Neural stem cell transplantation in the enteric nervous system: roadmaps and roadblocks.

The enteric nervous system (ENS) is vulnerable to a variety of genetic, metabolic or environmental threats, resulting in clinical disorders characterized by loss or malfunction of neuronal elements. These disorders have been difficult to treat and there is much enthusiasm for novel therapies such as neural stem cell (NSC) transplantation to restore ENS function in diseased segments of the gut. Recent research has indicated the potential for a variety of innovative approaches to this effect using NSC obtained from the central nervous system (CNS) as well as gut derived enteric neuronal progenitors. The main goal of this review is to summarize the current status of NSC research as it applies to the ENS, delineate a roadmap for effective therapeutic strategies using NSC transplantation and point out the numerous challenges that lie ahead.

Inhibitors of advanced glycation end-products prevent loss of enteric neuronal nitric oxide synthase in diabetic rats.

Gastrointestinal dysfunction is common in diabetes, and several studies indicate that loss of neuronal nitrergic inhibition may play an important role in its pathogenesis. However, the mechanisms responsible for this effect remain largely unknown. We have previously shown that advanced glycation end-products (AGEs) formed by non-enzymatic glycation dependent processes, can inhibit the expression of intestinal neuronal nitric oxide synthase (nNOS) in vitro acting via their receptor, receptor for AGEs. We now hypothesized that this effect may also be important in experimental diabetes in vivo. We aimed to evaluate the role of AGEs on duodenal nNOS expression and the effects of aminoguanidine (a drug that prevents AGE formation) and ALT-711 (AGE cross-link breaker) in experimental diabetes. Streptozotocin induced diabetic rats were randomized to no treatment, treatment with aminoguanidine (1 g L(-1) daily through drinking water) at the induction of diabetes, or treatment with ALT-711 (3 mg kg(-1) intraperitoneally), beginning at week 6. A fourth group was used as healthy controls. We performed real time polymerase chain reaction, Western blotting and immunohistochemistry to detect nNOS expression. AGE levels were analysed using sandwich ELISA. Diabetes enhanced accumulation of AGEs in serum, an effect that was prevented by treatment with aminoguanidine and ALT-711. Further, diabetic rats showed a significant reduction in duodenal nNOS expression by mRNA, protein and immunocytochemistry, an effect that was prevented by aminoguanidine. ALT-711 had similar effects on nNOS protein and immunohistochemistry (but not on mRNA levels). The generation of AGEs in diabetes results in loss of intestinal nNOS expression and may be responsible for enteric dysfunction in this condition. This study suggests that treatment directed against AGEs may be useful for the treatment of gastrointestinal complications of diabetes.

Suppression of nNOS expression in rat enteric neurones by the receptor for advanced glycation end-products.

Diabetes mellitus results in a loss of neuronal nitric oxide synthase (nNOS) expression in the myenteric plexus but the underlying mechanisms remain unknown. We hypothesized that this may be mediated by advanced glycation end-products (AGEs), a class of modified protein adducts formed by non-enzymatic glycation that interact with the receptor for AGE (RAGE) and which are important in the pathogenesis of other diabetic complications. Whole mount preparations of longitudinal muscles with adherent myenteric plexus (LM-MPs) from the duodenum of adult male rats were exposed to glycated bovines serum albumin (AGE-BSA) or BSA for 24 h. Western blotting, immunohistochemistry and real-time reverse transcriptase polymerase chain reaction (RT-PCR) for mRNA showed a significant reduction in nNOS expression in LM-MPs after exposure to AGE-BSA. NO release, as measured by the Griess reaction, was also significantly reduced by AGE-BSA. A neutralizing antibody against RAGE attenuated the reduction of nNOS protein caused by AGE-BSA. Immunohistochemistry revealed co-localization of RAGE expression with Hu, a marker for neuronal cells but not for S-100, a glial marker. Advanced glycation end-products reduce nNOS expression in the rat myenteric neurones acting via the receptor RAGE. Our results suggest novel pathways for disruption of the nitrergic phenotype in diabetes.

Caspase inhibition increases survival of neural stem cells in the gastrointestinal tract.

