Obligatory Activation of SRC and JNK by GDNF for Survival and Axonal Outgrowth of Postnatal Intestinal Neurons.

The neurotrophin GDNF acts through its co-receptor RET to direct embryonic development of the intestinal nervous system. Since this continues in the post-natal intestine, co-cultures of rat enteric neurons and intestinal smooth muscle cells were used to examine how receptor activation mediates neuronal survival or axonal extension. GDNF-mediated activation of SRC was essential for neuronal survival and axon outgrowth and activated the major downstream signaling pathways. Selective inhibition of individual pathways had little effect on survival but JNK activation was required for axonal maintenance, extension or regeneration. This was localized to axonal endings and retrograde transport was needed for central JUN activation and subsequent axon extension. Collectively, GDNF signaling supports neuronal survival via SRC activation with multiple downstream events, with JNK signaling mediating structural plasticity. These pathways may limit neuron death and drive subsequent regeneration during challenges in vivo such as intestinal inflammation, where supportive strategies could preserve intestinal function.

GDNF requires HIF-1α and RET activation for suppression of programmed cell death of enteric neurons by metabolic challenge.

Intestinal inflammation challenges both function and structure of the enteric nervous system (ENS). In the animal model of TNBS-induced colitis, an influx of immune cells causes early neuron death in the neuromuscular layers, followed by axonal outgrowth from surviving neurons associated with upregulation of the neurotrophin GDNF (glial cell line-derived neurotrophic factor). Inflammation could involve ischemia and metabolic inhibition leading to neuronal damage, which might be countered by a protective action of GDNF. This was examined in a primary co-culture model of rat myenteric neurons and smooth muscle, where metabolic challenge was caused by dinitrophenol (DNP), O-methyl glucose (OMG) or hypoxia. These caused the specific loss of 50% of neurons by 24 h that was blocked by GDNF both in vitro and in whole mounts. Neuroprotection was lost with RET inhibition by vandetanib or GSK3179106, which also caused neuron loss in untreated controls. Thus, both basal and upregulated GDNF levels signal via RET for neuronal survival. This includes a key role for upregulation of HIF-1α, which was detected in neurons in colitis, since the inhibitor chetomin blocked rescue by GDNF or ischemic pre-conditioning in vitro. In DNP-treated co-cultures, neuron death was not inhibited by zVAD, necrosulfonamide or GSK872, and cleaved caspase-3 or - 8 were undetectable. However, combinations of inhibitors or the RIP1kinase inhibitor Nec-1 prevented neuronal death, evidence for RIPK1-dependent necroptosis. Therefore, inflammation challenges enteric neurons via ischemia, while GDNF is neuroprotective, activating RET and HIF-1α to limit programmed cell death. This may support novel strategies to address recurrent inflammation in IBD.

Early inflammatory damage to intestinal neurons occurs via inducible nitric oxide synthase.

Intestinal inflammation affects the enteric nervous system (ENS) that lies adjacent to the smooth muscle layers. Previously, we showed that the loss of ENS neurons in animal models such as tri-nitrobenzene sulphonic acid (TNBS)-induced colitis was a limited and early event despite progressive worsening of inflammation. Here, we demonstrated that the rapid appearance of activated immune cells in the intestinal wall is selectively neurotoxic via iNOS-derived NO, using TNBS-induced colitis in both rats and mice, and a co-culture model of ENS neurons and smooth muscle. An influx of neutrophils and macrophages occurred within hours of initiation of rat colitis, correlating with iNOS expression, acutely elevated NO and neuronal death. In vitro, chemical donors of NO selectively caused axonal damage and neuronal death. These outcomes were similar to those seen with combined culture with either activated peritoneal immune cells or the immune cell lines RAW-264 and RBL-2H3. Immune cell-mediated neurotoxicity was blocked by the iNOS inhibitor L-NIL, and neuronal death was inhibited by the RIP-1 kinase inhibitor necrostatin. In a mouse model, the stereotypic loss of myenteric neurons by Day 4 post-TNBS was abrogated by the selective iNOS inhibitors L-NIL or 1400W without effect on other parameters of intestinal inflammation. Preservation of ENS neurons also ameliorated the hyperplasia of smooth muscle that is characteristic of intestinal inflammation, in line with prior work showing neural regulation of smooth muscle phenotype. This identifies a predominant pathway of immune cell damage to the ENS, where early, acute elevation of NO from iNOS can be cytotoxic to myenteric neurons.

