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PARADOXICAL EFFECTS OF IMMUNE CELLS ON THE ENTERIC NERVOUS SYSTEM IN INTESTINAL INFLAMMATION by Shriram Venkataramana A thesis submitted to the Department of Physiology In conformity with the requirements for the degree of Master of Science Queen‘s University Kingston, Ontario, Canada (November, 2009) Copyright © Shriram Venkataramana, 2009 Abstract Inflammatory bowel disease causes structural and functional alterations in the enteric nervous system (ENS). Since the onset of intestinal inflammation involves the activation of resident immune cells as well as rapid influx of infiltrating cells, we proposed that changes in the ENS are a result of the release of toxic inflammatory factors. We hypothesized that early damage to the ENS in inflammation is caused by harmful levels of nitric oxide (NO) generated by the enzyme inducible nitric oxide synthase (iNOS) found in immune cells. This was assessed in the 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-model of colitis in rats. Large increases in infiltrating granulocytes, particularly neutrophils and blood-derived monocytes were found in the muscularis layers adjacent to the ENS. A rapid increase in iNOS immunoreactivity in the muscularis regions during early stages of inflammation (6 – 24 hr) was observed. Whether high NO levels generated by chemical donors could be toxic to neurons was tested in a co-culture model of myenteric neurons, smooth muscle and glia enzymatically isolated from neonatal rats. Exposure of co-cultures to NO for 48 hr resulted in significant, concentration dependent decrease in neuron survival. We then developed a model that permitted the direct study of immune cell interactions with myenteric neurons. Myenteric neurons were co-cultured with activated peritoneal immune cells that expressed iNOS and generated high NO levels (49 + 6.2µM) for 48 hr. This caused significant neuronal death, reducing neuron number by 19 + 5%, and disruption of axons. Pre-treatment of immune cells with a selective iNOS-inhibitor, L-NIL resulted in neuron numbers that were not significantly different from control (96 + 2%) suggesting that NO played a central role in mediating the damaging effects of immune cells. Lastly, when direct contact between immune cells and neurons was ii prevented in the previous experiment through use of trans-wells, unanticipated neurotrophic effects were observed. Increased axon outgrowth (282 + 57%) was detected in addition to loss of the neurotoxic effects in spite of similar experimental conditions. We concluded that proximity and contact plays an important role in determining the nature of immune cell mediated alterations in enteric neurons. iii Acknowledgements I would like to express my deepest gratitude to Dr. Michael Blennerhassett. Thank you for providing me the opportunity to mature as a scientist under your guidance. Your genuine enthusiasm for research inspired me to pursue an MSc in your lab, and your exceptional guidance has made it a truly rewarding experience. Additionally, I would also like to thank my committee members Dr. Andrew Craig and Dr. Alan Lomax for their helpful suggestions. I would also like to acknowledge the wonderful people I have had the privilege of working with in this department. In particular, Dr. Sandra Lourenssen and Roger Stanzel for their assistance in teaching me the various techniques I employed throughout my degree. I would also like to thank other members of the Blennerhassett lab, especially Dr. Pierre-Yves Gougeon and Kurtis Miller for their guidance and support. And lastly, I would like express my gratitude to my family and friends. To my parents, thank you for your love and constant words of encouragement. Without your unwavering support, this dissertation would have been impossible. The work presented in this thesis could not have been completed without funding provided by the Crohn‘s and Colitis Foundation of Canada and the CIHR Training Grant awarded to Dr. Michael G Blennerhassett. iv Table of Contents Abstract ............................................................................................................................... ii Acknowledgements ............................................................................................................ iv Table of Contents .................................................................................................................v List of Figures ................................................................................................................... vii List of Abbreviations ........................................................................................................ viii Chapter 1 Introduction ........................................................................................................ 1 1.1 Characterizing intestinal injury using animal models of IBD .................................... 1 1.2 Detrimental role of immune cells in intestinal inflammation................................... 6 1.2.1 Nitric Oxide as a potential neurotoxic agent ........................................................7 1.2.2 NO inhibition in animal models of inflammation .............................................. 9 1.3 A neurotrophic role for immune cells in IBD? ......................................................... 11 1.3.1 Role of cytokines in axon regeneration .............................................................. 13 1.3.2 Cytokines stimulate secretion of neurotrophic factors ...................................... 13 1.3.3 Novel neurotrophic factors secreted by macrophages ....................................... 14 1.4 Conclusions and hypotheses..................................................................................... 15 Chapter 2 Materials and Methods ..................................................................................... 17 2.1 Animals ..................................................................................................................... 17 2.2 TNBS-Induced Colitis .............................................................................................. 17 2.3 MPO Assay ...............................................................................................................18 2.4 DAB Histochemistry ................................................................................................18 2.5 Isolation of Peritoneal Immune Cells ...................................................................... 19 2.6 NADPH-Diaphorase Labelling ................................................................................. 19 2.7 Griess Reaction ......................................................................................................... 19 2.8 Tissue culture .......................................................................................................... 20 2.9 NO Addition ............................................................................................................. 21 2.10 Immune cells + Neuron co-culture studies ............................................................ 21 2.11 Immunocytochemistry ........................................................................................... 23 2.12 Determination of neuron and axon number ......................................................... 24 2.13 Statistical Analysis ................................................................................................. 24 Chapter 3 Results .............................................................................................................. 25 3.1 Neurotoxic effects of immune cells ......................................................................... 25 v 3.1.1 Increased presence of infiltrating macrophages and neutrophils in proximity to the ENS ...................................................................................................................... 25 3.1.2 iNOS expression is upregulated in the early stages of TNBS-induced intestinal inflammation ............................................................................................................. 30 3.1.3 NO is cytotoxic for myenteric neurons in vitro ................................................. 32 3.1.4 NO toxicity is specific to neurons; ISMC and glia are unaffected .................... 38 3.1.5 A co-culture model of immune cells and myenteric neurons ........................... 40 3.2 Neurotrophic Effects of Immune cells .................................................................... 45 3.2.1 Activated immune cells exert neurotrophic effects on myenteric neurons in coculture ........................................................................................................................ 45 Chapter 4 Discussion ........................................................................................................ 47 4.1 Rapid influx of infiltrating immune cells in TNBS-induced colitis ......................... 48 4.2 Activated immune cells damage cultured myenteric neurons using NO mediated mechanisms................................................................................................................... 50 4.3 Immune cells on trans-wells: .................................................................................. 54 4.4 Conclusions ............................................................................................................. 56 References ..........................................................................................................................57 vi List of Figures Figure 1: Schematic representation of a transverse section through the intestine ............ 5 Figure 2: Schematic representation of co-culture protocol .............................................. 22 Figure 3: Early increase in the number of infiltrating macrophages in inflamed colonic tissue in TNBS-induced colitis .......................................................................................... 27 Figure 4: Marked increase in the number of infiltrating neutrophils in inflamed colonic tissue in TNBS-induced colitis .......................................................................................... 29 Figure 5: iNOS expression is upregulated in the early stages of TNBS-induced colitis .... 31 Figure 6: Representative immunofluorescent images of myenteric neurons .................. 33 Figure 7: Chemical NO donors decrease enteric neuron survival in vitro ....................... 36 Figure 8: Selective loss of neurons caused by SNP – visualized by PI staining ............... 39 Figure 9: iNOS expression and NO release by activated peritoneal immune cells ........... 41 Figure 10: Direct addition of activated immune cells causes loss of myenteric neurons . 44 Figure 11: Indirect addition of activated immune cells causes neurotrophic effects ....... 