Effects of probenecid and brilliant blue G on rat enteric glial cells following intestinal ischemia and reperfusion.

The P2X7 receptor participates in several intracellular events and acts with the pannexin-1 channel. This study examined the effects of probenecid (PB) and brilliant blue G (BBG), which are antagonists of the pannexin-1 channel and P2X7 receptor, respectively, on rat ileum enteric glial cells after on ischemia and reperfusion. The ileal vessels were occluded for 45 min with nontraumatic vascular tweezers, and reperfusion was performed for periods of 24 h and 14 and 28 days. After ischemia (IR groups), the animals were treated with BBG (BG group) or PB (PB group). The double-labeling results demonstrated the following: the P2X7 receptor was present in enteric glial cells (S100ß) and enteric neurons positive for HuC/D; enteric glial cells exhibited different phenotypes; some enteric glial cells were immunoreactive to only S100ß or GFAP; and the pannexin-1 channel was present in enteric glial cells (GFAP). Density (in cells/cm2) analyses showed that the IR group exhibited a decrease in the number of cells immunoreactive for the P2X7 receptor, pannexin-1, and HuC/D and that treatment with BBG or PB resulted in the recovery of the numbers of these cells. The number of glial cells (S100ß and GFAP) was higher in the IR group, and the treatments decreased the number of these cells to the normal value. However, the PB group did not exhibit recovery of S100ß-positive glia. The cell profile area (µm2) of S100ß-positive enteric glial cells decreased to the normal value after BBG treatment, whereas no recovery was observed in the PB group. The ileum contractile activity was decreased in the IR group and returned to baseline in the BG and PB groups. BBG and PB can effectively induce the recovery of neurons and glia cells and are thus potential therapeutic agents in the treatment of gastrointestinal tract diseases.

Enteric glial cells immunoreactive for P2X7 receptor are affected in the ileum following ischemia and reperfusion.

The aim of this study was to analyze the effect of ischemia and reperfusion injury (IS) on enteric glial cells (EGCs) and neurons immunoreactive for the P2X7 receptor. Intestinal ischemia was induced by obstructing blood flow in the ileal vessels for 35 min. Afterwards, the vessels were reperfused for 14 days. Tissues were prepared for immunohistochemical labeling of P2X7 receptor, HuC/D (Hu) (pan-neuronal marker) and S100ß (glial marker); HuC/D (Hu) and glial fibrillary acidic protein (GFAP, glial marker)/DAPI (nuclear marker); or S100ß and GFAP/DAPI. Qualitative and quantitative analyses of colocalization, density, profile area and cell proliferation were performed via fluorescence and confocal laser scanning microscopy. The quantitative analyses revealed that a) neurons and EGCs were immunoreactive for P2X7 receptor; b) the P2X7 receptor immunoreactive cells and Hu immunoreactive neurons were reduced after 0 h and 14 days of reperfusion; c) the S100ß and GFAP immunoreactive EGCs were increased; d) the profile area of S100ß immunoreactive EGCs was increased by IS; e) few GFAP immunoreactive proliferated at 14 days of reperfusion; f) distinct populations of glial cells can be discerned: S100ß+/GFAP+ cells, S100ß+/GFAP- cells and S100ß-/GFAP + cells; g) histological analysis revealed less alterations in the epithelium cells in the IS groups and h) myeloperoxidase reaction revealed increased of the neutrophils in the lamina propria in the IS groups. This study showed that IS is associated with significant neuronal loss, increase of glial cells and altered purinergic receptor expression and that these changes may contribute to intestinal disorders.

Blockage of the P2X7 Receptor Attenuates Harmful Changes Produced by Ischemia and Reperfusion in the Myenteric Plexus.