Neural stem cell (NSC) transplantation is a promising tool for the restoration of the enteric nervous system in a variety of motility disorders. Post-transplant survival represents a critical limiting factor for successful repopulation. The aim of this study was to determine the role of both immunological as well as non-immune-mediated mechanisms on post-transplant survival of NSC in the gut. Mouse CNS-derived NSC (CNS-NSC) were transplanted into the pylorus of recipient mice with and without the addition of a caspase-1 inhibitor (Ac-YVAD-cmk) in the injection media. In a separate experiment, CNS-NSC were transplanted in the pylorus of mice that were immunosuppressed by administration of cyclosporin A (CsA). Apoptosis and proliferation of the implanted cells was assessed 1 and 7 days post-transplantation. Survival was assessed 1 week post-transplantation. The degree of immunoresponse was also measured. The addition of a caspase-1 inhibitor significantly reduced apoptosis, increased proliferation and enhanced survival of CNS-NSC. CsA-treatment did not result in improved survival. Our results indicate that caspase-1 inhibition, but not immunosuppression, improves survival of CNS-NSC in the gut. Pre-treatment with a caspase-1 inhibitor may be a practical method to enhance the ability of transplanted CNS-NSC to survive in their new environment.

The proteinase-activated receptor 2 is involved in nociception.

The proteinase-activated receptor 2 is expressed on a subset of primary afferent neurons and may participate in the neurogenic component of inflammation. We hypothesized that this receptor may also play a role in neuronal sensitization and contribute to the pathogenesis of pain in inflammatory conditions such as pancreatitis. Using a specific proteinase-activated receptor 2 activating peptide, we found evidence of such sensitization in vitro in the form of enhanced capsaicin- and KCl-evoked release of calcitonin gene-related peptide, a marker for nociceptive signaling. We then demonstrated that injection of the proteinase-activated receptor 2 activating peptide into the pancreatic duct can activate and sensitize pancreas-specific afferent neurons in vivo, as measured by Fos expression in the dorsal horn of the spinal cord. These observations suggest that proteinase-activated receptor 2 contributes to nociceptive signaling and may provide a novel link between inflammation and pain.

Neural stem cells express RET, produce nitric oxide, and survive transplantation in the gastrointestinal tract.

BACKGROUND & AIMS: Transplantation of neural stem cells (NSC) has been shown to be successful in a variety of experimental models of nongastrointestinal diseases. The aim of this study was to assess the potential of NSC transplantation as a therapeutic strategy for neuronal replacement in disorders of the enteric nervous system. METHODS: Central nervous system-derived NSC (CNS-NSC) were obtained from the subventricular zone of rat brain (E17). Expression of RET, GFRalpha1, and neuronal nitric oxide synthase (nNOS) was assessed by Western blot and immunocytochemistry. Nitric oxide (NO) production was assessed using the NO-sensitive fluorescent indicator DAF-2. CNS-NSC (labeled with CM-DiI) were transplanted into the pylorus of mice and fluorescent double-labeling immunostaining for betaIII-tubulin or PGP 9.5 and nNOS was performed at 2, 4, and 8 weeks after transplantation. RESULTS: Our results show that CNS-NSC express both the receptors (RET and GFRalpha1) for the enteric neurotrophin, GDNF; GDNF, in turn, induces expansion of the RET-expressing CNS-NSC population. Furthermore, CNS-NSC express nNOS and produce NO in vitro. When transplanted into the gut, CNS-NSC differentiate into neurons, continue to express nNOS and survive at least 8 weeks. CONCLUSIONS: We conclude that transplantation of CNS-NSC bears promise as a potential cellular replacement strategy for enteric neurons.

Nerve growth factor expression is up-regulated in the rat model of L-arginine-induced acute pancreatitis.

BACKGROUND & AIMS: In somatic pain models, increases in nerve growth factor (NGF) are linked to the development of pain and hyperalgesia. The aim of this study was to examine a rat model of acute necrotizing pancreatitis for changes in NGF expression. METHODS: NGF protein and messenger RNA (mRNA) levels in the pancreas were correlated with histopathologic changes during the course of acute necrotizing pancreatitis in rats induced by the intraperitoneal injection of L-arginine. Immunohistochemistry for NGF localization was performed on the pancreatic tissue. RESULTS: Two phases of NGF production were observed in the inflamed pancreas: an early release from pancreatic islets at 2 and 6 hours and a later increase in mRNA (18-fold at maximum) at 3 days and in protein levels (7-fold at maximum) at 5 days coinciding with maximum parenchymal necrosis. The intense NGF-like immunoreactivity was observed predominantly in the ductal cells in pancreas from rats with pancreatitis at 5 days. CONCLUSIONS: The development of acute necrotizing pancreatitis in this model leads to a significant increase in NGF production and appears to shift the major cellular sites of NGF production from the islets to the ductal cells. It is conceivable that NGF production in the inflamed pancreas is responsible for plastic changes in the sensory neurons that mediate peripheral sensitization and contribute to the generation of pain.

