abdominal pain and diarrhoea are shared in IBD and IBS patients, and patients frequently report IBS-like symptoms before developing IBD [5]. The possibility that symptoms like dysmotility and pain, in both IBD and IBS patients, originate in dysregulation in the enteric nervous system (ENS) needs further exploration; several lines of evidence suggests that this is the case (for recent reviews see [6,7]). It has previously been shown that LPS exposure is harmful to porcine myenteric neurons in vitro causing an enhanced neuronal cell death [8]. LPS was, in this study, also found to increase the proportion of neurons expressing vasoactive intestinal peptide (VIP). The present study was undertaken in order to further test LPS effects on cultured myenteric neurons from rat small intestine. The possible presence of the selective LPS receptor Toll-like receptor-4 (TLR4) in myenteric neurons was also explored. Neuronal survival and the expression of VIP and TLR4 were examined by using immunocytochemistry and neuronal cell counting. VIP has been suggested a neuroprotective agent in a number of enteric adaptive and injurious situations [9–11]. The

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expression of VIP is up-regulated and VIP has been shown to promote neuronal survival in cultured myenteric neurons from rat and porcine small intestine [12,13]. Therefore the possibility that VIP may rescue myenteric neurons from LPS-induced cell death was tested. 2. Materials and methods 2.1. Myenteric neuronal cultures Primary cultures of myenteric neurons were prepared from adult female rats (Sprague Dawley, 160–200 g, n = 25), using a modification of previously described methods [12,14]. The Animal Ethics Committee, Lund and Malmö, approved the procedures. The rats were deeply anaesthetised by an i.p. injection of chloral hydrate (30 mg/100 g body weight). The small intestine was exposed via a midline incision, and longitudinal muscle with attached myenteric ganglia was dissected out, using scalpel and small tweezers under aseptic conditions, using a dissection microscope. Approximately 20 cm of the distal small intestine was stripped in this fashion from the serosal side without penetrating the gut thereby avoiding contamination by faecal material. The tissue was placed in ice-cold Krebs solution during preparation, further cut into smaller pieces and two times washed in Ca2+ and Mg2+ free Hanks' balanced salt solution (HBSS), thereafter incubated in HBSS containing collagenase II (1.5 mg/ ml) and protease (1.5 mg/ml) at 37 °C, for 20 min. The vial was vortexed, and the tissue was incubated in trypsin (1.25 mg/ml) for 20 min. The digested tissue was triturated with a Pasteur pipette allowing further mechanical dissociation of the neurons and foetal calf serum (FCS) 50% was added. The cell suspension was centrifuged at 700 rpm for 7 min and washed twice in HBSS. The cell pellet was diluted to 2 ml in Neurobasal A (NBA) supplemented with 10% FCS, 0.5 mM glutamine, and 50 U penicillin and 50 μg streptomycin sulphate per ml, and constantly mixed. Cultures were prepared by seeding 50 μl of the cell suspension together with 950 μl of NBA on laminin precoated glass cover slips (13 mm in diameter) in 4-well dishes (NUNC A/ S, Roskilde, Denmark). The dishes were kept in an incubator (37 °C, 5% CO2). The cultures were grown for 4 or 6 days, in the latter 500 μl of the medium was replaced with fresh medium on the third day. At the end of the culture period the cultures were fixed for 30 min in a mixture of 2% formaldehyde and 0.2% picric acid (Stefanini's fixative) in 0.1 M phosphate-buffer, pH 7.2, followed by rinsing twice in Tyrode's solution containing 10% sucrose, and frozen until being processed for immunocytochemistry. LPS effects on neuronal survival were tested by the addition of 0.1, 2 or 20 μg/ml LPS (E. coli serotype 026:B6) to the culture medium. LPS was present throughout the culture period. Evaluation was by neuronal cell counting after 6 days in culture. Parallel controls were cultured in NBA (supplemented as described above). To test if VIP attenuated LPS-induced neuronal cell loss VIP (10− 6 M) was added together with LPS (0.1 μg/ml) to the neuronal cultures. Evaluation was by neuronal cell counting after 4 days in culture. Parallel controls were cultured in NBA