INTRODUCTION: Our work analyzed the effects of a P2X7 receptor antagonist, Brilliant Blue G (BBG), on rat ileum myenteric plexus following ischemia and reperfusion (ISR) induced by 45 min of ileal artery occlusion with an atraumatic vascular clamp with 24 h (ISR 24-h group) or 14 d of reperfusion (ISR 14-d group). MATERIAL AND METHODS: Either BBG (50 mg/kg or 100 mg/kg, BBG50 or BBG100 groups) or saline (vehicle) was administered subcutaneously 1 h after ischemia in the ISR 24-h group or once daily for the 5 d after ischemia in the ISR 14-d group (n = 5 per group). We evaluated the neuronal density and profile area by examining the number of neutrophils in the intestinal layers, protein expression levels of the P2X7 receptor, intestinal motility and immunoreactivity for the P2X7 receptor, nitric oxide synthase, neurofilament-200, and choline acetyl transferase in myenteric neurons. RESULTS: The neuronal density and profile area were restored by BBG following ISR. The ischemic groups showed alterations in P2X7 receptor protein expression and the number of neutrophils in the intestine and decreased intestinal motility, all of which were recovered by BBG treatment. CONCLUSION: We concluded that ISR morphologically and functionally affected the intestine and that its effects were reversed by BBG treatment, suggesting the P2X7 receptor as a therapeutic target.

Morphological Characterization of the Myenteric Plexus of the Ileum and Distal colon of Dogs Affected by Muscular Dystrophy.

Duchenne-like muscular dystrophy (canine dystrophinopathy) is a hereditary degenerative disease characterized by muscle changes similar to those described for Duchenne muscular dystrophy (DMD) and by alterations in the smooth muscles of the gastrointestinal tract. Some authors have suggested that these abnormalities may be associated with intestinal motility. This study analyzed the nitrergic and cholinergic neurons and P2X7 receptor expression in the myenteric plexus of the ileum and distal colon of dogs with muscular dystrophy. Immunohistochemical techniques were used to detect nitric oxide synthase (NOS) and acetylcholine transferase (ChAT) expression and to label all HuC/D- and P2X7 receptor-immunoreactive (IR) neurons. Transmission electron microscopy and basic histology were performed for further analysis. The results showed that nitrergic neurons exhibited a Dogiel type I morphology in the ileum and distal colon. The neuronal profile results showed that there were fewer NOS-, ChAT-, and HuC/D-IR neurons in the ileum than in the distal colon in the dystrophic (DT) dogs. Additionally, there were more NOS-, ChAT- and HuC/D-IR neurons per ganglion in the distal colon than in the ileum. The P2X7 receptor-expressing neurons colocalized with nitrergic and cholinergic neurons. Transmission and light microscopy revealed collagen between the muscle fibers, between the circular and longitudinal muscle layers and within the myenteric ganglia of dogs with muscular dystrophy. These findings provide a morphological description of the myenteric neurons in the ileum and distal colon of these DT dogs and may contribute to a better understanding of the gastrointestinal disorders found in patients with DMD. Anat Rec, 301:673-685, 2018. © 2017 Wiley Periodicals, Inc.

Submucosal neurons and enteric glial cells expressing the P2X7 receptor in rat experimental colitis.

The aim of this study was to evaluate the effect of ulcerative colitis on the submucosal neurons and glial cells of the submucosal ganglia of rats. 2,4,6-Trinitrobenzene sulfonic acid (TNBS; colitis group) was administered in the colon to induce ulcerative colitis, and distal colons were collected after 24h. The colitis rats were compared with those in the sham and control groups. Double labelling of the P2X7 receptor with calbindin (marker for intrinsic primary afferent neurons, IPANs, submucosal plexus), calretinin (marker for secretory and vasodilator neurons of the submucosal plexus), HuC/D and S100ß was performed in the submucosal plexus. The density (neurons per area) of submucosal neurons positive for the P2X7 receptor, calbindin, calretinin and HuC/D decreased by 21%, 34%, 8.2% and 28%, respectively, in the treated group. In addition, the density of enteric glial cells in the submucosal plexus decreased by 33%. The profile areas of calbindin-immunoreactive neurons decreased by 25%. Histological analysis revealed increased lamina propria and decreased collagen in the colitis group. This study demonstrated that ulcerative colitis affected secretory and vasodilatory neurons, IPANs and enteric glia of the submucosal plexus expressing the P2X7 receptor.