46 vii List of Abbreviations APTEX 3-aminopropyl-triethoxysilane ATP adenosine triphosphate Calcein AM acetomethoxy derivative of calcein cAMP cyclic adenosine monophosphate CSM circular smooth muscle layer CNS central nervous system DAB diaminobenzidine DETA-NONOate diethylenetriamine NONOate DMEM dulbecco‘s modified Eagle medium DNBS dinitrobenzene sulfonic acid DRG dorsal root ganglion ED1 infiltrating macrophages; rat homologue of human CD68 ED2 resident macrophages; rat homologue of human CD163 eNOS endothelial nitric oxide synthase ENS enteric nervous system FCS fetal calf serum GDNF glial cell-line derived neurotrophic factor GFAP glial fibrillary acidic protein GI gastrointestinal tract Hanks‘ Hanks' balanced salt solution HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid IBD inflammatory bowel disease IBS inflammatory bowel syndrome IFNγ interferon gamma IL-1 interleukin 1 iNOS inducible nitric oxide synthase ISMC intestinal smooth muscle cell kD kiloDalton L-NAME L-NG-Nitroarginine methyl ester L-NIL L-iminomethyl lysine viii L-NMMA L-N-monomethyl arginine citrate LPS lipopolysaccharide MPO myeloperoxidase NADPH nicotinamide adenine dinucleotide phosphate NBF neutral buffered formalin NGF nerve growth factor nNOS neuronal nitric oxide synthase NO nitric oxide NOS nitric oxide synthase OCT optimal cutting temperature ONOO- peroxynitrite O2- superoxide anion PBS phosphate buffered saline PI propidium iodide PVDF polyvinylidene fluoride RNOS reactive nitric oxide species ROS reactive oxygen species SM/MP smooth muscle and myenteric plexus layers SNAP S-nitroso-amino-penicillamine SNAP25 synaptosome-associated protein 25kD SNP sodium nitroprusside TNBS 2, 4, 6-trinitrobenzene sulfonic acid TNFα tumor necrosis factor alpha TNP trinitrophenyl VIP vasoactive intestinal peptide ix Chapter 1 Introduction Inflammatory bowel disease (IBD) is a chronic incurable disease that damages the gastrointestinal tract of affected individuals. It is largely a disease of the industrialized world, especially North America and Europe, and is more common in urban areas and northern climates (Loftus, Jr. & Sandborn, 2002;Hanauer, 2006). There are two major types of IBD, Crohn‘s and ulcerative colitis, both of which have an unknown etiology. Ulcerative colitis results in characteristic ulceration of the colon and rectum and typically involves only the innermost lining or mucosa, manifesting as continuous areas of inflammation (Head & Jurenka, 2003). In contrast, Crohn‘s disease can affect any part of the gastrointestinal tract from mouth to anus, although it most commonly affects the distal ileum (Head & Jurenka, 2004). It is characterized by skip lesions - areas of transmural inflammation with segments of normal lining in between (Sartor, 1995). Although the exact cause of IBD remains undetermined, the disease appears to be related to a combination of genetic and environmental factors. While IBD has a known genetic component, with 25% of Crohn‘s and 20% of ulcerative colitis patients having a family member with some form of IBD, this falls well short of predicting its occurrence (Head & Jurenka, 2004). Whether these are fundamentally different diseases or part of a mechanistic continuum remains an unanswered question. 1.1 Characterizing intestinal injury using animal models of IBD Although the cause of IBD is unknown, there is evidence that it involves interactions between the immune system, genetic susceptibility, and the environment 1 (Hanauer, 2006). Experimental animal models allow study of early events, permitting dissection of the interactions among different components and genes that determine susceptibility in ways that are not possible in humans. These models of intestinal inflammation can be divided into three main categories: inducible colitis models in animals with a normal immune system, adoptive transfer models in immunocompromised hosts, and genetically engineered knockouts and transgenic mice (Hibi et al., 2002;Elson et al., 2005). Among these, the 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced model of colitis has been extensively used to characterize the changes that occur in intestinal inflammation. This model involves the intra-rectal administration of the contact sensitizing hapten TNBS, which causes delayed hypersensitivity reactions by haptenating mucosal proteins with trinitrophenyl (TNP) groups, rendering such proteins immunogenic (Elson et al., 1996). This results in a cell-mediated immune response consistent with a type I helper T-lymphocyte. Furthermore, the exposure of infiltrating immune cells to ubiquitous mucosal antigens i.e., antigens that exist in the bacterial microflora, results in inflammation that persists even after the TNP-haptenated proteins have disappeared (Elson et al., 1996). Studies using the TNBS model of induced colitis demonstrated that the intestine undergoes significant structural and functional changes over the course of inflammation (Wirtz & Neurath, 2000). TNBS-induced inflammation is characterized by ulceration, necrosis of epithelial tissue, and increased permeability of the mucosa (Morris et al., 1989). In addition to the changes in mucosa, the muscularis layers are also significantly affected. A profound thickening of the intestinal wall is observed, which is a result of significant hyperplasia 2 and hypertrophy of the intestinal smooth muscle cells (ISMC) (Blennerhassett et al., 1999). Numerous groups have demonstrated that TNBS-induced colitis causes striking alterations to the enteric nervous system (ENS) (Poli et al., 2001;Boyer et al., 2005;Linden et al., 2005). The ENS has often been described as the ‗brain of the gut‘ and comprises of neurons and glia that are found in the wall of the gut. The neurons of the ENS are collected into two types of ganglia: myenteric (Auerbach's) and submucosal (Meissner's) plexuses (Fig. 1A, B – obtained from Smout et al, 1992; (Heanue & Pachnis, 2007). Neurons in the myenteric plexus are located between the inner and outer layers of the muscularis externa, while those of the submucosal plexus are located in smaller ganglia within the submucosa. Dinotrobenzene sulfonic acid (DNBS) induced colitis in rats, a model similar to TNBS-induced colitis, caused significant neuronal loss in the inflamed region by 24 hours with only 49% of neurons remaining by days 4 to 6 and thereafter, when inflammation had subsided (Sanovic et al., 1999) (Fig. 1C – obtained and modified with permission from Sanovic et al, 1999). Furthermore, Poli et al observed significant changes in the architecture of the myenteric plexus of TNBS-treated rats that closely paralleled the severity of lesions (Poli et al., 2001). Major reductions in neuronal marker expression were observed in seriously damaged areas of colons, further confirming previous studies showing that different populations of enteric nerves (cholinergic, adrenergic, nitrergic, VIPergic and tachykinergic) are strongly affected by inflammatory processes (Kimura et al., 1994;Mizuta et al., 2000a). Linden et al. reported an indiscriminant loss of about 20% of myenteric neurons in guinea pigs, most of which occurred within 12 hr of TNBS-administration (Linden et al., 2005). In mouse DNBS-induced colitis, a 60% decrease in the number of myenteric neurons was detected 3 in whole mount tissue while a smaller reduction in cell numbers (40%) was detected in cross-sections (Boyer et al., 2005). 4 Figure 1 A B Source – Heanue and Pachnis, 2007 Nature Reviews| Neuroscience D PGP Axons/section Neuron number (/mm) C Source – Smout et al, 1992 Wrightson Biomedical Publishing, Limited Timeline of TNBS Colitis Acute Phase ENS Damage Reinnervation Resolution Fig 1. A-B: Schematic representation of a transverse section through the intestine. Enteric neurons are organized into ganglia (grey) found within two main plexi. The myenteric (Auerbach‘s) plexus occupies a position between the longitudinal and circular muscle layers. An inner submucosal (Meissner‘s) plexus resides within the submucosa (Source – Heanue and Pachnis, 2007). C-D: Pattern of neuron loss and axon changes in chemically induced colitis. (C) Reduction in neuron number is observed in the early stages of inflammation in the DNBS-model of colitis in rats (Source – Sanovic et al, 1999). (D) TNBS induced colitis causes a transient decrease in the number of axons innervating the circular smooth muscle, followed by an increase that persists post-resolution of inflammation (Source – Lourenssen et al, 2005). 5 That transient or even permanent structural and functional alterations in the ENS occur in IBD is supported by a number of observations. In patients with IBD, alterations in enteric ganglia, such as neuronal hypertrophy and hyperplasia are regularly reported (Geboes & Collins, 1998). Other structural abnormalities including ganglion cell and axonal degeneration and necrosis have also been observed in IBD (Steinhoff et al., 1988;Brewer et al., 1990;Dvorak et al., 1993). It appears that the nature of the alterations of the ENS appear to be dependent on the disease (Crohn‘s vs. ulcerative colitis), the region of the intestinal wall and whether or not the tissue was from a site of active inflammation, but it is difficult to interpret these results because they involved evaluation of cross-sections and the time course of the disease process was unclear. Nevertheless, the substantial impact of intestinal inflammation on the ENS may be involved in the alterations to sensory perception, gastric emptying, orocecal transit and intestinal motility that have been observed in animals as well as patients with IBD (Collins, 1996;Wells & Blennerhassett, 2004).More importantly, most of these changes represent long-lasting or even permanent alterations to the intestinal wall, which may contribute to post-inflammatory conditions such as irritable bowel syndrome (IBS). 1.2 Detrimental role of immune cells in intestinal inflammation The inappropriate activation of the immune system is known to play a major role in the inflammatory process. The onset of intestinal inflammation involves the activation of resident immune cells as well as the rapid influx of infiltrating cells, and there is a corresponding increase in the levels of inflammatory mediators in the inflamed mucosa and muscularis layers (James & Klapproth, 1996). Once activated by pro-inflammatory stimuli, immune cells display substantial changes in protein synthesis that contribute to inflammation. For example, activated neutrophils and macrophages are known to 6 synthesize proinflammatory cytokines like tumor necrosis factor-α (TNF-α), interleukin1 (IL-1) and interferon-γ (IFN-γ) and reactive metabolites of nitrogen and oxygen such as nitric oxide (NO), superoxide radical (O2-), peroxynitrite (ONOO-)among others (Papadakis & Targan, 2000). In patients with IBD, the infiltration by immune cells into the intestinal mucosa was found to be a prominent cause of tissue injury and clinical manifestations of the disease (Foell et al., 2003). Furthermore, animal studies using the TNBS model of colitis demonstrated that inhibition of neutrophil activation or depletion of the circulating neutrophils is beneficial to the mucosa and deeper layers of the intestine (Wallace et al., 1998). The application of a topical steroid budesonide in a DNBS model in rats achieved a dose dependent reduction of neuron loss, providing further evidence for direct involvement of immune cells in causing neuron loss. In a clinical setting, the antiinflammatory effects of corticosteroids in the treatment of IBD have been ascribed at least in part to their ability to reduce neutrophil infiltration (Cronstein et al., 1992). Similarly, beneficial effects of the commonly used drug 5-aminosalicylic acid have been attributed to its ability to inhibit the formation of reactive free radicals from granulocytes (Cronstein et al., 1992). Taken together, these studies suggest that the early tissue damage observed in intestinal inflammation can be ascribed to proinflammatory mediators released by infiltrating immune cells. 