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(supplemented as described above) with or without addition of VIP (10− 6 M) or LPS (0.1 μg/ml). 2.2. Immunocytochemistry As general neuronal marker antibodies against HuC/HuD (a mouse monoclonal anti-human neuronal protein) were used (code no A-21271; Molecular Probes, Eugene, OR, USA; dilution 1:800; [14]). For the detection of VIP- and TLR4containing neurons a polyclonal VIP antiserum against human synthetic VIP raised in guinea pig (code no B-GP 340-1; EuroDiagnostica AB, Malmö, Sweden; dilution 1:1280; [15]) in combination with a polyclonal antiserum against synthetic peptide sequence, amino acids 796–812, of mouse TLR4 raised in rabbit (code no 222-25-1TLR4C; ImmunoKontact, AMS Biotechnology Ltd, United Kingdom; dilution 1:400) were used in triple staining procedures with anti-Hu. After incubation with the primary antibodies overnight at 4 °C in a moist chamber the preparations were exposed (60 min) to a mixture of fluorescein isothiocyanate conjugated goat anti-mouse IgG antiserum (Jackson Immunoresearch Laboratories, USA; diluted 1:100), Texas Red conjugated swine anti-rabbit IgG antiserum (DAKO, Glostrup, DK; dilution 1:400), and 7-amino-4-methyl coumarin-3-acetic acid conjugated anti-guinea pig antiserum (Jackson Immunoresearch Laboratories, USA; diluted 1:400). After a final rinse in phosphate buffer-saline (PBS) the preparations were mounted and analysed using a fluorescence microscope with appropriate filter settings. In order to assess the specificity of the VIP antibodies inactivation with excess amount of antigen (100 μg of synthetic peptide per ml diluted antiserum) was performed. Since synthetic antigens for HuC/D and TLR4 antibodies are not commercially available omission of primary antibodies was used as control. Controls did not exhibit any immunostaining. 2.3. Chemicals HBSS, Neurobasal A, FCS and penicillin-streptomycin were purchased from GibcoBRL, Life Technologies AB, Täby, Sweden. LPS, collagenase II, protease, trypsin and poly-D-lysine were obtained from Sigma-Aldrich Sweden AB, Sweden. 2.4. Cell counting and statistical analysis The numbers of surviving neurons (i.e. HuC/HuD-immunoreactive cells) after LPS and/or VIP treatment were calculated and expressed in percentage of the control run in parallel. The mean from 3 slides from each animal (n = 5–11 in each group) was determined. The proportions of neurons labelled for VIP, TLR4, or VIP and TLR4 (VIP/TLR4) were estimated by counting cultures triple immunostained with anti-Hu, anti-VIP and anti-TLR4. The following protocol was used: starting from one defined point of the preparation and moving across the slide in a systematic way, at least 100 Hu-immunoreactive nerve cell bodies on each slide were examined for simultaneous staining of VIP and/or TLR4. The results were expressed in percentage

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the neurons survived, as compared to parallel controls cultured in NBA. Simultaneous presence of VIP (10− 6 M) and LPS (0.1 μg/ml) in the culture media resulted in a neuronal survival (90.5 ± 4.2%) similar to that of controls run in parallel with NBA only as culture media. Addition of VIP (10− 6 M), without presence of LPS, resulted in an increased neuronal survival (128.0 ± 6.6%) as compared to controls run in parallel. Results are summarised in Fig. 4. 4. Discussion

Fig. 1. Neuronal survival, expressed as the number of Hu-immunoreactive neurons in % of controls run in parallel, of small intestinal myenteric neurons cultured for 6 days with or without addition of LPS to the media. LPS enhanced neuronal cell death in a concentration dependent manner. ⁎p b 0.01 as compared to controls, #p b 0.01 as compared to LPS 0.1 μg/ml, §p b 0.01 as compared to LPS 2 μg/ml; (n = 5).

of the Hu-immunoreactive cells. The mean from 3 slides from each animal (n = 5 in each group) was determined. Statistical differences were determined using one-way analysis of variance test (ANOVA) followed by Bonferroni's post hoc test.

