Estrogen receptor ß controls proliferation of enteric glia and differentiation of neurons in the myenteric plexus after damage.

Injury to the enteric nervous system (ENS) can cause several gastrointestinal (GI) disorders including achalasia, irritable bowel syndrome, and gastroparesis. Recently, a subpopulation of enteric glial cells with neuronal stem/progenitor properties (ENSCs) has been identified in the adult ENS. ENSCs have the ability of reconstituting the enteric neuronal pool after damage of the myenteric plexus. Since the estrogen receptor ß (ERß) is expressed in enteric glial cells and neurons, we investigated whether a selective ERß agonist, LY3201, can influence neuronal and glial cell differentiation. Myenteric ganglia from the murine muscularis externa were isolated and cultured in either glial cell medium or neuronal medium. In glial cell medium, the number of glial progenitor cells (Sox10+) was increased by fourfold in the presence of LY3201. In the neuronal medium supplemented with an antimitotic agent to block glial cell proliferation, LY3201 elicited a 2.7-fold increase in the number of neurons (neurofilament+ or HuC/D+). In addition, the effect of LY3201 was evaluated in vivo in two murine models of enteric neuronal damage and loss, namely, high-fat diet and topical application of the cationic detergent benzalkonium chloride (BAC) on the intestinal serosa, respectively. In both models, treatment with LY3201 significantly increased the recovery of neurons after damage. Thus, LY3201 was able to stimulate glial-to-neuron cell differentiation in vitro and promoted neurogenesis in the damaged myenteric plexus in vivo. Overall, our study suggests that selective ERß agonists may represent a therapeutic tool to treat patients suffering from GI disorders, caused by excessive neuronal/glial cell damage.

Alterações morfométricas no plexo mioentérico do cólon menor equino distendido experimentalmente / Morphometrical alterations in myoenteric plexus of experimentally distended equine small colon

O cólon menor dos equinos é frequentemente acometido por afecções obstrutivas, sendo a disfunção da motilidade uma complicação comum após o tratamento cirúrgico. Este transtorno pode estar relacionado com lesões no plexo mioentérico ocorridas durante a distensão intestinal, contudo pouco se sabe sobre sua fisiopatologia. O objetivo deste estudo foi avaliar as alterações morfológicas na inervação mioentérica em segmentos de cólon menor de eqüinos submetidos à distensão intraluminal com pressão suficiente para promover redução da perfusão microvascular (isquemia parcial) da parede intestinal. Nove eqüinos foram submetidos à distensão do cólon menor por 4h. Fragmentos da parede intestinal foram colhidos antes e ao final da distensão, após 1,5 e 12 horas de reperfusão no segmento experimental e ao final do procedimento em segmento distante. As amostras foram fixadas e processadas rotineiramente e secções histológicas foram coradas com cresil violeta para a morfometria. Por meio de um software de análise de imagens, obtiveram-se a área, o perímetro e os diâmetros mínimo e máximo do corpo neuronal, do núcleo e do nucléolo dos neurônios e as áreas do citoplasma e do nucleoplasma. Verificou-se redução significativa (P<0,05) das áreas do corpo neuronal e do citoplasma ao final da distensão, retornando aos valores equivalentes aos iniciais durante a reperfusão. Conclui-se que a distensão intraluminal alterou morfologicamente os neurônios do plexo mioentérico. Essas modificações morfológicas podem estar associadas e contribuir para explicar a disfunção da motilidade freqüentemente observada em casos clínicos.

The equine small colon is frequently affected by obstruction, and intestinal motility dysfunction is a common complication after its surgical treatment. This fact may be related to myoenteric plexus lesion caused by distention; however, little is known about the pathophysiology of this condition. The objective of this study was to evaluate the morphological alterations in the myoenteric inervation of segments of small colon of horses subjected to intraluminal distension with reduction of the microvascular perfusion (partial ischemia) of the intestinal wall. Nine horses were used to promote distension of on segment of small colon for 4 hours. Samples of intestinal wall were collected before and at the end of the distension, after 1.5 and 12 hours of reperfusion in the experimental segment and at the end of the procedure in a different distant segment. Samples were processed and histological sections were stained with cresyl violet for the morphometric studies. An image analyzer software was used to measure perimeter, diameter, and area of the neuronal body, nucleus and nucleolus of the neurons and the areas of the cytoplasm and nucleoplasm. Significant reductions (P<0.05) in the areas of the neuronal body and cytoplasm were detected at the end of intestinal distension, returning to the basal values during the reperfusion. In conclusion, intraluminal distension promoted changes in the morphology of the neurons of myoenteric plexus. These morphological modifications may be associated to the motility dysfunction frequently observed in clinical cases.