1.2.1 Nitric Oxide as a potential neurotoxic agent The observation that loss of enteric neurons may be associated with the infiltration of activated immune cells at the early stages of intestinal inflammation led us to consider immune cell-generated factors as a possible cause of ENS damage. These 7 inflammatory mediators might act directly on the ENS or induce the release of proinflammatory cytokines from the neighboring cells. We proposed that the early damage to the ENS involving both the loss of axons and neurons is due to the effects overproduction of NO by immune cells. The activation of neutrophils and macrophages by either bacterial lipopolysaccharide (LPS), cytokines, or oxidative stress results in the synthesis of the enzyme inducible nitric oxide synthase (iNOS) (Vallance et al., 2004). De novo synthesis of the iNOS protein occurs as a result of the re-programming of gene expression via various transcriptional factor pathways, chief among which is the nuclear factor-κβ (NF- κβ)-pathway (Kleinert et al., 2004). Unlike the constitutive isoforms of NOS – endothelial and neuronal NOS (eNOS and nNOS, respectively), which generate nanomolar quantities of NO, iNOS maintains micromolar concentrations of NO synthesis for hours or days depending on how long the enzyme is present in the cells (Nussler & Billiar, 1993;Nathan & Xie, 1994). At such concentrations, NO rapidly reacts with other free radicals to form reactive nitrogen oxide species (RNOS) that mediate most of the effects of iNOS-derived NO (Kubes & McCafferty, 2000). Peroxynitrite is one such product of free radical reactions, formed as a result of NO oxidation by O2-. It can react with all the major classes of biomolecules and, therefore, has the potential to mediate cytotoxicity independently of NO or O2- (Beckman & Koppenol, 1996). RNOS can S-nitrosate thiols to modify key signaling molecules such as kinases and transcription factors. Several key enzymes in mitochondrial respiration are also inhibited by RNOS and this leads to a depletion of ATP and cellular energy (Beckman et al., 1990). These actions of NO do not occur through a receptor—its target cell specificity depends on the concentration, chemical reactivity, nature of the neighboring cells and the way that they are programmed to respond. A combination of 8 these interactions may explain the tissue damage and alterations in morphology and function, some of the characteristic features of intestinal inflammation. Several studies have identified increased proportions of iNOS expressing cells in inflamed intestines in both animal models and patients with ulcerative colitis. Inflamed mucosal tissue shows a wide variety of iNOS expressing cells and correspondingly elevated NO concentrations (McCafferty et al., 1999). The muscularis layers also show increased presence of infiltrating immune cells in addition to the dense network of resident muscularis macrophages (Hori et al., 2008). The presence of such high levels of NO producing cells in the vicinity of the submucosal and myenteric plexuses led us to propose that NO is responsible for the ENS damage. 1.2.2 NO inhibition in animal models of inflammation The potential role of NO as a mediator of inflammation in the gut has fuelled many studies in animal models of IBD, using NOS inhibitors and iNOS-/- animals to address the role of iNOS-derived NO in these experimental settings. Early studies, which used nonspecific NOS isoform inhibitors such as L-NG-Nitroarginine methyl ester (LNAME), have produced ambiguous results (Miller et al., 1993;Grisham et al., 1994;Hogaboam et al., 1995;Kiss et al., 1997). For example, Miller et al demonstrated the beneficial effects of NOS inhibition on the mucosa using L-NAME in a TNBS-model of ileal inflammation (Miller et al., 1993). Whether these protective effects of NOS inhibition extended to the deeper neuromuscular layers of the intestine was addressed by oral administration L-NAME shortly after the induction of colitis. This therapy significantly attenuated epithelial permeability, granulocyte infiltration, ISMC hyperplasia and a marked reduction in damage to NADPH-diaphorase containing nerves 9 in the myenteric plexus (Miller et al., 1993;Hogaboam et al., 1995;Kiss et al., 1997). Since iNOS is the predominant NO generating isoform at this stage in inflammation, the decreased macroscopic damage could be a result of attenuation of iNOS activity. Kiss et al conducted a series of critical experiments attempting to elucidate the role played by iNOS. Administration of L-NAME 6 hr after TNBS injection caused reduction in iNOS activity and macroscopic lesions. In contrast, pre-administration of L-NAME (2 days prior to TNBS injection) was found to augment macroscopic damage and increased colonic iNOS activity at 6, 12, 24 and 72 hr post induction of colitis (Kiss et al., 1997). Such pre-treatment with L-NAME would lead to an inhibition of constitutive NO, known to be protective in the intestinal mucosa, thereby exacerbating the subsequent damage following challenge (Lopez-Belmonte & Whittle, 1995). In contrast, when the administration of L-NAME is delayed until the time of iNOS expression, colonic injury and inflammation is reduced. Since early work in the field of NOS inhibitors used compounds such as L-NAME, L-N-monomethyl arginine citrate (L-NMMA) and other L-arginine analogs, they inadvertently inhibited constitutive NOS (eNOS and nNOS) which are critical to regulating tissue perfusion, microvascular permeability, and platelet and leukocyteendothelial interactions (Miller et al., 1993;Grisham et al., 1994;Hogaboam et al., 1995;Rachmilewitz et al., 1995a;Cross & Wilson, 2003). Therefore, recent work has focused on using more selective iNOS inhibitors like aminoguanidine and L-iminomethyl lysine (L-NIL) (Ribbons et al., 1995). Yamaguchi et al showed that administration of aminoguanidine in TNBS-induced colitis prevented weight loss, decreased colonic damage scores and myeloperoxidase activity (Yamaguchi et al., 2001). The use of L-NIL 10 in other inflammatory models showed some benefits such as reduced diarrhea but did not alter the morphologic features of the disease (Ribbons et al., 1995). The development of iNOS deficient mice provided an elegant model to study its importance in intestinal inflammation. McCafferty et al reported that in acetic acid induced colitis, a model of acute mucosal injury, the iNOS-deficient mice healed less effectively than their wild-type counterparts (Yamasaki et al., 1998;McCafferty et al., 1999;Zingarelli et al., 1999). This indicates that iNOS may offer some protection in the early phase of the acute, non-specific chemically induced colitis. On the other hand, in a more chronic model of colitis, Zingarelli et al reported that genetic ablation of iNOS suppressed nitrosative and oxidative damage and resulted in a significant resolution of mice with TNBS colitis in comparison to controls (Zingarelli et al., 1999). In light of the above studies, it is apparent that NO might have a dichotomous function as both a beneficial and detrimental molecule in the gut. In numerous models of intestinal inflammation, inhibition of NO increased tissue dysfunction and injury, whereas in other models, inhibition of NO proved beneficial (Miller et al., 1993;Kubes & McCafferty, 2000). The role of constitutive and inducible NO production during healing is currently under investigation, while the reliability of existing models for the experimental evaluation of healing processes is still questionable. 1.3 A neurotrophic role for immune cells in IBD? Numerous studies have reported decreased axon numbers in the intestine in the early stages of TNBS-induced colitis (Sanovic et al., 1999;Lourenssen et al., 2002;Lourenssen et al., 2005). The examination of axons within the smooth muscle layers at later time points to determine whether axonal growth had occurred to 11 compensate for the initial loss revealed interesting results. By Day 6 of colitis and thereafter, a striking and consistent increase in the number of axon profiles per section was observed, to a level more than double that measured in control tissue, and four times the lowest values seen earlier in the time course. The values on Day 25 and Day 35 post colitis were similar, and both were significantly greater than control (p<0.05). This suggested that the axonal number had stabilized at the increased level, after a period of expansion over Days 4 and 6 of TNBS-colitis that occurred despite the presence of continued severe inflammation (Lourenssen et al., 2005) (Fig. 1D – obtained and modified with permission from Lourenssen et al, 2005). We proposed that while infiltrating immune cells cause the initial damage, they are also responsible, either directly or indirectly, for the compensation for the initial loss by increasing neurite outgrowth. The idea that inflammation and, more specifically, factors secreted from macrophages might enhance neuron survival and promote axonal regeneration arose over a decade ago. Resident and infiltrating immune cells present in the inflamed tissue generate a large number of cytokines and growth factors in addition to the potentially toxic NO. For example, a study demonstrated that injection of macrophages into dorsal root ganglia could enhance axon outgrowth (Lu & Richardson, 1991). It was suggested that macrophages migrate to the damaged area of the peripheral nervous system and participate in the removal of axon and myelin debris, stimulate the proliferation of neighboring non-neuronal cells and augment the synthesis of growth factors and promote neovascularization (Lu & Richardson, 1991). The exact process by which macrophages induce axon regeneration, and the specific factors involved in it are currently an area of intense research. 12 1.3.1 Role of cytokines in axon regeneration There are several possible mechanisms that could contribute to the neurotrophic environment observed in the later stages of intestinal inflammation. Cytokines have been thought to play an important role in neural development and repair in the CNS. Tissue levels of the proinflammatory cytokine IL-1ß are known to increase within 24 hr of nerve injury increased within 24 hr of nerve injury and remain high for days (Stoll & Muller, 1999). Further evidence for its involvement in repair was found when the application of an IL-1 receptor antagonist impeded peripheral nerve regeneration (Guenard et al., 1991). Another study demonstrated that application of exogenous TNF-α has regenerative effects on CNS axons post injury (Schwartz et al., 1991). These studies suggest that ‗cytotoxic‘ cytokines released by macrophages can have diverse consequences on the inflamed tissue, which might even extend to neurotrophic effects. Further research is needed to identify whether cytokines enhance axon regeneration through direct effects on the neuronal cell bodies or by acting on the accessory cells in the vicinity of neurons. 