In the present study we have shown that addition of LPS to cultured myenteric neurons from rat small intestine decreased neuronal survival in a concentration-dependent manner. LPSinduced neuronal cell death has previously been shown to occur also in cultured porcine myenteric neurons [8] as well as in retinal [16] and central neurons [17]. An interesting difference between central and enteric neurons is the present finding of TLR4 expression in a subpopulation of myenteric neurons. LPS-induced neurodegeneration, in vivo as well as in vitro, of central neurons, is generally considered to be by way of activation of microglia. Microglia, but not central neurons, express TLR4 [17,18]. Although an ultrastructural study has suggested the presence of a small population of glia resembling

3. Results 3.1. LPS effects on neuronal survival Presence of LPS during culturing of myenteric neurons enhanced neuronal cell death in a concentration dependent manner (Fig. 1). The highest LPS concentration, 20 μg/ml, caused a reduction in neuronal survival by approx 50%, as compared to controls run in parallel. 3.2. LPS effects on neuronal expression of VIP and TLR4 The proportion of VIP-immunoreactive neurons cultured in control media was 26 ± 2.8% while myenteric neurons cultured in the presence of LPS, all concentrations tested, approximately 34% were VIP immunoreactive (Fig. 2). The proportion of TLR4immunoreactive myenteric neurons was approximately 35% regardless of if LPS was added or not to the culture media (Fig. 2). The proportions of cultured myenteric neurons expressing both VIP and TLR4 (VIP+/TLR4+) and neurons expressing VIP only (VIP + /TLR4 − ) or TLR4 only (VIP − /TLR4 + ) were estimated in 6 days cultures with or without the addition of 2 μg/ml LPS. LPS induced a higher proportion of VIP+/TLR4+ expressing neurons and a lower proportion of VIP−/TLR4+ neurons while no change in VIP+/TLR4− neurons was detected, as compared to controls run in parallel (Fig. 3). 3.3. VIP effects on LPS induced neuronal cell death Culturing myenteric neurons for four days in 0.1 μg/ml LPS caused a reduced number of surviving neurons; 74.0 ± 3.9% of

Fig. 2. Percentage of VIP- (upper panel) and TLR4- (lower panel) immunoreactive (IR) myenteric neurons after culturing for 6 days with or without the addition of LPS to the culture media. Presence of LPS increased the proportion of VIP-IR neurons while the proportion of TLR4-IR neurons was unchanged. ⁎p b 0.01 as compared to controls run in parallel; (n = 5).

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Fig. 3. Upper panel. Proportions of myenteric neurons, cultured for 6 days with or without the addition of 2 μg/ml LPS, expressing VIP and TLR4 (VIP+/TLR4+), TLR4 only (VIP−/TLR+), or VIP only (VIP+/TLR4−). LPS induced a higher proportion of VIP+/TLR4+-IR neurons; a lower proportion of VIP−/TLR+-IR neurons while VIP+/TLR−-IR neurons were unaffected. ⁎p b 0.05 as compared to controls run in parallel without addition of LPS; (n = 5). Lower panel. Micrographs showing myenteric neurons cultured for 6 days and triple immunostained for Hu, VIP and TLR4. A subpopulation of the Hu-IR neurons expressed VIP and TLR4. Magnification X200.