Proliferation modulates intestinal smooth muscle phenotype in vitro and in colitis in vivo.

Intestinal inflammation causes an increased intestinal wall thickness, in part, due to the proliferation of smooth muscle cells, which impairs the contractile phenotype elsewhere. To study this, cells from the circular muscle layer of the rat colon (CSMC) were isolated and studied, both in primary culture and after extended passage, using quantitative PCR, Western blot analysis, and immunocytochemistry. By 4 days in vitro, both mRNA and protein for the smooth muscle marker proteins α-smooth muscle actin, desmin, and SM22-α were reduced by >50%, and mRNA for cyclin D1 was increased threefold, evidence for modulation to a proliferative phenotype. Continued growth caused significant further decrease in expression, evidence that phenotypic loss in CSMC was proportional to the extent of proliferation. In CSMC isolated at day 2 of trinitrobenzene sulfonic acid-induced colitis, flow cytometry and Western blotting showed that these differentiated markers were reduced in mitotic CSMC, while similar to control in nonmitotic CSMC. By day 35 post-trinitrobenzene sulfonic acid, when inflammation has resolved, CSMC were hypertrophic, but, nonetheless, showed markedly decreased expression of smooth muscle protein markers per cell. In vitro, day 35 CSMC displayed an accelerated loss of phenotype and increased thymidine uptake in response to serum or PDGF-BB. Furthermore, carbachol-induced expression of phospho-AKT (a marker of cholinergic response) was lost from day 35 CSMC in vitro, while retained in control cells. Therefore, proliferation reduces the expression of smooth-muscle-specific markers in CSMC, possibly leading to altered contractility. However, inflammation-induced proliferation in vivo also causes lasting changes that include unexpected priming for an exaggerated response to proliferative stimuli. Identification of the molecular mechanisms of intestinal smooth muscle cell phenotypic modulation will be helpful in reducing the detrimental effects of inflammation.

Analgesia and mouse strain influence neuromuscular plasticity in inflamed intestine.

BACKGROUND: Mouse models of inflammatory bowel disease (IBD) identify an impact on the enteric nervous system (ENS) but do not distinguish between Crohn's disease and ulcerative colitis phenotypes. In these models, analgesia is required, but its influence on different strains and disease outcomes is unknown. Therefore, changes to the ENS and intestinal smooth muscle were studied in trinitrobenzene sulfonic acid (TNBS) and dextran sodium sulfate (DSS) induced colitis to identify the effects of analgesia, and compared between two mouse strains. METHODS: Colitis was induced in CD1 or BALB/c mice receiving analgesia with either buprenorphine or tramadol. Euthanasia was on Day 8 (DSS) or Day 4 (TNBS). Outcomes were Disease Activity Index and cytokine assay, and quantitative histology and immunocytochemistry were used to evaluate effects of inflammation on neurons and smooth muscle. KEY RESULTS: In BALB/c mice, both models of colitis caused >2-fold increase in smooth muscle cell number. DSS caused axon proliferation without neuron loss while TNBS caused significant neuron loss and axonal damage. Buprenorphine (but not tramadol) was generally anti-inflammatory in both strains, but correlated with lethal outcomes to TNBS in BALB/c mice. CONCLUSIONS AND INFERENCES: Smooth muscle growth is common to both models of colitis. In contrast, ENS damage in TNBS is correlated with the severe response of a Crohn's disease-like phenotype, while DSS correlates with a milder, ulcerative colitis-like outcome in the deeper tissues. Analgesia with tramadol over buprenorphine is supported for mouse studies of IBD.