1.3.2 Cytokines stimulate secretion of neurotrophic factors Proinflammatory cytokines have been thought to mediate some of their effects via the release of neurotrophic factors from the accessory cells in the ENS. Von Boyen et al demonstrated that administration of either LPS, tumor necrosis factor-α (TNF-α) or interleukin-1ß (IL-1ß) to enteric glial cells caused the release of glial-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) (Yin et al., 2003;von Boyen et al., 2006a;von Boyen et al., 2006b). Similarly, studies in the CNS have demonstrated that these changes are accompanied by the upregulation of the corresponding 13 neurotrophin receptors (Stoll & Muller, 1999). Considering the above studies, it is evident that the infiltration of a large number of activated macrophages can induce the release of neurotrophins in the inflamed intestine. Since neurotrophic factors, especially GDNF and NGF, are known to play an important role in post-natal plasticity of the ENS (Giaroni et al., 1999), it is conceivable that an increase in neurotrophin secretion in IBD could have a major impact in ENS survival and repair. 1.3.3 Novel neurotrophic factors secreted by macrophages The existence of novel neurotrophic factors in the inflammatory milieu was proposed when macrophage induced axon growth in the injured optic nerve was not blocked by antibodies to several known neurotrophins, nor by inhibitors to downstream signaling pathways activated by those factors (Filbin, 2006). Yin et al. first identified a small ~12kDa active component of macrophage-released soluble factors, and then used mass spectroscopy to unveil a novel neurotrophin – oncomodulin (Yin et al., 2006). Oncomodulin was shown to be abundantly expressed and secreted by activated macrophages, and when added in combination with cAMP, it was found to strongly potentiate axon outgrowth in optic nerve injury. The same study also demonstrated similar neurotrophic effects of oncomodulin on neurons from the dorsal root ganglia (DRG) (Yin et al., 2006). Since this novel neurotrophic factor is secreted in large amounts from activated macrophages and enhances axon regeneration in diverse neural populations, it is plausible that it plays a critical role in resolving intestinal inflammation by establishing an environment conducive to ENS repair. 14 1.4 Conclusions and hypotheses It is clear that in intestinal inflammation, the activation of the innate immune system can serve as a double edged sword. It appears that the release of inflammatory mediators and cytokines in the acute stages of inflammation by infiltrating immune cells is responsible for a majority of ENS injury seen in IBD. While the role of NO in mediating tissue injury in IBD is a well researched topic, unequivocal evidence linking NO and ENS damage is still lacking. Extensive work in models of inflammation with inhibited NO production, using both, chemical NO inhibitors and iNOS-/- animals, has provided inconclusive evidence for the role iNOS in causing ENS damage. Inconsistencies in the results of these studies may have arisen due to differences in species, strains, housing conditions, models and the execution. Despite these shortcomings, the studies have provided some insight into the relative contributions of each isoform in intestinal inflammation. It seems that constitutive production of small amounts of NO is beneficial in settings of acute mucosal injury, but chronic overproduction via iNOS overexpression may be detrimental. Moreover, the end result of pharmacological interventions in NO production during the course of colitis depends strongly on the time-points of intervention, the combination of pharmacological agents used, as well as the bioavailability of these agents. We believe that instead of attempting to understand the role of iNOS in whole animal studies, using a simplified culture model of the intestinal environment will provide more information on the role of NO in causing ENS damage. The early damage experienced by the enteric neurons is repaired in the later stages by the secretion of various neurotrophic factors and cytokines conducive to axon regeneration. Numerous studies in animal models of colitis have demonstrated the 15 increase in axons in the later stages of inflammation, but none have attempted to understand the cause (Lourenssen et al., 2005). Research in other models of nerve injury such as sciatic nerve injury, optic nerve lesions and in DRG has demonstrated the potential of activated macrophages to arrest neuron loss and enhance axon regeneration. Whether these activated macrophages have similar effects on the damaged ENS is an interesting question. Based on the above review of literature we adopted a combination of in vivo and in vitro approaches to achieve the following goals: To characterize the time course of immune cell infiltration within the inflamed colon in the proximity of ENS To study iNOS expression adjacent to ENS at times when neuron and axon damage is apparent To determine whether elevated NO levels generated by chemical NO donors are toxic to cultured myenteric neurons To study the effect of iNOS generated NO on cultured neurons using peritoneal immune cells. Additionally, the role of proximity of immune cells to cultured neurons will also be investigated using semi-permeable trans-wells. 16 Chapter 2 Materials and Methods 2.1 Animals All primary cell cultures were derived from 3 to 10-day old (neonatal) male Sprague-Dawley rats (Charles River, Montreal, PQ, Canada). Adult male Sprague-Dawley rats were used to obtain immune cells for culture work or for experiments with TNBSinduced colitis. All work with animals was carried out according to a protocol approved by Queen‘s University Animal Care Committee. 2.2 TNBS-Induced Colitis A rat model of chemically induced colitis was used, as described previously (Morris et al., 1989;Wells & Blennerhassett, 2004;Lourenssen et al., 2005). Adult (225300g) male Sprague-Dawley rats were anaesthetized with halothane, and then 500 µl of 0.2 M 2, 4, 6-trinitrobenzene sulfonic acid (TNBS; FLUKA) dissolved in 50% ethanol in phosphate buffered saline (PBS) was administered rectally using a PE-50 catheter into the distal 8 cm of the colon. Control rats received no treatment because previous studies have established the lack of effects with either ethanol or PBS administration (Morris et al., 1989). During the first 4 days following TNBS infusion, a period in which the severity of the inflammation is greatest, rats were provided with additional fluid (2% NaCl and 0.9% sucrose) and received subcutaneous injections of buprenorphine hydrochloride (0.3 mg/kg; Reckitt and Colman Products, Ltd.) twice a day to minimize pain. Some rats were sacrificed at the 6hr, 24hr, 2 day and 4 day time point, and tissue from the middescending colon was removed. This tissue was either snap frozen in dry ice or embedded paraffin wax for sectioning. 35 days after the initial treatment, when the overt 17 signs of inflammation had disappeared, the remaining rats were sacrificed and the middescending colon was removed. 2.3 MPO Assay Samples for the MPO assay included tissue from mid to distal colon (adjacent to tissue used for histology) and were obtained either from control or TNBS-treated animals. Samples were removed from the inflamed region or from areas 1.5 to 2.0 cm proximal to the affected region, cleaned of mesentery and luminal contents, rinsed with cold PBS, blotted dry, and immediately frozen with dry ice. The samples were stored at -80°C until thawed for MPO activity determination using the O-dianisidine method described previously (Boughton-Smith et al., 1988). The change in absorbance was read at 460 nm in a spectrophotometer (EL-800 - Universal Microplate Reader, Bio-Tek Instruments). MPO activity was expressed as the amount of enzyme necessary to produce a change in absorbance of 1.0 per min per gram of wet weight of tissue. 2.4 DAB Histochemistry To obtain frozen sections of intestine for histology, segments of colon were removed from control and inflamed adult rats, quickly washed in PBS, covered in OCT (Sakura Finetek) and frozen on dry ice. Tissues were stored at -80°C before sectioning (10 μm) onto APTEX-coated slides (Sigma). The sections were then dried at room temperature for 1 hr, and kept frozen at -80°C until further analysis took place. Neutrophils were located using the diaminobenzidine (DAB) reaction. Before the reaction, frozen slides were defrosted and washed twice for 2 min with phosphate buffered saline (PBS) in order to remove the OCT from the slides. In preparing the DAB reaction solution, 5 mg DAB (SIGMA) was dissolved into 10 ml of PBS and 300 µl of 18 0.1% H2O2. Slides were incubated in this solution for 15 min at room temperature. Following incubation, slides were counter-stained with Fast Green FCF for 30 s, and cover-slip using Permount. 2.5 Isolation of Peritoneal Immune Cells Immune cells were collected from adult male Sprague-Dawley rats (Charles River, Montreal, PQ, Canada), by peritoneal lavage. 15 ml of sterile medium (DMEM; GIBCO) was injected in to the peritoneal cavity of animals that were sacrificed by overdose of anesthetic (Halothane: Sigma, St. Louis, MO). The animals were gently massaged to liberate immune cells and the lavage fluid was retrieved from the peritoneal cavity. The lavage fluid was centrifuged (350 x g; 5 min) (MultiRF Centrifuge; ThermoIEC) to increase the density of cells. The total cell number was determined using a hemocytometer, and the suspension was then adjusted to a concentration of 106 cells per ml. All the steps were performed under sterile conditions. 2.6 NADPH-Diaphorase Labelling Immune cells were incubated at 37°C in medium (DMEM; GIBCO) for 1, 2, 3 and 6 hrs post extraction from the animal. After the incubation period, the cells were fixed in neutral buffer formalin for 15 mins, washed with 0.1 M Tris buffer and reacted for NADPH diaphorase staining using 0.25 mg/ml nitroblue tetrazolium, 1 mg/ml NADPH, 0.5 % Triton X-100 in 0.1 M Tris buffer, pH 7.6, for 20-30 min at 37°C. 2.7 Griess Reaction In order to study the levels of NO released by the NO donors, S-nitroso-aminopenicillamine (SNAP), sodium nitroprusside (SNP) and diethylenetriamine NONOate 19 (DETA-NONOate) (SIGMA, St. Louis, MO), samples were collected from co-cultures at 2 hr intervals after addition. Since NO is highly unstable in solution and rapidly converts to nitrates and nitrites, and their relative proportion cannot be predicted with certainty, the best index of total NO production is a sum of both nitrates and nitrites (Granger et al., 1996). The enzyme nitrate reductase was used to convert nitrates to nitrites, and the nitrite concentrations were measured using a colorimetric assay. The absorbance at 540nm of each sample was measured in a microplate reader. Nitrate reductase and other reagents used for this assay were obtained from a commercially available kit and the manufacturer‘s instructions were followed (NO colorimetric assay kit, Cayman Chemical Co). 2.8 Tissue culture To obtain cultures of intestinal smooth muscle cells and neurons from the ENS, the small intestine of 3 to 7-day-old Sprague-Dawley rats (Charles River, Montreal, Quebec, Canada) was isolated and the mesentery completely removed. The mucosa with the attached muscularis mucosa was separated from the smooth muscle layers and discarded. The remaining longitudinal and circular smooth muscle layers and the enclosed myenteric plexus were dissociated using 0.4% trypsin II (Sigma, St. Louis, MO) in HEPES-buffered Hanks saline (pH 7.35) as described previously (Blennerhassett & Lourenssen, 2000;Lourenssen et al., 2009). Cell suspensions were plated onto glassbottomed culture plates (15-mm diameter) previously coated with collagen (SIGMA, St. Louis, MO). Medium (DMEM; GIBCO) containing 5% fetal calf serum (FCS) was then added and the co-cultures were allowed to grow to confluence. A schematic diagram of co-culture protocol is displayed in Fig. 2. These were called "co-cultures" to reflect the 20 predominance of neurons and ISMC, although other cell types such as glial cells are present in low numbers. 