microglia within enteric ganglia [19] microglia is definitely not a prominent cell type in the peripheral nervous system, if present at all. This and, in particular, the finding of an expression of TLR4 in a substantial proportion of the myenteric neurons suggest that LPS directly targets myenteric neurons. The link between LPS recognition by neuronal TLR4 and neuronal cell death is, however, still not revealed. Expression of TLR4 in peripheral neurons is not unique to subpopulations of myenteric neurons since TLR4 expression is also reported to occur in trigeminal nociceptive neurons [20], and in nodose ganglia [21]. The significance of LPS recognition by enteric neurons can only be speculated on at present. Since the ENS is crucial in normal regulation of gastro-intestinal functions the LPS-induced neurodegeneration may play a significant role in the development of gastro-intestinal dysmotility and dysregulation in diseases like IBD and IBS. The aetiology of these two large groups of intestinal disorders is as yet unknown. The cause of IBD has been put forward as due to a defective immune tolerance to the enteric microflora in combination with a genetic predisposition of the affected individuals [2,22]. TLR4 is expressed by human intestinal epithelium, although to a low extent, and TLRs are key mediators in epithelial tolerance to commensal bacteria [22]. From a microanatomical point of view neuronal TLR4 might take part in this response since subpopulations of myenteric neurons issue mucosal projections [23] and thus may well come into contact with luminal TLR ligands. This suggestion is corrobo-

rated by preliminary findings on cryostat sections of whole intestinal wall from normal healthy rats, showing a mucosal innervation of TLR4-immunoreactive nerve fibres extending high up in the mucosa (own unpublished). The possibility of luminal or subepithelial recognition of bacterial products by non-epithelial

Fig. 4. Neuronal survival, expressed as the number of Hu-immunoreactive neurons in % of controls run in parallel, of small intestinal myenteric neurons cultured for 4 days in culture media only (contr), in the presence of LPS (0.1 μg/ ml), LPS (0.1 μg/ml) and VIP (10− 6 M), or VIP (10− 6 M) in the media. Presence of LPS reduced neuronal survival while the simultaneous presence of VIP and LPS resulted in a survival comparable to that of control. Culturing in the presence of VIP only promoted neuronal survival. ⁎p b 0.01 as compared to controls, #p b 0.01 as compared to neurons cultured in the presence of LPS, §p b 0.01 as compared to controls; (n = 11).

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cells is further strengthened by the finding [24] of a direct activation of cultured intestinal myofibroblasts by LPS, as well as of lipoteichoic acid (LTA), illustrating the complexity of the innate response. The concentration-dependent LPS-induced neurodegeneration of myenteric neurons may reflect a hyperexcitation causing cell death. Excitotoxicity is considered to cause a selective cell death through excessive stimulation by excitatory transmitters e.g. glutamate [25]. Glutamate-induced excitotoxicity has been suggested to occur in enteric neurons [26] and, given that LPS exposure sensitises the enteric neurons to glutamate or another excitatory transmitter, this may thus be a putative mechanism. A marked enteric neuronal cell loss is reported to occur in experimentally induced colitis [27–29]. The mechanisms behind have been ascribed as, at least partly, mediated by neutrophils [28,29] or as non-specific [7]. The here presented findings contradict that enteric neuronal cell death is secondary to the inflammation. We suggest that neuronal plasticity and neurodegeneration are central components of intestinal inflammation since a subpopulation of myenteric neurons are directly targeted by LPS. This further strengthens and highlights the assumption that neurodegenerative changes of the ENS are linked to the altered gut functions experienced in IBD. Findings revealing such links may open up new possible targets for pharmacological interventions. Electrophysiological recordings from enteric neurons in colitis models have revealed a hyperexcitability mainly in the type of enteric neurons designated as AH neurons. Such neurons comprise intrinsic sensory neurons. However, also subpopulations of neurons belonging to the class of S neurons are affected; S neurons comprise inter- and motor neurons (For a recent review see [30]). The mechanisms behind and the cause of such alterations in enteric neurophysiology are unknown but the possibility that they are induced by neuronal LPS exposure needs further exploration. If so, this may also provide an explanation to the high incidence of post infectious IBS. IBS is characterised by an increased visceral sensitivity and neuronal hypersensitivity is probably a crucial event in the generation of symptoms like hyperalgesia and diarrhoea [6]. The finding that presence of LPS during culturing of myenteric neurons increased the proportion of VIP+/TLR4+immunoreactive neurons, while the proportion of VIP−/TLR4+immunoreactive neurons decreased, is of interest. VIP has been put forward as a neuroprotective neurotransmitter in the enteric [11,12] as well as in the central nervous system [31]. VIP has also been found to attenuate the inflammatory response in intestinal inflammation [32,33]. The higher proportion of VIPimmunoreactive neurons after culturing in the presence of LPS may be due either to the fact that LPS recognition per se induces an increased expression of VIP or that VIP-negative neurons are more vulnerable. The readiness by which VIP expression is upregulated in enteric neurons in a number of injurious situations [10,11,14], including intestinal inflammation [34] and LPS treatment [8] speaks in favour of the first suggestion. However, a combined action of the two suggested events is to be regarded. Notable is that presence of LPS did not seem to induce any increased expression of TLR4 in cultured myenteric neurons as has been found to be the case in LPS-exposed myofibroblasts