Stress increases descending inhibition in mouse and human colon.

BACKGROUND: A relationship between stress and the symptoms of irritable bowel syndrome (IBS) has been well established but the cellular mechanisms are poorly understood. Therefore, we investigated effects of stress and stress hormones on colonic descending inhibition and transit in mouse models and human tissues. METHODS: Stress was applied using water avoidance stress (WAS) in the animal model or mimicked using stress hormones, adrenaline (5 nM), and corticosterone (1 µM). Intracellular recordings were obtained from colonic circular smooth muscle cells in isolated smooth muscle/myenteric plexus preparations and the inhibitory junction potential (IJP) was elicited by nerve stimulation or balloon distension oral to the site of recording. KEY RESULTS: Water avoidance stress increased the number of fecal pellets compared to control (p < 0.05). WAS also caused a significant increase in IJP amplitude following balloon distension. Stress hormones also increased the IJP amplitude following nerve stimulation and balloon distension (p < 0.05) in control mice but had no effect in colons from stressed mice. No differences were observed with application of ATP between stress and control tissues, suggesting the actions of stress hormones were presynaptic. Stress hormones had a large effect in the nerve stimulated IJP in human colon (increased >50%). Immunohistochemical studies identified alpha and beta adrenergic receptor immunoreactivity on myenteric neurons in human colon. CONCLUSIONS & INFERENCES: These studies suggest that WAS and stress hormones can signal via myenteric neurons to increase inhibitory neuromuscular transmission. This could lead to greater descending relaxation, decreased transit time, and subsequent diarrhea.

Intestinal smooth muscle phenotype determines enteric neuronal survival via GDNF expression.

Intestinal inflammation causes initial axonal degeneration and neuronal death, as well as the proliferation of intestinal smooth muscle cells (ISMC), but subsequent axonal outgrowth leads to re-innervation. We recently showed that expression of glial cell-derived neurotrophic factor (GDNF), the critical neurotrophin for the post-natal enteric nervous system (ENS) is upregulated in ISMC by inflammatory cytokines, leading us to explore the relationship between ISMC growth and GDNF expression. In co-cultures of myenteric neurons and ISMC, GDNF or fetal calf serum (FCS) was equally effective in supporting neuronal survival, with neurons forming extensive axonal networks among the ISMC. However, only GDNF was effective in low-density cultures where neurons lacked contact with ISMC. In early-passage cultures of colonic circular smooth muscle cells (CSMC), polymerase chain reaction (PCR) and western blotting showed that proliferation was associated with expression of GDNF, and the successful survival of neonatal neurons co-cultured on CSMC was blocked by vandetanib or siGDNF. In tri-nitrobenzene sulfonic acid (TNBS)-induced colitis, immunocytochemistry showed the selective expression of GDNF in proliferating CSMC, suggesting that smooth muscle proliferation supports the ENS in vivo as well as in vitro. However, high-passage CSMC expressed significantly less GDNF and failed to support neuronal survival, while expressing reduced amounts of smooth muscle marker proteins. We conclude that in the inflamed intestine, smooth muscle proliferation supports the ENS, and thus its own re-innervation, by expression of GDNF. In chronic inflammation, a compromised smooth muscle phenotype may lead to progressive neural damage. Intestinal stricture formation in human disease, such as inflammatory bowel disease (IBD), may be an endpoint of failure of this homeostatic mechanism.

Defects in sensory nerve numbers and growth in mutant Kit and Steel mice.