The Effect of Ischemia and Reperfusion on Enteric Glial Cells and Contractile Activity in the Ileum.

BACKGROUND: We investigated the effects of ischemia followed by different periods of reperfusion (I/R) on immunoreactive S100ß-positive glial and Hu-immunoreactive neurons co-expressing the P2X2 receptor in the myenteric plexus of the rat ileum. METHODS: The ileal artery was occluded for 35 min with an atraumatic vascular clamp. The animals were killed 24 h, 72 h, and 1 week after ischemia. Sham animals were not submitted to ileal artery occlusion. The relative density, size, and co-localization of P2X2 receptor-expressing cells in relation to S100ß-immunoreactive glial and Hu-immunoreactive neuronal cells were evaluated. Additionally, we analyzed the effects of I/R on gastrointestinal transit and ileum contractile activity. RESULTS: The cellular density of P2X2 receptor and neuronal Hu immunoreactivity/cm(2) decreased after I/R, whereas glial S100ß immunoreactivity/cm(2) increased. No significant differences between sham and I/R groups were observed regarding the perikarya area of Hu-positive neurons. The area of S100ß-immunoreactive glial cells increased by 24.1 % 1 week after I/R compared with the 24 h group. Methylene blue progression along the small intestine decreased (P < 0.05) from 24.5 ± 2.3 % in the sham group to 17.2 ± 2.0 % 1 week post-ischemia. We noted a significant (P < 0.05) decrease in the maximal contraction amplitude triggered by electrical field stimulation in the presence of ATP in preparations submitted to 24 h of I/R. CONCLUSIONS: Changes in the P2X2 receptor density parallel myenteric neuronal loss following I/R of the rat ileum. This, together with the increase in the activated (oversized) glial cells, may contribute to decreased GI motility after I/R.

Differential effects of experimental ulcerative colitis on P2X7 receptor expression in enteric neurons.

The digestive tracts of ulcerative colitis and Crohn's disease patients present with pathophysiological processes and intestinal necrosis. This study examined the P2X7 receptor and changes in the distal colon in enteric neurons of rats with experimental ulcerative colitis. The analysis was performed in the distal colons of rats with ulcerative colitis induced by the administration of 2,4,6-trinitrobenzene sulfonic acid (colitis group). The survival time after colitis induction was 24 h. The treated animals were compared to sham rats injected with phosphate-buffered saline and to animals with no intervention (control group). Tissues were prepared for immunohistochemical double-staining methods to examine P2X7 receptor, choline acetyltransferase (ChAT), calbindin, calretinin, anti-HuC/D (pan-neuronal) and S100ß (pan-glial). The colocalization of the P2X7 receptor-immunoreactive (IR) cells was observed in the myenteric plexus with nitric oxide synthase (NOS)-, ChAT-,calbindin-, calretinin- and HuC/D-IR neurons and S100ß-IR cells in the control, sham and colitis groups. The neuronal density (cell bodies/cm(2)) decreased in the myenteric plexus by 11, 18, 34, 22 and 60% in the P2X7 receptor, NOS-, ChAT-, calbindin- and calretinin-IR neurons, respectively. In addition, the densities (cell bodies/cm(2)) of HuC/D-IR neurons and S100ß-IR enteric glial cells decreased by 33 and 29%, respectively. The profile areas were reduced by 6.8 and 21% in NOS- and ChAT-IR neurons, respectively. There was also a 20% increase of calbindin-IR neurons. Morphological changes were observed, such as increased neutrophils, disintegration of the intestinal epithelium and goblet cells and decreased collagen. This study demonstrated that colitis differentially affects P2X7 receptor-expressing enteric neurons based on their chemical codes and may cause changes in morphology and motility.