2.9 NO Addition S-nitroso-amino-penicillamine (SNAP), sodium nitroprusside (SNP) and diethylenetriamine NONOate (DETA-NONOate) solutions were prepared in medium (DMEM; GIBCO) to yield stock concentrations of 1.25, 5, 12.5, and 25 mM. 20 µl of each, when added to a 1 ml culture well, gave final concentrations of 25, 100, 250, and 500 µM. NO donor treated co-cultures were incubated for 48 hrs, after which immunocytochemistry was performed to stain for neuronal cell bodies and axons (Fig. 2 - schematic). 2.10 Immune cells + Neuron co-culture studies Peritoneal immune cells were harvested as described above. For studies involving direct addition of peritoneal cells to neuronal co-cultures, 106 immune cells were added to confluent myenteric neuron and ISMC co-cultures described above. Trans-well studies involved placing inserts (0.4µm Pore Polycarbonate Membrane Inserts, Corning) on cocultures. Immune cells were placed on these inserts, allowing soluble factors to diffuse through the semi-permeable membrane on to the co-cultures. After a 48 hr incubation period, culture wells in either condition were washed with phosphate buffered saline (PBS), followed by NBF fixation and immunocytochemistry (Fig. 2 – schematic). 21 Figure 2 Fig 2. Schematic representation of co-culture protocol. Myenteric neurons, glia and intestinal smooth muscle cells were obtained from neo-natal rats and cultured in 5% foetal calf serum. 72 hr after set up, these co-cultures were incubated with NO donors or peritoneal immune cells for 48 hr, followed immunohistochemical analysis for changes in neurons and axons. 22 2.11 Immunocytochemistry Immunocytochemistry with was used to study the effect of chemical NO donors and LPS-activated immune cells on neuron survival in co-cultures. Control and various test conditions were compared, and the neuron numbers counted using a 40X objective. Co-cultures were fixed in neutral buffered formalin for 20 minutes, followed by washing in PBS. They were then blocked in 0.2% Tween-20 in PBS containing 1% goat serum for 1 hr. This was followed by incubation with primary antibodies to either HuD (mouse monoclonal; 1:1000; Molecular Probes), SNAP-25 (rabbit polyclonal; 1:1000; Molecular Probes) or nNOS (rabbit polyclonal; 1:500; Eurodiagnostica). All primary antibodies were made in antibody diluting buffer (DAKO). Culture wells were washed with PBS following removal of primary antibody and exposed to the following secondary antibodies conjugated to fluorescent tags: goat anti-rabbit (1/1000) labelled with Alexa555 and goat anti-mouse (1/500) labelled with Alexa-488 (Molecular Probes). After 2 hr incubation with secondary antibody, a few drops of 50% glycerol were applied to the center of the dual-label co-cultures, which were then coverslipped and preserved at 4°C. Staining was visualized with a fluorescent microscope (Olympus BX60 or BX51) and images were digitally captured using a Coolsnap FX camera (Roper Scientific) and Image-Pro Plus 6.0 (Media Cybernetics) software. For detection of iNOS, ED1 and ED2, paraffin sections (4µm) were cut using a microtome (Fisher) and dried overnight. These were then deparaffinised, sections were blocked for 1h at room temperature in PBS containing 1% goat serum followed by incubation with mouse rabbit anti-iNOS (1:500; BD Biosciences) or mouse anti-ED2 (1:100; Serotec) or rabbit anti ED1 (1:100; Serotec) at 4°C overnight. After washing in 23 PBS (3 x 5min) coverslips were incubated in secondary antibody for 1 hr at room temperature in a humidity chamber. Secondary antibodies included goat anti-mouse IgG conjugated with Alexa Fluor 488 (1:500; Molecular Probes) and goat anti-rabbit Alexa Fluor 555 (1:500; Molecular Probes) Images were obtained under fluorescent illumination with an Olympus BX-51 fluorescent microscope using a Coolsnap FX camera (Roper Scientific) and Image Pro Plus 6.0 (Media Cybernetics) software. 2.12 Determination of neuron and axon number Neuron number was determined following immunocytochemistry for HuD by counting all of the positively labelled neurons in every third field of view of a strip spanning the dish diameter, using a x40 objective, and this was then repeated in the perpendicular axis. The area analyzed was 5.5 x 104/µm2 per culture, which was 3.1% of the total area. For axon counts in the same culture dish, all SNAP-25 labelled axons in a vertical strip along the diameter of the culture dish were counted using a x60 water immersion objective This was summed across fields to give a number representative of the axon number per culture well. 2.13 Statistical Analysis Data analysis was performed using Microsoft Excel and the XL Toolbox software. All values are expressed as the mean + standard deviation (SD). A standard one-way analysis of variance (ANOVA) was used to evaluate the differences between multiple populations followed by unpaired, two-tailed Student‘s t-tests corrected using the Bonferroni method to compare differences between two populations. 24 Chapter 3 Results 3.1 Neurotoxic effects of immune cells 3.1.1 Increased presence of infiltrating macrophages and neutrophils in proximity to the ENS A single administration of TNBS/EtOH in the rat distal colon caused regional inflammation that was characterized by ulceration, hyperaemia, adhesions and oedema, and was similar to that described in previous reports (Wells & Blennerhassett, 2004;Lourenssen et al., 2005). Since major alterations in structure and function of the inflamed colon occurred early in the time course of colitis, we proposed that the infiltration of activated immune cells could be responsible for the rapid onset of tissue damage and specifically, of damage to the ENS. Therefore, we focused on the number and nature of infiltrating immune cells in the regions surrounding ENS at early time points in TNBS-colitis. The resident, tissue-specific population of macrophages were labelled with the anti-ED2 antibody. Cross sections of colon from control animals displayed small numbers of resident macrophages in the muscularis regions (10.2 + 2.6 macrophages/mm2) (Fig. 3A). In animals with TNBS-induced colitis, there was no significant differences in ED2-positive cells at 6, 12 and 24 hr when compared with untreated controls (Fig. 3A-C) (p=o.46; n=3). In contrast, blood derived monocytes, which were labelled with anti-ED1 (a marker of infiltrating macrophages) were initially present in very low numbers (1 + 0.6 macrophages/mm2) but demonstrated a rapid and marked rise in the CSM region upon initiation of inflammation (Fig. 3D-E), with a 2025 fold increase observed at 12 hr after induction of inflammation (p<0.01) and a further doubling by 24 hr (Fig. 3F) (p<0.01, n>3). It is evident that in the non-inflamed colon, resident macrophages outnumber the blood derived monocytes. In contrast, the number of infiltrating macrophages greatly exceeds that of tissue specific macrophages in inflammation. 26 Figure 3 Fig 3. Early increase in the number of infiltrating macrophages in inflamed colonic tissue in TNBS-induced colitis: A-C: Fluorescence micrographs of resident, tissue-derived macrophages (ED2-positive) in cross sections of control colon (A), and 24 hr post induction of inflammation (B). Arrows indicate ED2-immunoreactive macrophages in the CSM region. No significant changes are observed by 24 hr (p=0.46, n=3). D-F: Cross sections of colons from control (D) and inflamed animals 24 hr postinduction of colitis (E) are labelled for infiltrating, blood-derived macrophages in the CSM region. A significant increase is observed within 12 hr of inflammation and which further increases by 24 hr. Treatment groups reaching statistical significance compared to control by a two-tailed student‘s t-test corrected for multiple comparisons (p < 0.016) are indicated (*). 27 Neutrophils as well as macrophages contribute to the rapid influx of immune cells into sites of inflammation. To study the infiltration of neutrophils at early time points of inflammation, histochemistry was used to identify peroxidase activity, present in high levels within neutrophils, through precipitation of diaminobenzidine (DAB) in cross sections of control and inflamed animals. The CSM region in control animals displayed low levels of neutrophil infiltration (0.4 + 0.25 neutrophils/mm2). No significant changes were detected by 6 hr, but a 30-fold increase was observed by 12 hr following induction of colitis (Fig. 4A) (p<0.025, n=3). No further changes were noticed by 24 hr, suggesting that neutrophils largely arrived at the site of inflammation between 6 and 12 hr. To further confirm the rapid accumulation of neutrophil granulocytes in the vicinity of smooth muscle/myenteric plexus (SM/MP), homogenates of the SM/MP were assayed for myeloperoxidase (MPO) activity. MPO is a marker enzyme with activity proportional to neutrophil accumulation in tissue samples (Schierwagen et al., 1990). While control levels were uniformly low (1.2 + 0.23 units/mg), tissue from inflamed animals revealed a trend of increasing neutrophil activity by 12 hr post-induction of TNBS-colitis which plateaued by 24 hr (Fig. 4B) (p<0.025, n=3). This is consistent with our histochemical findings that suggest that neutrophil infiltration in the SM/MP region occurs primarily between 12 and 24 hr. 28 Figure 4 Fig 4. Increase in the number of infiltrating neutrophils in inflamed colonic tissue in TNBS-induced colitis: DAB histochemistry performed on cross-sections of colon from control and TNBS-treated animals revealed a significant increase in neutrophils number in the CSM within 12 hr post-induction of inflammation with no further increase by 24 hr (B) (p<0.025, n=3). MPO assay on smooth muscle and myenteric plexus layers from control and inflamed animals revealed a significant increase in neutrophil infiltration by12 hr of inflammation (B). Treatment groups reaching statistical significance compared to control by a two-tailed student‘s t-test corrected for multiple comparisons (p < 0.025) are indicated (*). 