[24]. In the present study this was, however, judged by estimating the proportion of TLR4-immunoreactive myenteric neurons; a qualitative, and not a quantitative method. Culturing myenteric neurons in the presence of a low dose of LPS reduced neuronal survival to approximately 75%. Simultaneous presence of VIP rescued the neurons. Administration of VIP to the neuronal cultures caused per se an increased survival, as has been previously demonstrated [12]. The mechanisms behind the neuroprotective effect of VIP on LPSinduced neuronal cell death are at present unknown. VIP has in a number of systems been suggested to act as an immunomodulator in both innate and adaptive immune responses. It inhibits the production of proinflammatory cytokines [33] and causes down-regulation of both TLR2 and TLR4 expressions [35]. VIP also exerts neuroprotective effects in non-inflammatory insults, as has been shown to be the case in VIP-induced protection against excitotoxic white matter damage in newborn mice [36]. If VIP, in the present study, rescues enteric neurons from LPSinduced cell death in a general way, due to its ability to promote neuronal survival, or by interacting with LPS binding or LPSinduced secondary signalling mechanism is at present not possible to answer. The activation of VPAC1 receptors, and several different transduction pathways and transcription factors has been described to be involved in the anti-inflammatory effects of VIP exerted on macrophages [37]. Further studies on myenteric neurons aiming at e.g. identifying by which receptor the VIP effects are mediated and, not least, clarifying by which mechanisms the LPS-induced neurodegeneration is executed are needed. The significance of results generated in various in vitro systems to the in vivo situation must by all means be questioned. With regard to studies on intestinal inflammation a number of experimental in vivo models exist ranging from transgenic animals lacking crucial genes like interleukin-10 and methods using adoptive transfer of activated lymphocytes to exposure by agents like trinitrobenzene sulfonic acid (TNBS) or dextran sulphate sodium (DSS), and irradiation (for a review see [38]). All of these methods produce robust inflammatory reactions; some also manifest extragastrointestinal symptoms. These models are valuable and have, together with clinical studies on patients, generated the bulk of current knowledge in the field. However, when it comes to the elusive neuroimmune mechanisms the array of inflammatory mediators released and the diversity of simultaneous ongoing cellular activities make it difficult to extract in depth information from the above-mentioned in vivo systems. In that respect the use of in vitro models aiming to unravel detailed mechanisms like cellular and pharmacological interactions are necessary. The in vitro model system used in the present study was primary culturing of myenteric neurons from rat small intestine. This model has previously been characterised in terms of neurotransmitter expression and neuronal survival [12,14] and has been found to contain dissociated neurons that gradually coalesce to form ganglionic like formations. The neurons grow extensive varicose nerve terminal networks and are supported by a sizeable supportive network of glial fibrillary acidic protein (GFAP) positive enteric glia. The cultures contain no immune cells and thus direct effects of e.g. LPS may be tested. Studies on