The roles in the nervous system of the receptor tyrosine kinase Kit and its ligand, Steel factor, are unclear. We have now found first, that sensory nerve populations are reduced in mutant Kit and Steel mice, implicating Steel-Kit interactions in neuronal development. Second, sensory axonal regeneration (which occurs independently of nerve growth factor, or NGF) is impaired, while collateral sprouting (NGF dependent) is normal. Therefore, there is a selective involvement of Kit signal transduction pathways in nerve growth; supporting this, in wild-type animals Kit was up-regulated in regenerating, but unchanged in sprouting, sensory neurons. The receptor tyrosine kinase Kit thus contrasts with the receptor tyrosine kinase trkA, which is activated by the sprouting stimulus (NGF) but not by the axonal regeneration signal.

Early damage of sympathetic neurons after co-culture with macrophages: a model of neuronal injury in vitro.

Since activated immune cells may damage peripheral nerves during inflammation, we developed a co-culture model that permits the direct study of macrophage-induced neuronal damage. Sympathetic neurons were enzymatically isolated from neonatal mice and co-cultured with increasing numbers of peritoneal macrophages for 24 h. This caused rapid neuronal cell death, reducing neuronal number by 24.1 +/- 4% with the addition of 11.5 x 10(3) macrophages, representing a ratio of 8 macrophages per neuron. Nuclear analysis showed that cell death occurred by both apoptosis and necrosis. These effects were not mimicked by addition of macrophage-conditioned medium, and were prevented by 10 microM dexamethasone. Although no appreciable neuronal death occurred beyond 24 h, the density of neurites was decreased between 1 and 2 days of co-culture (p < 0.05). There is, therefore, a rapid induction of cytotoxicity by macrophages after their addition to the neuronal cultures, followed by axonal damage without neuronal cell death.

Lysophosphatidylserine potentiates nerve growth factor-induced differentiation of PC12 cells.

Since lysophosphatidylserine (LPS) is required for nerve growth factor (NGF)-induced secretion of histamine from rat mast cells, we investigated whether LPS might potentiate the effects of NGF in inducing neural differentiation of PC12 cells. Cell morphology was evaluated 48 h after addition of NGF, LPS or NGF + LPS. LPS alone was ineffective, but strongly promoted NGF-induced differentiation to give rise to cells that more closely resembled neurons in primary culture. LPS increased the number of PC12 cells that developed neurites in response to NGF (0.01-40 ng/ml), with the response to 1.0 ng/ml increasing from 17.8 +/- 2.2 to 50.8 +/- 4.1% when LPS was also present. Neurite length was also greater in PC12 cells receiving NGF + LPS: 17.8 +/- 2.2% of cells had neurites longer than three cell body diameters with 1.0 ng/ml NGF + 1 microg/ml LPS, compared to 1.6 +/- 1.6% with NGF alone. Further, cells responding to NGF + LPS typically developed only 1-2 neurites per cell (90.9%, 1 microg/ml LPS), compared with the multipolar appearance with NGF alone (71.1% with 3-6 neurites, 10 ng/ml NGF). LPS occurs at sites of tissue damage where NGF can also be present, and therefore may be a naturally-occurring modifier of neuronal structure and/or function.

Absence of the GluR2 receptor sensitizes mouse sympathetic neurons to nerve growth factor deprivation.

Over-activation of glutamate receptors is implicated in neurodegeneration. Using mice with a deletion in the GluR2 gene, we studied the sensitivity of sympathetic neurons to reduced levels of nerve growth factor (NGF), which can cause neuronal cell death. Under standard culture conditions of 50 ng/ml NGF, neurons from the superior cervical ganglion survived and grew equally well compared with wild type controls. However, the subsequent reduction of NGF levels caused significantly poorer survival among mutant neurons by 48 h, at 44+/-13% of control at 10 ng/ml NGF, and dropping further to 14+/-6% at 0.05 ng/ml NGF. These results suggest that the absence of GluR2 impairs the ability of these NGF-sensitive neurons to survive under limiting amounts of this neurotrophic factor.