29 3.1.2 iNOS expression is upregulated in the early stages of TNBS-induced intestinal inflammation A principal agent of the cytotoxic actions of activated neutrophils and macrophages is NO derived from iNOS (MacMicking et al., 1997). Immunocytochemistry was used to detect the presence and location of iNOS-expressing immune cells in the SM/MP, in cross sections from control and inflamed colon (Fig. 5AB). While the control animals displayed no iNOS presence in the muscularis region, a large number of iNOS-positive cells were distributed throughout the longitudinal and circular smooth muscle layers in the inflamed animals, with many positive cells immediately adjacent to the ganglia of myenteric and submucosal neurons (p<0.05; n>5 animals). Fig. 5C displays the increasing levels of iNOS expression in the first 2 days of colitis, plotted as a ratio of iNOS positive cells/mm2 in the CSM region. These findings show that myenteric neurons and axons could be exposed to high levels of NO in the early stages of TNBS-induced colitis, and justified the further study of its potential involvement in the colitis-induced damage to the ENS. 30 Figure 5 Fig 5. iNOS expression is upregulated in the early stages of TNBS-induced colitis: Fluorescence micrographs of iNOS immunoreactivity in a cross-section of the colon from control animal (A), and 24 hr post induction of inflammation (B). Arrows indicate iNOS immunoreactive cells in proximity to the myenteric plexus. Quantification of iNOS positive cells reveals a significant increase in iNOS expression in the CSM region within 6 hr of induction of inflammation, with further increases at 12 and 24 hr (C). Treatment groups reaching statistical significance compared to control by a two-tailed student‘s t-test corrected for multiple comparisons (p < 0.016) are indicated (*). 31 3.1.3 NO is cytotoxic for myenteric neurons in vitro Since a large number of iNOS expressing cells were found in the vicinity of enteric ganglia at a time when neuron death was evident, we chose to assess whether NO alone is damaging to cultured neurons. This took advantage of a co-culture model of myenteric neurons, smooth muscle cells and glia with well-described structural and functional properties that support its relevance to the study of colitis (Lourenssen et al., 2009). To establish co-cultures, the SM/MP of neo-natal rats was enzymatically dissociated and the resulting cell suspension was incubated in DMEM with 5% FCS at 37°C as described previously (Lourenssen et al., 2009). Following incubation, immunocytochemistry was performed to detect myenteric neurons. In addition to ISMC and glia, neurons were present in these co-cultures, identified using antibodies to the pan-neuronal marker HuD (Fig. 6A). Axons were identified using SNAP-25, a synaptosomal-associated protein present in neuron cell bodies and axons (Fig. 6B). A composite image of Fig. 6A and B shows co-localization of HuD and SNAP-25. This served as an excellent environment to test the effect of NO on myenteric neurons in vitro. 32 Figure 6 Fig 6. Representative immunofluorescent images of myenteric neurons from primary co-cultures of smooth muscle and myenteric plexus of rats (post-natal day 3 – 5) are displayed in A-C. Myenteric neurons are identified using HuD, a neuronal cell body specific marker (arrows)(A). Axons are detected using SNAP-25, a synaptosomal associated protein present in both, the cell body and axons (arrowheads) (B). Composite image of A and B displays co-localization of HuD and SNAP-25 (C). A, B and C represent the same field of view. 33 Three chemical NO donors – SNP, SNAP and DETA-NONOate were added individually to neuronal co-cultures and following 48 hr incubation, immunocytochemistry for HuD and SNAP-25 was performed to detect neuronal cell bodies and their axons, respectively. A minimum of 3 animals were used for each experiment with duplicate wells per treatment, and systematic analysis at highmagnification (see Materials and Methods) was employed to quantify changes in neuron numbers and axons. Control conditions displayed numerous HuD labelled neurons with long axon extensions and multiple branch points. Addition of NO donors at high concentrations resulted in significant decrease in the number of HuD expressing neurons in all three donors. While the addition of 100µM SNAP or SNP resulted in significant neuron loss (p=0.03 and p=0.001 respectively), 250µM was the threshold concentration for DETA-NONOate (p=0.003) (Fig. 7A-C). Since the nitrergic (nNOS) neurons constitute a large proportion of the total neurons within the ENS and release low levels of NO constitutively, it has been proposed that these may be differentially responsive to high NO concentrations (Kurjak et al., 1999). In order to assess whether high NO levels cause a uniform loss of cultured neurons, the specific effects on the nNOS population were assessed. Following a 48 hr treatment with the NO donor SNAP, anti-nNOS antibodies were used to identify nitrergic neurons in culture. Neuron death was observed when NO donors were administered at concentrations higher than 100µM (100 µM: 83.4 + 0.3%; 250 µM: 68.4 + 8.7%; 500 µM: 65.2 + 23.2%; values compared to untreated control). The loss of nitrergic neurons in response to NO addition was similar to the loss of total neurons, suggesting that exposure to high levels of NO results in non-selective loss of neurons in vitro. 34 While the addition of SNAP, SNP or DETA-NONOate in high concentrations resulted in the loss of cultured neurons, the results were not identical. This led us to postulate that these subtle differences were a result of differential NO generation by the donors. To better characterize the release of NO from the chemical donors SNAP, SNP and DETA-NONOate, NO levels were assayed by Griess reaction at increasing times after addition to media. This was unsuccessful for determination of NO levels generated by DETA-NONOate, where it is possible that the parent compound of this donor, diethylenetriamine, interfered with the Griess reaction. The concentrations of NO in media containing either SNAP or SNP were measured 48 hr post addition (Fig. 7A-B), which showed that significant neuron loss was only detected when NO concentrations were higher than 25 µM, with the highest incidence of neuronal death occurring when NO concentrations approached 60 µM. Overall, it was clear that NO, when derived from either of two different chemical donors in vitro, was associated with rapid and extensive neuronal death. This further supported the hypothesis of a central role of NO in causing damage to the ENS in vivo. 35 Figure 7 36 Fig 7. Chemical NO donors decrease enteric neuron survival in vitro: Significant reduction in neuron survival was observed after 48 hr treatment of cocultured myenteric neurons with varying concentrations of (A) SNP, (B) SNAP and (C) DETA-NONOate. Neuron numbers were obtained by immunohistochemical analysis of HuD-labelled neurons in co-cultures. This data is represented on the left axis, expressed as percentage of HuD-labelled neurons in untreated time-matched control. NO concentrations generated by addition of SNP (A) or SNAP (B) are displayed as a line graph on the right axis. Treatment groups reaching statistical significance compared to control by a two-tailed student‘s t-test corrected for multiple comparisons (p < 0.01) are indicated (*). 37 3.1.4 NO toxicity is specific to neurons; ISMC and glia are unaffected Since elevated NO levels reduced neuron survival in co-cultures, it was important to elucidate whether the toxic effects of NO caused indiscriminate damage to all cell types in the co-cultures or whether the injury was restricted to neurons alone. To assess the viability of smooth muscle cells and enteric glia after NO treatment, control cultures or cohorts exposed to NO donors as above were incubated with propidium iodide (PI), a nuclear fluorochrome whose uptake is a marker of cell death. At this point, immunocytochemistry was used to identify neuron cell bodies. Control co-cultures showed consistent exclusion of PI by all three predominant cell types – ISMC, glia and neurons. In contrast, 30 min of treatment with the NO donor SNP (250 µM) revealed colocalization of PI with HuD staining, suggesting selective loss of viability in some neurons (Fig. 8A-E). By 1.5 hr of treatment with SNP, this outcome was still evident, with the selective uptake of PI by some, but not all, neurons while all adjacent nonneuronal cells remained PI-negative (Fig. 8C-E). In conclusion, it appears that nonneuronal cells are not adversely affected by exposure to NO donors, and suggested that neuronal cell bodies and axons might be selectively susceptible to the toxic effects of NO. 38 Figure 8 Fig 8. Fluorescence photomicrographs showing rapid onset of selective neuronal cell death with exposure to the NO donor SNP (250 µM). A-B: Low power images of myenteric neurons in co-culture exposed to SNP for 30 min in the presence of 10-8M propidium iodide (A) followed by immunocytochemistry for HuD to detect neuronal cell bodies (B). The co-localization of propidium iodide (red) with HuD staining (green) shows selective loss of viability in some neurons (arrows). C, D, E: Higher magnification images of a co-culture exposed to SNP for 1.5 hr, showing overlap of propidium iodidepositive nuclear staining (C) with HuD staining of one neuron (D; arrow). E, merged images combined with bright field image to show other neurons and non-neuronal cells also present in co-culture, but propidium iodide negative. 39 3.1.5 A co-culture model of immune cells and myenteric neurons While the study of the inflamed intestine in vivo can provide insight into the variety of immune cell types present and time course of their appearance and potential actions, there is limited scope in dissecting the process of inflammatory damage. Therefore, we chose to model these events in vitro by exposing the cellular components of the intestine to activated immune cells and determining the consequences. To develop this in vitro system, the co-culture model of myenteric neurons characterized above was used in conjunction with peritoneal immune cells. Peritoneal immune cells were harvested from adult rats by peritoneal lavage and cultured at various densities (103 to 106 cells/mL) in DMEM. NADPH-diaphorase histochemistry was performed at various times post-seeding to evaluate NOS activity. Widespread NOS activity was observed among these cells within 2 hr in vitro (Fig. 9A-B). Furthermore, assay of culture supernatants by Griess reaction showed that NO concentration increased in proportion to the number of immune cells (Fig. 9C, n=3). These data suggested that the number of NOS-positive immune cells directly determined the concentration of NO in media in vitro. Therefore, it was possible to create a unique model of aspects of inflammation in vitro through addition of these cells to neuronal cocultures. 40 Figure 9 Fig 9. iNOS expression and NO release by activated peritoneal immune cells in vitro: Representative phase contrast micrographs of NADPH-diaphorase histochemistry in activated peritoneal immune cells reveal a low level of NOS activity 30 min post extraction (A), and an upregulation by 2 hr (B). Arrows indicate cells with active NOS identified by NADPH-diaphorase staining. Inset – 20X magnification. (C) Supernatants from immune cell cultures seeded at varying densities (103-106 cells/ml) were assayed for NO production using the Griess reaction. Consistent increase in NO levels was observed with increasing cell numbers. All data are reported as means (n=3, +SD). 