Differential effects of intestinal ischemia and reperfusion in rat enteric neurons and glial cells expressing P2X2 receptors.

BACKGROUND: Intestinal ischemia followed by reperfusion (I/R) may occur following intestinal obstruction. In rats, I/R in the small intestine leads to structural changes accompanied by neuronal death. AIM: The objective was to analyze the impact of I/R injury on different neuronal populations in the myenteric plexus of the rat ileum after different periods of reperfusion. METHODS: The superior mesentery artery was occluded for 45 minutes, and animals were euthanized after 24 hours and 1 week of reperfusion. Immunohistochemical analyses were performed with antibodies against the P2X2 receptor in combination with antibodies against nitric oxide synthase (NOS), choline acetyltransferase (ChAT), calbindin, calretinin, the pan-neuronal marker anti-HuC/D, or S100ß (glial marker). RESULTS: Dual immunolabeling demonstrated that approximately 100% of NOS-, ChAT-, calbindin-, and calretinin-immunoreactive neurons in all groups expressed the P2X2 receptor. Following I/R, the neuronal density decreased in the P2X2 receptor-, ChAT-, calretinin-, and HuC/D-immunoreactive neurons at 24 hours and 1 week following injury compared to the densities in the control and sham groups. The calbindin-immunoreactive neuron density was not reduced in any of the groups. The density of enteric glial cells increased by 40% in the I/R group compared to the density in the sham groups. We also observed increases of 12%, 16%, and 23% in the neuronal cell body profile areas of the NOS-, ChAT-, and calbindin-immunoreactive neurons, respectively, at 1 week following I/R. However, the average size of the calretinin-immunoreactive neurons was reduced by 12% in the I/R group at 24 hours. CONCLUSIONS: This work demonstrates that I/R is associated with a significant loss of different classes of neurons in the myenteric plexus accompanied by morphological changes and an increased density of enteric glial cells; all of these effects may underlie conditions related to intestinal motility disorder.

Effects of ischemia and reperfusion on subpopulations of rat enteric neurons expressing the P2X7 receptor.

BACKGROUND: Intestinal ischemia followed by reperfusion (I/R) may occur following intestinal obstruction. In rats, I/R in the small intestine leads to structural changes accompanied by neuronal death. AIM: To analyze the impact of I/R injury on different neuronal populations in the myenteric plexus of rat ileum. METHODS: The ileal artery was occluded for 35 min and animals were euthanized 6, 24, and 72 h, and 1 week later. Immunohistochemistry was performed with antibodies against the P2X7 receptor as well as nitric oxide synthase (NOS), calbindin, calretinin, choline acetyltransferase (ChAT), or the pan-neuronal marker anti-HuC/D. RESULTS: Double immunolabeling demonstrated that 100% of NOS-, calbindin-, calretinin-, and ChAT-immunoreactive neurons in all groups expressed the P2X7 receptor. Following I/R, neuronal density decreased by 22.6% in P2X7 receptor-immunoreactive neurons, and decreased by 46.7, 38, 39.8, 21.7, and 20% in NOS-, calbindin-, calretinin-, ChAT-, and HuC/D-immunoreactive neurons, respectively, at 6, 24, and 72 h and 1 week following injury compared to the control and sham groups. We also observed a 14% increase in the neuronal cell body profile area of the NOS-immunoreactive neurons at 6 and 24 h post-I/R and a 14% increase in ChAT-immunoreactive neurons at 1 week following I/R. However, the average size of the calretinin-immunoreactive neurons was reduced by 12% at 6 h post-I/R and increased by 8% at 24 h post-I/R. CONCLUSIONS: This work demonstrates that I/R is associated with a significant loss of different subpopulations of neurons in the myenteric plexus accompanied by morphological changes, all of which may underlie conditions related to intestinal motility disorder.