Myenteric plexus injury and apoptosis in experimental colitis.

Intestinal inflammatory conditions are associated with structural and functional alterations of the enteric nervous system (ENS). While injury to the enteric nervous system is well described, the mechanisms of neuronal injury and neuronal cell loss remain unclear. The aim of the present study was to examine the neural consequences of distal colitis and to assess the role of neutrophil granulocytes in mediating these changes. Colitis was induced in C3H/HEN female mice with dinitrobenzene sulfonic acid. The mice were then sacrificed at 0.5, 1, 1.5, 2, 3, 4, 6, 12, 24, 120 h post instillation of dinitrobenzene sulfonic acid. The inflammatory response was assessed by macroscopic damage score, myeloperoxidase activity and histology. HuC/D and PGP 9.5 immunostaining was used to examine myenteric plexus density and structure, neural cell body numbers and distribution in cross-section and whole mount preparations. Apoptosis was investigated in whole mount preparations double stained with HuC/D and activated caspase-3 or cleaved poly (ADP-ribose) polymerase (PARP). Dinitrobenzene sulfonic acid-induced colitis was associated with a rapid and significant loss of HuC/D immunoreactive myenteric plexus neuronal cell bodies (42% decrease relative to control) that remained unchanged between 6 and 120 h. No change in myenteric plexus density was observed with PGP 9.5 immunostaining. Neuronal apoptosis was evident between 0.5 and 3 h. PARP immunoreactive neurons ranged between 1% and 2.5%. Colitis was associated with significant impairment in colonic propulsive function. Pre-treatment of mice with anti-neutrophil serum attenuated the inflammatory response and partially reduced the extent of myenteric plexus neuronal cell loss. Taken together, these data suggest that acute colitis is associated with loss of myenteric plexus neurons that is partly mediated by neutrophil granulocyte infiltration and is accompanied by impairment of colonic motility.

Profile of the cell body of nadh-diaphorase positive myenteric neurons from the stomach of rats (Rattus norvegicus) subjected to chronic alcoholism

O objetivo do trabalho foi comparar o perfil dos corpos celulares dos neurônios mioentéricos NADH-diaforase positivos do estômago de ratos. Utilizou-se 10 animais (Rattus norvegicus), provenientes dos grupos: a) controle (n=5), que durante 210 dias receberam, ad libitum, dieta com teor protéico normal (22 por cento) e água; e b) experimental (n=5), que durante 210 dias receberam, ad libitum, ração com teor protéico normal (22 por cento) e aguardente-de-cana diluída a 30 Gay Lussac (30º v/v). Os estômagos coletados foram submetidos à técnica de evidenciação neuronal. A mensuração do perfil dos corpos celulares dos neurônios (n=1.000) foi através de um Sistema Computadorizado de Análise de Imagem. O perfil dos corpos celulares dos neurônios do grupo controle ficou entre 60,16 a 638,64 m2. No grupo experimental variou de 40,84 a 599,15 µm2. Constatamos redução significante no tamanho do corpo celular, aumento de neurônios pequenos e diminuição de neurônios grandes

Estudo morfoquantitativo do plexo mioentérico do duodeno de ratos submetidos a carência de proteínas e vitaminas do complexo B / Morphoquantitative study of the duodenum myenteric plexus from rats subjected to lack of proteic and complex B vitamins

Este estudo teve como objetivo avaliar os efeitos das carências protéica e vitamínica sobre os neurônios do plexo mioentérico do duodeno de ratos. 24 animais, aos 90 dias de idade, foram divididos em dois grupos: controle (n=12) e experimental (n=12). Durante 120 dias, o grupo controle recebeu ração com teor protéico de 22 por cento, vitaminas e minerais; o grupo experimental, 8 por cento, sem suplementação de vitaminas. Após período experimental, coletou-se o sangue dos animais e retirou-se o duodeno para análise ultraestrutural e morfoquantitativa (NADH-d; NADPH-d). O grupo experimental apresentou proteínas totais e albumina plasmática reduzidas e peso inferior aos do grupo controle. A densidade (NADPH-d) foi maior e significante para o grupo experimental; na técnica da NADH-d, o perfil neuronal diferiu entre os grupos. Os aspectos ultraestruturais mostraram-se semelhantes entre os grupos. Os resultados permitem concluir que a dieta imposta promoveu um quadro moderado de desnutrição, não provocando alterações quantitativas, porém levou a alterações no perfil neuronal

Distribution of pituitary adenylyl cyclase activating peptide (PACAP) immunoreactivity in neurons of the guinea-pig digestive tract and their projections in the ileum and colon.