Proteinase-activated receptor-2 activation evokes oesophageal longitudinal smooth muscle contraction via a capsaicin-sensitive and neurokinin-2 receptor-dependent pathway.

BACKGROUND: Intraluminal acid evokes sustained oesophageal longitudinal smooth muscle (LSM) contraction and oesophageal shortening, which may play a role in oesophageal pain and the aetiology of hiatus hernia. In the opossum model, this reflex has been shown to involve mast cell activation and release of neurokinins from capsaicin-sensitive neurons. The aim of this study was to determine whether proteinase-activated receptor-2 (PAR-2) activation evokes reflex LSM contraction via similar mechanisms. METHODS: Tension recording studies were performed using opossum oesophageal LSM strips in the presence and absence of pharmacological agents. In addition, the effect of trypsin on single isolated LSM cells was determined using videomicroscopy, and the expression of PAR-2 in oesophageal tissue was examined using immunohistochemistry. KEY RESULTS: The PAR-2 agonist trypsin evoked sustained, concentration-dependent contraction of LSM muscle strips, but had no effect on isolated LSM cells. The trypsin-induced contraction was blocked by capsaicin desensitization, substance P (SP) desensitization or application of the selective neurokinin-2 (NK-2) receptor antagonist MEN 10376. Immunohistochemistry revealed co-localization of SP, calcitonin gene-related peptide and PAR-2 in axons of opossum oesophageal LSM. CONCLUSIONS & INFERENCES: Longitudinal smooth muscle contraction induced by trypsin involves capsaicin-sensitive neurons and subsequent activation of NK-2, which is identical to the pathway involved in acid-induced LSM contraction and oesophageal shortening. This suggests that acid-induced LSM contraction may involve mast cell-derived mediators that activate capsaicin-sensitive neurons via PAR-2.

Selective loss of NGF-sensitive neurons following experimental colitis.

Nerve growth factor (NGF) enhances neuronal survival during injury to the mature central and peripheral nervous systems, but its potential as a neuroprotective factor in the enteric nervous system (ENS) has not been examined. We used the trinitrobenzene sulfonic acid (TNBS)-induced model of colitis to examine if NGF-sensitive neurons were selectively spared from inflammation-induced cell loss. Immunocytochemistry of whole mounts of the rat colon showed that total myenteric neuronal number decreased by 32.9% +/- 1.4% by 35 days after inflammation. At this time, the proportion of neurons expressing both the p75 and trkA receptor decreased to 38.4% from a control value of 62.0%. The distribution of expression of neural phenotypes among the NGF receptor-expressing population was differentially affected by inflammation, with selective decrease among cholinergic excitatory neurons and calbindin-expressing neurons, and a trend to increase among inhibitory nitrergic neurons. This is evidence of a novel mechanism whereby intestinal inflammation can give rise to a permanent imbalance between excitatory and inhibitory neural pathways, thus tending to compromise intestinal function.

Neural regulation of intestinal smooth muscle growth in vitro.

The loss of intrinsic neurons is an early event in inflammation of the rat intestine that precedes the growth of intestinal smooth muscle cells (ISMC). To study this relationship, we cocultured ISMC and myenteric plexus neurons from the rat small intestine and examined the effect of scorpion venom, a selective neurotoxin, on ISMC growth. By 5 days after neuronal ablation, ISMC number increased to 141+/-13% (n = 6) and the uptake of [(3)H]thymidine in response to mitogenic stimulation was nearly doubled. Atropine caused a dose-dependent increase in [(3)H]thymidine uptake in cocultures, suggesting the involvement of neural stimulation of cholinergic receptors in regulation of ISMC growth. In contrast, coculture of ISMC with sympathetic neurons increased [(3)H]thymidine uptake by 45-80%, which was sensitive to propranolol (30 microM) and was lost when the neurons were separated from ISMC by a permeable filter. Western blotting showed that coculture with myenteric neurons increased alpha-smooth muscle-specific actin nearly threefold to a level close to ISMC in vivo. Therefore, factors derived from enteric neurons maintain the phenotype of ISMC through suppression of the growth response, whereas catecholamines released by neurons extrinsic to the intestine may stimulate their growth. Thus inflammation-induced damage to intestinal innervation may initiate or modulate ISMC hyperplasia.