41 Addition of peritoneal immune cells to co-cultures (5 x 105 – 106 per well) resulted in numerous close associations between neurons and activated immune cells. Following immunocytochemistry to detect neuronal cell bodies and axons, it was evident that the addition of immune cells caused marked damage to the myenteric neurons (Fig. 10A, B). Fig. 10C shows an example of ED1 positive macrophages surrounding ISMC and neurons. In these co-cultures, the quantification of neuron number showed that the addition of immune cells caused a significant decrease in neuron survival by 48 hr, to 81 + 5% of untreated control cultures (Fig. 10A-B) (p<0.05, n=3). Further, SNAP-25 immunocytochemistry revealed a disruption of axon structure in immune cell-treated cocultures, with the appearance of fragmented axons and decreased contacts with glial and smooth muscle cells (Fig. 10B). To validate a potential role of NO among the diverse cytotoxic actions of immune cells, the selective iNOS inhibitor L-NIL was used. This allowed determination of the extent of damage to neurons and axons that was a result of elevated NO levels. Immune cells were treated with 100 µM L-NIL, a concentration shown maximally effective elsewhere (Boucher et al., 1999), prior to addition to the co-cultures. Pre-treatment with L-NIL prevented the significant neuron death observed in immune-cell treated cultures (p>0.05; 96 + 2% of untreated controls) (Fig. 10C). Overall, the outcomes of study using this co-culture model indicated that the addition of immune cells caused neural damage in vitro that closely mimicked that seen in vivo. Further, immune cell-mediated damage to myenteric neurons in vitro was predominantly an iNOS-dependent process, suggesting that NO may be the key mediator 42 of neural damage and death. This was substantiated by the fact that chemical NO donors decreased neuron survival, further implicating a central role of NO in inflicting neuronal damage. 43 Figure 10 Fig 10. Direct addition of activated immune cells causes loss of myenteric neurons in vitro. A, B. Co-cultures of ENS neurons were treated with activated peritoneal immune cells. Composite images of neuronal cell bodies (HuD: green) and axons (SNAP-25: red) from a time-matched control co-culture (A), and one treated with 106 peritoneal immune cells (B) reveal neuronal damage in immune cell-treated cocultures. SNAP-25 labelling revealed discontinuous axon segments (arrowheads) suggesting axon fragmentation and degeneration. (C) ED1 positive macrophages (red) were found in close proximity to HuD labelled neurons (green). (D) Changes in neuron number were quantified and expressed as a percentage of HuD-positive neurons in untreated, time-matched controls. Exposure to 106 immune cells caused a significant loss of neurons, which was prevented by pre-treatment of immune cells with L-NIL, an iNOS specific inhibitor. Treatment groups reaching statistical significance by a two-tailed student‘s t-test corrected for multiple comparisons (p<0.025) are indicated (*). 44 3.2 Neurotrophic Effects of Immune cells 3.2.1 Activated immune cells exert neurotrophic effects on myenteric neurons in co-culture We had previously determined that the addition of activated peritoneal immune cells directly to myenteric neuron co-cultures reduced neuron survival and caused axon fragmentation (Fig. 10). Next, we studied the importance of proximity and contact between immune cells and neurons by placing activated immune cells on trans-wells directly above the co-cultures and assessed neurite outgrowth and neuron survival 48 hr later (Fig. 11A-B). In stark contrast to the ‗direct-addition‘ studies, the addition of 106 immune cells via trans-wells resulted in strong potentiation of axon outgrowth (282 ± 57%; p<0.05, n>5) as well as unimpaired neuron survival (117 ± 8.3%; p>0.05, n>5). (Fig. 11C) Immunocytochemistry showed that proximity to immune cells in this case promoted extensive non-directed, random outgrowth of most axons which individually appeared longer and with more branch points. This suggests that diffusible factors secreted from immune cells have the ability to exert a novel neurotrophic effect on cultured ENS neurons, a phenomenon that warrants further study. 45 Figure 11 Fig 11. Activated immune cells on trans-wells exert neurotrophic effects on cultured myenteric neurons: Co-cultures of ENS neurons were treated with activated peritoneal immune cells placed 0.4µm trans-wells. Composite images of neuronal cell bodies (HuD: green) and axons (SNAP-25: red) from control co-cultures (A) and cultures treated with 106 peritoneal immune cells placed on trans-wells (B) reveal unaltered neuron numbers (p>0.05). SNAP-25 labelling revealed a robust increase in neurite outgrowth in treated cultures (p<0.025). (C) These changes were quantified and expressed as percentage of HuD-labelled neurons and SNAP-25 labelled axons in time-matched, untreated control. Treatment groups reaching statistical significance by a two-tailed student‘s t-test corrected for multiple comparisons (p<0.025) are indicated (*). 46 Chapter 4 Discussion The enteric nervous system (ENS) is now well recognized by gastroenterologists and gastrointestinal physiologists alike as the ―brain of the gut‖. This is because it controls gut function - including motility, secretion, absorption, blood flow, and aspects of the local immune system (Hansen, 2003). The ENS exhibits a high degree of functional autonomy, and contains an extremely diverse population of neurons in terms of neurochemistry, projections and functions (Furness, 2000). Due to the unique environment of the GI tract, there are a number of instances in which the enteric neurons display plasticity and undergo regeneration. Neuroplastic changes in the ENS may be observed in physiological states, such as development and aging, or occur as a consequence of pathological conditions such as intestinal inflammation. In humans, the effects of intestinal inflammation on the ENS include structural modifications which can range from axon damage to neuronal cell death, as well as the loss of entire ganglia; functional changes to the ENS include altered synthesis, storage and release of neurotransmitters as well altered regulation of receptor systems (Brewer et al., 1990;Geboes & Collins, 1998;Sharkey & Kroese, 2001;De et al., 2004). The outcome of these changes to the ENS typically include sensory-motor and secretory impairment of gut function. Various studies have attempted to elucidate the nature and mechanism of damage to the ENS caused by inflammatory conditions such as Crohn‘s disease and ulcerative colitis, but the causes of neuron loss and axon injury are poorly understood 47 (Hogaboam et al., 1995;Marlow & Blennerhassett, 2006;Zandecki et al., 2006;Lourenssen et al., 2009;Sand et al., 2009). The purpose of this study was to provide insight into some of the factors responsible for the inflammation mediated changes in the ENS. Specifically, the role of immune cell generated mediators in causing neuron loss and axon plasticity was assessed. Using a combination of in vivo and in vitro approaches, this study evaluated the hypothesis that high levels of NO released by the enzyme iNOS, present in inflammatory cells such as macrophages and neutrophils, are responsible for the detrimental effects of inflammation of the ENS. Furthermore, we tested our hypothesis that the initial inflammatory insult to the neurons is followed by a compensatory regeneration of axons that is also dependent on a complex array of immune cell-generated factors. 4.1 Rapid influx of infiltrating immune cells in TNBS-induced colitis The gastrointestinal tract is said to display a state of ―physiological inflammation,‖ manifested by presence of abundant leukocytes in the intraepithelial and sub-epithelial compartments (Fiocchi, 1997) . These cells are ideally situated to sample luminal antigens, and respond to the plethora of chemical and biological challenges encountered by the gut on a daily basis. In contrast to the ‗normal‘ inflammation, when a true inflammatory response, or ―pathological inflammation,‖ occurs in the intestine, such a response is dominated by infiltrating immune cells, primarily represented by neutrophils, macrophages, and cytotoxic T cells (Fiocchi, 1997). These cells are known to play the role of aggressors that attack and destroy nearby cells, either directly through physical contact or indirectly through the release of soluble factors such as reactive 48 oxygen and nitrogen metabolites, cytotoxic proteins, lytic enzymes, or cytokines (Larrick et al., 1980;Beckman et al., 1990;Alvarez et al., 2004). We proposed that the release of such toxic factors in intestinal inflammation in the vicinity of the ENS causes the cellular damage observed in IBD and animal models of inflammation. In order to observe the location and number of infiltrating immune cells near the ENS, immunocytochemistry and MPO assays were used. A large increase in the number of neutrophils was detected in the SM/MP regions using both techniques. This is consistent with other studies that demonstrated a similar rapid increase in neutrophils to the site of inflammation (McCafferty et al., 1997;Sanovic et al., 1999). Blood derived monocytes, another component of the body‘s innate immune response, were studied in the inflamed tissue using a specific marker for infiltrating macrophages. A similar trend of increasing numbers hours after induction of inflammation was observed, suggesting that the activation of the innate immune system and the subsequent response is immediate. A close temporal association is obvious between the inflammatory response and the ENS damage observed in our studies. This suggests that cytotoxic factors secreted by infiltrating immune cells may well be responsible for the loss of ENS neurons and axons. Among the various toxic chemicals generated by activated immune cells, iNOS derived NO is a principal cytotoxic agent that mediates tissue damage (Cross & Wilson, 2003). We noticed a rapid increase in iNOS expression in the CSM region adjacent to the myenteric and submucosal plexuses. This time course of this upregulation correlates with the arrival of infiltrating macrophages and neutrophils into the CSM, suggesting that these cells are responsible for increased iNOS expression. These data are consistent 49 with other studies that report similar observations regarding iNOS activity in the early stages of inflammation (Kankuri et al., 1999;McCafferty et al., 1999;Vallance et al., 2004). iNOS protein expression was shown to be increased within the inflammatory infiltrate of the lamina propria in patients with ulcerative colitis, whereas age-matched controls demonstrated no iNOS immunoreactivity (Godkin et al., 1996;Kimura et al., 1998). Localization of iNOS to areas of intense inflammation has also been reported in Crohn‘s disease (Boughton-Smith et al., 1993;Rachmilewitz et al., 1995b). Interestingly, a close temporal correlation is observed between the timing of increase in iNOS, and that of neuronal death in the TNBS model. Examination of the time course of TNBS treatment showed that neuron number was significantly decreased by 24 hr, with further decrease until day 4. This closely parallels the rapid increase in iNOS expression in the early stages of inflammation. Furthermore, no neuron loss is observed past day 4, when iNOS expression is not at peak levels, but damage scores are still high. This suggests that high NO levels generated by iNOS are responsible for neuron death in inflammation. 