Maria Adelaide Micci

Acute pancreatitis results in referred mechanical hypersensitivity and neuropeptide up-regulation that can be suppressed by the protein kinase inhibitor k252a ☆

Although pain is a cardinal feature of pancreatitis, its pathogenesis is poorly understood and treatment remains difficult. Nociceptive sensitization in several somatic pain models has been associated with activation of protein kinases including trkA, protein kinase C, and protein kinase A. We therefore tested the hypothesis that systemic treatment with a kinase inhibitor, k252a, known to inhibit all of these kinases would alleviate pain in an animal model of pancreatitis. Von Frey filament testing of somatic referral regions was evaluated as a method to measure referred pain in a rat model of acute necrotizing pancreatitis induced by L-arginine. Rats with pancreatitis showed increased sensitivity to abdominal stimulation with Von Frey filament. This referred mechanical sensitivity was associated with an 8-fold increase in levels of phosphorylated trkA in the pancreas and with significant up-regulation of both calcitonin gene-related peptide and preprotachykinin mRNA expression in thoracic dorsal root ganglia and with increased calcitonin gene-related peptide and substance P immunoreactivity in spinal cord segment T10. Treatment with the kinase inhibitor k252a suppressed the phosphorylation of trkA in the pancreas as well as reversed both the behavioral changes and the increase in neuropeptide expression associated with pancreatitis.

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

Abstract  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.

Neural stem cells for the treatment of disorders of the enteric nervous system: Strategies and challenges

The main goal of this review is to summarize the status of the research in the field of stem cells transplantation, as it is applicable to the treatment of gastrointestinal motility. This field of research has advanced tremendously in the past 10 years, and recent data produced in our laboratories as well as others is contributing to the excitement on the use of neural stem cells (NSC) as a valuable therapeutic approach for disorders of the enteric nervous system characterized by a loss of critical neuronal subpopulations. There are several sources of NSC, and here we describe therapeutic strategies for NSC transplantation in the gut. These include using NSC as a relatively nonspecific cellular replacement strategy in conditions where large populations of neurons or their subsets are missing or destroyed. As with many other recent “breakthroughs” stem cell therapy may eventually prove to be overrated. However, at the present time, it does appear to provide the hope for a true cure for many currently intractable diseases of both the central and the peripheral nervous system. Certainly more extensive research is needed in this field. We hope that our review will encourage new investigators in entering this field of research ad contribute to our knowledge of the potentials of NSC and other cells for the treatment of gastrointestinal dysmotility. Developmental Dynamics 236:33–43, 2007.

Improvement of gastric motility with gastric electrical stimulation in STZ-induced diabetic rats

Aims: The aims of this study were to observe whether gastric motility was impaired in streptozotocin (STZ)-induced diabetic rats and whether gastric electrical stimulation was able to restore the impaired motility. Methods: Ten control rats and 30 STZ-induced diabetic rats were used in this study. Gastric slow waves were recorded at baseline and 0, 1, 2, 3 and 4 weeks after the injection of STZ or vehicle. Gastric emptying with (long or short pulses) or without gastric electrical stimulation was measured 6 weeks after STZ injection in a group of 10 diabetic rats each. Results: (1) STZ injection resulted in hyperglycemia and weight loss. (2) Gastric motility was impaired in the diabetic rats. The percentage of normal slow waves was progressively reduced 2 weeks after STZ injection. Compared with the control rats, gastric emptying in the diabetic rats was significantly delayed 6 weeks after STZ injection (60 ± 3 vs. 79 ± 2%, p < 0.02). (3) Gastric electrical stimulation with either long or short pulses accelerated gastric emptying in the diabetic rats. (4) Gastric electrical stimulation with long but not short pulses was capable of normalizing gastric dysrhythmia in the diabetic rats. Conclusion: Our data show that gastric motility is impaired in STZ-induced diabetic rats as reflected by a progressive reduction in the percentage of normal gastric slow waves and delayed gastric emptying. Moreover, here we show that gastric electrical stimulation normalizes delayed gastric emptying in diabetic rats and this normalization is not attributed to the effect of gastric electrical stimulation on gastric slow waves.

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

Characteristics of Alzheimers 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 Alzheimers disease.

Traumatic Brain Injury-Induced Dysregulation of the Circadian Clock

Circadian rhythm disturbances are frequently reported in patients recovering from traumatic brain injury (TBI). Since circadian clock output is mediated by some of the same molecular signaling cascades that regulate memory formation (cAMP/MAPK/CREB), cognitive problems reported by TBI survivors may be related to injury-induced dysregulation of the circadian clock. In laboratory animals, aberrant circadian rhythms in the hippocampus have been linked to cognitive and memory dysfunction. Here, we addressed the hypothesis that circadian rhythm disruption after TBI is mediated by changes in expression of clock genes in the suprachiasmatic nuclei (SCN) and hippocampus. After fluid-percussion TBI or sham surgery, male Sprague-Dawley rats were euthanized at 4 h intervals, over a 48 h period for tissue collection. Expression of circadian clock genes was measured using quantitative real-time PCR in the SCN and hippocampus obtained by laser capture and manual microdissection respectively. Immunofluorescence and Western blot analysis were used to correlate TBI-induced changes in circadian gene expression with changes in protein expression. In separate groups of rats, locomotor activity was monitored for 48 h. TBI altered circadian gene expression patterns in both the SCN and the hippocampus. Dysregulated expression of key circadian clock genes, such as Bmal1 and Cry1, was detected, suggesting perturbation of transcriptional-translational feedback loops that are central to circadian timing. In fact, disruption of circadian locomotor activity rhythms in injured animals occurred concurrently. These results provide an explanation for how TBI causes disruption of circadian rhythms as well as a rationale for the consideration of drugs with chronobiotic properties as part of a treatment strategy for TBI.

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

Abstract  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.

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

Abstract  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.

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

Abstract  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.

Divergent fate and origin of neurosphere-like bodies from different layers of the gut

Enteric neural stem cells (ENSCs) are a population of neural crest-derived multipotent stem cells present in postnatal gut that may play an important role in regeneration of the enteric nervous system. In most studies, these cells have been isolated from the layer of the gut containing the myenteric plexus. However, a recent report demonstrated that neurosphere-like bodies (NLBs) containing ENSCs could be isolated from mucosal biopsy specimens from children, suggesting that ENSCs are present in multiple layers of the gut. The aim of our study was to assess whether NLBs isolated from layers of gut containing either myenteric or submucosal plexus are equivalent. We divided the mouse small intestine into two layers, one containing myenteric plexus and the other submucosal plexus, and assessed for NLB formation. Differences in NLB density, proliferation, apoptosis, neural crest origin, and phenotype were investigated. NLBs isolated from the myenteric plexus layer were present at a higher density and demonstrated greater proliferation, lower apoptosis, and higher expression of nestin, p75, Sox10, and Ret than those from submucosal plexus. Additionally, they contained a higher percentage of neural crest-derived cells (99.4 ± 1.5 vs. 0.7 ± 1.19% of Wnt1-cre:tdTomato cells; P < 0.0001) and produced more neurons and glial cells than those from submucosal plexus. NLBs from the submucosal plexus layer expressed higher CD34 and produced more smooth muscle-like cells. NLBs from the myenteric plexus layer contain more neural crest-derived ENSCs while those from submucosal plexus appear more heterogeneous, likely containing a population of mesenchymal stem cells.