Intestinal inflammation modulates expression of the synaptic vesicle protein neuronal calcium sensor-1.

The calcium-binding protein neuronal calcium sensor 1 (NCS-1) is involved in modulation of neurotransmitter release in the peripheral and central nervous systems. Since intestinal inflammation impairs neurotransmitter release, we evaluated the expression of NCS-1 in the normal rat colon and in dinitrobenzene sulfonic acid (DNBS)-induced colitis. Immunocytochemistry and Western blots showed high levels of NCS-1 in the myenteric plexus and in axons in the smooth muscle layers; 23 +/- 2% of myenteric neurons were NCS-1 positive, with staining restricted to the largest neurons. NCS-1-positive axons decreased to 13.3 +/- 0.4% of total axons by day 2 and dropped further to 7.0 +/- 0.1% by day 4, returning to control levels by day 16. Dual-label Western blot analysis showed that the expression of NCS-1 relative to PGP 9.5 decreased by 50% on day 4 but returned to control by day 16. The selective loss of NCS-1 during colitis may underlie the altered neural function seen in the inflamed intestine.

NGF deprivation of adult rat brain results in cholinergic hypofunction and selective impairments in spatial learning.

Cholinergic hypofunction has often been correlated with a variety of behavioural impairments. In the present study, adult Wistar rats were intraventricularly infused with antibodies to nerve growth factor (anti-NGF) to examine the effects on cholinergic neurons of the basal forebrain, and on behavioural performance. Immunocytochemical techniques indicated that chronically infused anti-NGF penetrates into the basal forebrain, cortex, striatum, corpus callosum and hippocampus, confirming previous findings after a single injection. Treatment with anti-NGF for 1 or 2 weeks resulted in a significant decrease of 27-33% in density of choline acetyltransferase immunostaining of the cholinergic cell bodies in the medial septum and vertical diagonal band, and a 26% reduction in choline acetyltransferase enzyme activity in the septal area. An array of spatial learning Morris water maze tasks was used to distinguish between acquisition skills and the flexible use of learned information in novel tests. Rats subjected to the spatial learning paradigm received anti-NGF infusion for 2 weeks prior to and for another 2 weeks during the behavioural testing. The anti-NGF-treated animals were found to be no different from those receiving control serum in the Morris water maze acquisition task, either in the latency to find the platform or in the time spent searching in the training quadrant when the platform was removed. However, in consecutive extinction trials, anti-NGF rats continued to search in the empty training quadrant, suggesting the occurrence of perseveration; control rats expanded their search over other areas of the pool.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of inflammation-induced hypertrophy of rat intestinal smooth muscle cell.

Inflammation of the human intestine causes thickening of the smooth muscle layers, and studies in rats infected with Trichinella spiralis (Tsp) have shown hyperplasia of the intestinal smooth muscle cells (ISMC). We have shown that Tsp-induced inflammation caused a fivefold increase in total protein per ISMC over control, while ISMC from the noninflamed distal ileum also showed a threefold increase. The amount of alpha-smooth muscle (SM) actin per ISMC increased nearly 500% over control by postinfection (PI) day 6. The proportion of alpha-SM actin in the total cellular protein increased 200% by day 6 PI, indicating a higher density of alpha-SM actin in the hypertrophied ISMC. Gamma-SM actin mRNA increased sharply and was matched by an increased fractional content of gamma-SM actin protein. These increases in the smooth muscle-specific actins may affect force production and further demonstrate the plasticity of smooth muscle in the inflamed intestine.