4.2 Activated immune cells damage cultured myenteric neurons using NO mediated mechanisms To assess whether high NO levels are in fact neurotoxic, we tested whether the addition of chemical NO donors to cultured neurons caused a reduction in survival. This took advantage of the co-culture model developed in the lab that combined myenteric neurons with smooth muscle cells and glia. This model has well described structural and functional properties, and provided a unique method to parallel inflammation in an in vitro system (Lourenssen et al., 2009). To better understand the culture model, we first 50 characterized the rate of NO release from the donors using the Griess reaction. This method of studying NO concentrations has been used previously to quantify NO levels from various sources (Hogaboam et al., 1995;Morales-Ruiz et al., 1997). Our data suggests that NO concentrations peak within minutes of addition to medium, with further increases in levels over the time course of our study. This data is consistent with results from other studies that demonstrated near instantaneous NO release by SNAP and SNP (Zaragoza et al., 1997;Zou & Cowley, Jr., 1997). Incubation of co-cultures with various concentrations of SNAP, SNP and DETANONOate for 48 hr resulted in a dose dependent decrease in the number of neurons and a concurrent reduction in axon outgrowth. Since the toxic effects of the NO donors are exerted primarily through NO mediated pathways, it can be concluded that high NO concentration exhibit neurotoxicity. Studies have assessed the nature of neuronal loss in the ENS after inflammation, but it is yet unclear whether this occurs proportionally among all neural phenotypes or there is a selective loss of a certain neuron population (Mizuta et al., 2000a;Linden et al., 2005). One study found that TNBS induced inflammation leads to a non-specific loss of about 20% of the myenteric neurons in guinea pigs (Linden et al., 2005). Another study in a rat model of DSS-induced colitis demonstrated specific loss of nNOS neurons (Mizuta et al., 2000b). In the order to clarify whether these neurons are more susceptible to damage than others to NO mediated damage, we investigated the effect of high NO concentrations on cultured myenteric neurons. Results showed a substantial decrease in neuron survival with elevated NO levels, which is similar to the loss sustained by the HuD stained neurons. Since nitrergic neurons only account for one 51 third of enteric neurons, a 31.6% loss of nNOS-positive neurons would not explain the 32.5% loss in total neurons. The possibility of more subtle and selective outcomes among the large diversity of neurons present in the ENS requires further research. In the next phase of the study, we tested whether high levels of NO generated by iNOS in immune cells could damage myenteric neurons in vitro. Cultured myenteric neurons from neonatal rats were used to model the ENS in an in vitro system. Additionally, immune cells from the peritoneal cavity of adult rats were used to replicate the actions of infiltrating immune cells in inflamed tissue. The peritoneal cells contain large numbers of macrophages and neutrophils, which act as the first line of defense against invasion of micro-organisms in the peritoneum (Morales-Ruiz et al., 1997). These cells contain the enzyme iNOS, which on activation produces large amounts of NO (Chinen et al., 1999). Therefore, they represented an ideal NO source for use in our in vitro experiments. Before using peritoneal cells as an NO source, it was necessary to confirm iNOS expression and the subsequent NO generation by these cells. Cultured peritoneal cells were stained with NADPH-diaphorase after activation to study iNOS expression. NOS requires the presence of nicotinamide adenine dinucleotide phosphate (NADPH) as an electron donor, and under defined fixation conditions the NADPH-diaphorase reaction is a histochemical marker for NOS (Downing, 1994). iNOS activity in the immune cells was minimal 1 hr post extraction, but was much higher by the 4 hr mark as indicated by the strong purple labeling. This data is consistent with the other studies that demonstrate elevated iNOS activity 4-8 hr after activation (Lorsbach et al., 1993). As no known immune cell-activators had been used to induce iNOS expression, we concluded that mechanical disturbance and possible exposure to bacteria from surroundings during 52 peritoneal lavage resulted in activation of peritoneal cells. In tandem with histochemical labeling, supernatants from wells containing immune cells were assayed for NO production at 24 hr using the Griess reaction. Immune cells were found to generate micromolar quantities of NO. Moreover, it has been shown that once activated, iNOS can generate micromolar quantities of NO for up to 5 days (Nussler & Billiar, 1993). Such concentrations have been shown to have cytotoxic effects in a variety of cell types (as reviewed in (MacMicking et al., 1997). The relative ease with which iNOS can be induced in vitro, and its capacity to produce high levels of NO for sustained durations offered a convenient method to examine whether iNOS affects ENS neuron survival. The addition of activated immune cells resulted in markedly reduced neuron survival and disrupted neurites. This was interpreted as a result of high NO levels, as the simultaneous inhibition of iNOS using L-NIL significantly attenuated neuron loss. A similar study, using peritoneal immune cells on a culture of myenteric neurons, was published in 2009 by the Ekblad group (Sand et al., 2009). They reported a similar reduction in neuron survival over a course of 48 hr, but the loss was attributed to the purportedly high levels of mast cells in the immune cell population. While mast cells are present, they represent only a very small proportion of the total cell number in normal animals, about 2-5% (Enerback & Svensson, 1980), and are thus unlikely to have a significant effect. Further, the addition of a mast cell stabilizer, doxantrazole, only prevented a portion of neuron loss (Sand et al., 2009), and might be attributed to the well-established role of mast cells in initiating the activation of other immune cells. In contrast, the use of the specific iNOS inhibitor L-NIL caused a near total prevention of neuron death following addition of immune cells to our neuronal co-culture model; strongly suggesting that iNOS from macrophages and neutrophils is the primary cause of 53 neuron loss. Other studies using CNS neurons have made similar observations, where locally high concentrations of NO generated by brain macrophages or astroglia caused neuron death (Chao et al., 1992;Mander et al., 2005). 4.3 Immune cells on trans-wells: Peritoneal immune cells were activated using LPS, and co-cultures were exposed to them using the trans-well model. The high levels of iNOS-generated NO diffused into the co-culture medium through the semi-permeable trans-well, while direct immune cell contact was prevented. Considering the nature of reactive oxygen and nitrogen intermediates, and other inflammatory cytokines such as TNF-α, IL-1β and IFN-γ secreted by macrophages and neutrophils, major decreases in neuron survival and axon outgrowth were anticipated. Instead, the diffusible immune derived factors exerted highly unexpected neurotrophic effects on cultured neurons. A substantial increase in axon outgrowth was observed in addition to total neuron survival after 48 hr incubation. Similar results were obtained when labeled for nitrergic neurons, indicating a possibly non specific neurotrophic effect of immune cell addition. These results seemingly contradict the previous ‗neurotoxic‘ effects observed by direct addition of immune cells to cultured neurons (Fig. 10). While the two experiments are essentially identical in terms of cell types used, period of incubation etc, the use of trans-wells to separate immune cells from neurons could possibly explain the conflicting results. Direct addition places neurons in close contact with immune cells, exposing them to locally high concentrations of NO, ONOO-, O2- and other reactive metabolites, thereby creating a neurotoxic environment. Instead, trans-wells separate the neurons 54 from immune cells and the aforementioned cytotoxic agents by a distance of 800900µm. This acts as a physical barrier for peroxynitrite, which is known to have short half-life in biological milieu (i.e., 10–20 ms) and is rapidly scavenged by a variety of extracellular reactions, most notably with CO2 (Alvarez et al., 2004). Additionally, the relatively restricted diffusion distance (<10-20µM) of peroxynitrite would further limit its capacity to cause oxidative damage to cells (Romero et al., 1999). Therefore, it can be inferred that the toxicity of iNOS generated NO would decrease as a function of distance between immune cells and neurons. As an interesting correlate, a study using neurons from the superior cervical ganglion reported a 25% loss when co-cultured with activated macrophages from the peritoneal cavity (Arantes et al., 2000). When direct contact between the two cell types was prevented by use of conditioned medium from cultured macrophages, neuron death was prevented. This suggests that direct cell to cell contact is necessary for the iNOS generated respiratory burst to cause neuron damage. The neuroprotective effects exerted by immune cell generated factors could be due to their ability to mediate production of neurotrophic factors from smooth muscle and accessory cells. Recently, a group showed secretion of glial-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) from enteric glia cells in response to the proinflammatory cytokines TNF and IL1 (von Boyen et al., 2006a;von Boyen et al., 2006b). Several lines of evidence indicate that neurotrophic factors like GDNF and NGF could counteract the detrimental effects of inflammation (Reinshagen et al., 2000). Thus, production of neurotrophins from cytokine-stimulated glia may play an important role in limiting the damage caused by the activated immune cells used in this study. 55 4.4 Conclusions Using a combination of in vivo and in vitro approaches, this study tested the hypothesis that infiltrating immune cells are responsible for alterations to the enteric nervous system in intestinal inflammation. In the TNBS model of induced colitis in rats, we have documented the presence of large number of infiltrating macrophages and neutrophils adjacent to the ENS in the early stages of inflammation. A rapid and pronounced increase in iNOS expression was also detected in the circular smooth muscle layers adjacent to the myenteric and submucosal plexuses. While previous work has characterized immune cell infiltration and iNOS expression in to sites of inflammation in the gut, mucosa and lamina propria regions were the primary sites of investigation (McCafferty et al., 1999). To the best of our knowledge, we are the first group to evaluate infiltrating neutrophils, macrophages and iNOS expression adjacent to ENS at early time-points following TNBS administration. Additionally, through the development and analysis of an intestinal co-culture model, we have determined that elevated NO levels generated by chemical donors are toxic to neurons in vitro. iNOS generated NO from activated peritoneal exudates cells was also shown to cause a similar reduction in neuron survival, which was prevented by the use of selective iNOS inhibitor L-NIL. While the direct addition of immune cells displayed neurotoxic effects, the use of trans-wells in these experiments to separate immune cells and neurons revealed unanticipated neurotrophic effects. We propose that these contradictory outcomes can be attributed to the difference in proximity of the two cell types. In conclusion, this study demonstrates the central role played by NO in mediating damage to neurons. 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