Differential effects of undernourishment on the differentiation and maturation of rat enteric neurons.

The colocalization, number, and size of various classes of enteric neurons immunoreactive (IR) for the purinergic P2X2 and P2X7 receptors (P2X2R, P2X7R) were analyzed in the myenteric and submucosal plexuses of control, undernourished, and re-fed rats. Pregnant rats were exposed to undernourishment (protein-deprivation) or fed a control diet, and their offspring comprised the following experimental groups: rats exposed to a normal diet throughout gestation until postnatal day (P)42, rats protein-deprived throughout gestation and until P42, and rats protein-deprived throughout gestation until P21 and then given a normal diet until P42. Immunohistochemistry was performed on the myenteric and submucosal plexuses to evaluate immunoreactivity for P2X2R, P2X7R, nitric oxide synthase (NOS), choline acetyltransferase (ChAT), calbindin, and calretinin. Double-immunohistochemistry of the myenteric and submucosal plexuses demonstrated that 100% of NOS-IR, calbindin-IR, calretinin-IR, and ChAT-IR neurons in all groups also expressed P2X2R and P2X7R. Neuronal density increased in the myenteric and submucosal plexuses of undernourished rats compared with controls. The average size (profile area) of some types of neurons in the myenteric and submucosal plexuses was smaller in the undernourished than in the control animals. These changes appeared to be reversible, as animals initially undernourished but then fed a normal diet at P21 (re-feeding) were similar to controls. Thus, P2X2R and P2X7R are present in NOS-positive inhibitory neurons, calbindin- and calretinin-positive intrinsic primary afferent neurons, cholinergic secretomotor neurons, and vasomotor neurons in rats. Alterations in these neurons during undernourishment are reversible following re-feeding.

Effects of ischemia and reperfusion on P2X2 receptor expressing neurons of the rat ileum enteric nervous system.

PURPOSE: We investigated the effects of ischemia/reperfusion in the intestine (I/R-i) on purine receptor P2X2-immunoreactive (IR) neurons of the rat ileum. METHODS: The superior mesenteric artery was occluded for 45 min with an atraumatic vascular clamp and animals were sacrificed 4 h later. Neurons of the myenteric and submucosal plexuses were evaluated for immunoreactivity against the P2X2 receptor, nitric oxide synthase (NOS), choline acetyl transferase (ChAT), calbindin, and calretinin. RESULTS: Following I/R-i, we observed a decrease in P2X2 receptor immunoreactivity in the cytoplasm and surface membranes of neurons of the myenteric and submucosal plexuses. These studies also revealed an absence of calbindin-positive neurons in the I/R-i group. In addition, the colocalization of the P2X2 receptor with NOS, ChAT, and calretinin immunoreactivity in the myenteric plexus was decreased following I/R-i. Likewise, the colocalization between P2X2 and calretinin in neurons of the submucosal plexus was also reduced. In the I/R-i group, there was a 55.8% decrease in the density of neurons immunoreactive (IR) for the P2X2 receptor, a 26.4% reduction in NOS-IR neuron, a 25% reduction in ChAT-IR neuron, and a 47% reduction in calretinin-IR neuron. The density of P2X2 receptor and calretinin-IR neurons also decreased in the submucosal plexus of the I/R-i group. In the myenteric plexus, P2X2-IR, NOS-IR, ChAT-IR and calretinin-IR neurons were reduced in size by 50%, 49.7%, 42%, and 33%, respectively, in the I/R-i group; in the submucosal plexus, P2X2-IR and calretinin-IR neurons were reduced in size by 56% and 72.6%, respectively. CONCLUSIONS: These data demonstrate that ischemia/reperfusion of the intestine affects the expression of the P2X2 receptor in neurons of the myenteric and submucosal plexus, as well as density and size of neurons in this population. Our findings indicate that I/R-i induces changes in P2X2-IR enteric neurons that could result in alterations in intestinal motility.