Pituitary adenylyl cyclase activating peptide (PACAP) is a novel hypothalamic peptide that is widely distributed in neurons, including those of the gastrointestinal tract. In this study, a polyclonal antiserum directed against PACAP-27 was used to investigate the localisation of PACAP throughout the gut and to determine the projections of PACAP-immunoreactive (IR) neurons in the guinea-pig small and large intestines. PACAP-IR fibres were seen in the myenteric and submucous plexuses, in the longitudinal and circular muscle layers and around blood vessels of the submucosa throughout the gut. In both the small and large intestine, PACAP-IR cell bodies, most with Dogiel type-I morphology, were seen in the myenteric ganglia following colchicine treatment. Lesion studies (myotomy and myectomy operations) revealed that PACAP-IR interneurons projected anally in the ileum and colon. Myectomy operations resulted in a loss of PACAP-IR fibres in the circular muscle under the operation, whereas PACAP-IR fibres remained in the submucosa and around blood vessels. Following extrinsic denervation of the ileum, the number of PACAP-IR fibres in the submucosal ganglia and around blood vessels decreased. This suggests that a portion of PACAP-IR fibres supplying the submucosal ganglia and blood vessels have an extrinsic source. To investigate this, immunohistochemical studies were performed on sympathetic and dorsal root ganglia. Numerous reactive cells were seen in the dorsal root ganglia, but none was seen in sympathetic pre- or paravertebral ganglia.

Neuroplasticity in the smooth muscle of the myenterically and extrinsically denervated rat jejunum.

The objective of this study was to examine the effects of two different denervation procedures on the distribution of nerve fibers and neurotransmitter levels in the rat jejunum. Extrinsic nerves were eliminated by crushing the mesenteric pedicle to a segment of jejunum. The myenteric plexus and extrinsic nerves were eliminated by serosal application of the cationic surfactant benzyldimethyltetradecylammonium chloride (BAC). The effects of these two denervation procedures were evaluated at 15 and 45 days. The level of norepinephrine in whole segments of jejunum was initially reduced by more than 76% after both denervation procedures, but by 45 days the level of norepinephrine was the same as in control tissue. Tyrosine hydroxylase (noradrenergic nerve marker) immunostaining was absent at 15 days, but returned by 45 days. However, the pattern of noradrenergic innervating axons was altered in the segment deprived of myenteric neurons. Immunohistochemical studies showed protein gene product 9.5 (PGP 9.5)-immunoreactive fibers in whole-mount preparations of the circular smooth muscle in the absence of the myenteric plexus and extrinsic nerves. At 45 days, the number of nerve fibers in the circular smooth muscle increased. Vasoactive intestinal polypeptide (VIP)-immunoreactive fibers, a subset of the PGP 9.5 nerve fibers, were present in the circular smooth muscle at both time points examined. Choline acetyltransferase (CAT) activity and VIP and leucine enkephalin levels were measured in separated smooth muscle and submucosa-mucosal layers of the denervated jejunum. VIP and leucine-enkephalin levels were no different from control in tissue that was extrinsically denervated alone. However, the levels of these peptides were elevated two-fold in the smooth muscle 15 and 45 days after myenteric and extrinsic denervation. In the submucosa-mucosa, VIP and leucine enkephalin levels also were elevated two-fold at 15 days, but comparable to control at 45 days. CAT activity was equal to control in the smooth muscle but elevated two-fold in the submucosa-mucosa at both times. These results provide evidence for innervation of the circular smooth muscle by the submucosal plexus. Moreover, these nerve fibers originating from the submucosal plexus proliferate in the absence of the myenteric plexus. Furthermore, the myenteric neurons appear to be essential for normal innervation of the smooth muscle by the sympathetic nerve fibers. It is speculated that the sprouting of the submucosal plexus induced by myenteric plexus ablation is mediated by increased production of trophic factors in the hyperplastic smooth muscle.