Ascorbic acid prevents beta-amyloid-induced intracellular calcium increase and cell death in PC12 cells.

Characteristics of Alzheimer's disease include loss of brain neurons associated with the deposit of beta-amyloid protein (A beta) which is thought to be toxic to neurons possibly via induction of intracellular calcium and generation of free radicals. On this basis, we have determined the effect of ascorbic acid on the cell death and calcium increase induced by A beta in PC12 cells. We found that ascorbic acid completely abolished A beta-induced calcium increase and cell death in PC12 cells, indicating that calcium elevation and cell death are associated phenomena induced by A beta that can be rescued by antioxidants. These results are important to understand the mechanisms by which A beta is toxic to neurons and suggest that antioxidants may be part of future treatments for Alzheimer's disease.

Na+/Ca2+ exchange in catfish retina horizontal cells: regulation of intracellular Ca2+ store function.

The role of the Na+/Ca2+ exchanger in intracellular Ca2+ regulation was investigated in freshly dissociated catfish retinal horizontal cells (HC). Ca2+-permeable glutamate receptors and L-type Ca2+ channels as well as inositol 1,4,5-trisphosphate-sensitive and caffeine-sensitive intracellular Ca2+ stores regulate intracellular Ca2+ in these cells. We used the Ca2+-sensitive dye fluo 3 to measure changes in intracellular Ca2+ concentration ([Ca2+]i) under conditions in which Na+/Ca2+ exchange was altered. In addition, the role of the Na+/Ca2+ exchanger in the refilling of the caffeine-sensitive Ca2+ store following caffeine-stimulated Ca2+ release was assessed. Brief applications of caffeine (1-10 s) produced rapid and transient changes in [Ca2+]i. Repeated applications of caffeine produced smaller Ca2+ transients until no further Ca2+ was released. Store refilling occurred within 1-2 min and required extracellular Ca2+. Ouabain-induced increases in intracellular Na+ concentration ([Na+]i) increased both basal free [Ca2+]i and caffeine-stimulated Ca2+ release. Reduction of external Na+ concentration ([Na+]o) further and reversibly increased [Ca2+]i in ouabain-treated HC. This effect was not abolished by the Ca2+ channel blocker nifedipine, suggesting that increases in [Na+]i promote net extracellular Ca2+ influx through a Na+/Ca2+ exchanger. Moreover, when [Na+]o was replaced by Li+, caffeine did not stimulate release of Ca2+ from the caffeine-sensitive store after Ca2+ depletion. The Na+/Ca2+ exchanger inhibitor 2',4'-dimethylbenzamil significantly reduced the caffeine-evoked Ca2+ response 1 and 2 min after store depletion.

Distribution of the inositol trisphosphate receptor in the catfish retina.

Inositol 1,4,5-trisphosphate (InsP3) mobilizes intracellular stored Ca2+ by binding to specific receptors that are similar to the ryanodine receptor of skeletal and cardiac muscle. We have immunolocalized the InsP3 receptor to the inner nuclear layer and limiting membranes of the catfish retina. Immunocytochemistry on dissociated retinal cells further localized the receptor in the horizontal, bipolar and Müller glial cells. Immunostaining of the rat retina localized the InsP3 receptor to the plexiform layers. These data show a different distribution of InsP3 receptor in the catfish retina compared to that of other vertebrates, that may be suggestive of a different functional role for this receptor in different species.

Isolation of an egg-laying hormone-binding protein from the gonad of Aplysia californica and its localization in oocytes.

A protein solubilized from a membrane preparation of the gonad of Aplysia californica has been isolated by affinity chromatography, using bag cell egg-laying hormone (ELH) as the bound ligand, and partially purified and characterized by gel electrophoresis. The protein has an apparent molecular weight of 52 kDa and consists of two disulfide-linked subunits of about 30 kDa each. The protein is glycosylated and has an acidic pI. Approximately 10-15 micrograms of this protein can be isolated from a single ovotestis, representing less than 1% of the total protein in the gonad; but the protein could not be detected in buccal mass or body wall, tissues which do not have apparent response to ELH. Antibodies generated against this ELH-binding protein (ELHBP) were used to localize sites in the ovotestis which might contain this molecule and thus represent targets for egg-laying hormone. Immunocytochemical results indicate that the oocytes are a rich source of this protein, since their cytoplasm was the only detectable site of immunoreactivity. Whether this binding protein represents an egg-laying hormone receptor is uncertain, but its prevalence in oocytes suggests that ELH plays a signaling role on these gametes.