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survival of enteric neurons in response to agents like LPS or in inflamed tissue are almost impossible to accurately interpret in vivo. This is mainly due to the fact that intestines are hollow organs with a high capability of adaptation. Intricate morphometry taking into account possible intestinal changes in wall thickness, length as well as of circumference are needed in order to correctly compare control versus treated or inflamed intestine. Thus to ultimately reveal all aspects on inflammatory mechanisms a combination of knowledge generated from both in vivo and in vitro methods are needed. Few in vitro models, rendering studies on the neuronal contribution and consequences in intestinal inflammation possible, exist. Primary neuronal culturing has been extensively used to reveal inflammatory mechanisms in central neurons and now we introduce a similar approach in order to uncover the enteric neuronal involvement in IBD and IBS. Acknowledgements This work was supported by the Swedish Medical Research Council (project no K2005-72X-13406-06A), the Swedish Animal Welfare Agency (project no 2005-2277), Lund University Medical Faculty and the Ihre, 9-m-liv, and Crafoord Foundations. References [1] Cario E, Podolsky DK. Toll-like receptor signalling and its relevance to intestinal inflammation. Ann NY Acad Sci 2006;1072:332–8. [2] Harris G, KuoLee R, Wangxue C. Role of toll-like receptors in health and disease of gastrointestinal tract. World J Gastroenterol 2006;12:2149–60. [3] Gwee KA, Graham JC, McKendrick MW, Collins SM, Marshall JS, Walters JS, Read NW. Psychometric scores and persistence of irritable bowel after infectious diarrhoea. Lancet 1996;347:150–3. [4] Spiller RC. Inflammation as a basis for functional GI disorders. Best Pract Res Clin Gastroenterol 2004;18:641–61. [5] Bercik P, Verdu EF, Collins SM. Is irritable bowel syndrome a low-grade inflammatory bowel disease? Gastroenterol Clin North Am 2005;34:235–45. [6] Grundy D, Al-Chaer ED, Aziz Q, Collins SM, Ke M, Taché Y, Wood JD. Fundamentals of neurogastroenterology: basic science. Gastroenterology 2006;130:1391–411. [7] Vasina V, Barbara G, Talamonti L, Stanghellini V, Corinaldesi R, Tonini M, De Ponti F, De Giorgio R. Enteric neuroplasticity evoked by inflammation. Auton Neurosci 2006;126–127:264–72. [8] Arciszewski MB, Pierzynowski S, Ekblad E. Lipopolysaccharide induces cell death in cultured porcine myenteric neurons. Dig Dis Sci 2005;50:1661–8. [9] Ekblad E, Mulder H, Sundler F. Vasoactive intestinal peptide expression in enteric neurons is upregualted by both colchicine and axotomy. Regul Pept 1996;63:113–21. [10] Ekblad E, Sjuve R, Arner A, Sundler F. Enteric neuronal plasticity and a reduced number of interstitial cells of Cajal in hypertrophic rat ileum. Gut 1998;42:836–44. [11] Ekblad E, Bauer AJ. Role of vasoactive intestinal peptide and inflammatory mediators in enteric neuronal plasticity. Neurogastroenterol Motil 2004;16:123–8. [12] Sandgren K, Lin Z, Fex-Svenningsen Å, Ekblad E. Vasoactive intestinal peptide and nitric oxide promote survival of adult rat myenteric neurons in culture. J Neurosci Res 2003;72:595–602. [13] Arciszewski MB, Ekblad E. Effects of vasoactive intestinal peptide and galanin on survival of cultured porcine myenteric neurons. Regul Pept 2005;125:185–92. [14] Lin Z, Sandgren K, Ekblad E. Increased expression of vasoactive intestinal polypeptide in cultured myenteric neurons from adult rat small intestine. Auton Neurosci 2003;107:9–19.

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