Myenteric neurons and intestinal mucosa of diabetic rats after ascorbic acid supplementation

AIM: To investigate the effect of ascorbic acid (AA) dietary supplementation on myenteric neurons and epithelial cell proliferation of the jejunum of adult rats with chronic diabetes mellitus. METHODS: Thirty rats at 90 d of age were divided into three groups: Non-diabetic, diabetic and diabetic treated with AA (DA) (1 g/L). After 120 d of treatment with AA the animals were killed. The myenteric neurons were stained for myosin-V and analyzed quantitatively in an area of 11.2 mm2/animal. We further measured the cellular area of 500 neurons per group. We also determined the metaphasic index (MI) of the jejunum mucosa layer of about 2500 cells in the intestinal crypts, as well as the dimensions of 30 villi and 30 crypts/animal. The data area was analyzed using the Olympus BX40 microscope. RESULTS: There was an increase of 14% in the neuronal density (792.6 ± 46.52 vs 680.6 ± 30.27) and 4.4% in the cellular area (303.4 ± 5.19 vs 291.1 ± 6.0) respectively of the diabetic group treated with AA when compared to control diabetic animals. There were no signifi cant differences in MI parameters, villi height or crypt depths among the groups.CONCLUSION: Supplementation with AA in the diabetic animal promoted moderate neuroprotection. There was no observation of alteration of the cellular proliferation of the jejunum mucosa layer of rats with chronic diabetes mellitus with or without supplementation with AA.

Expression of the P2X_2 receptor in different classes of ileum myenteric neurons in the female obese ob/ob mouse

AIM:To examine whether the ob/ob mouse model of obesity is accompanied by enteric nervous system ab-normalities such as altered motility METHODS:The study examined the distribution of the P2X 2 receptor (P2X 2 R) in myenteric neurons of female ob/ob mice. Specifically, we used immunohistochemistry to analyze the co-expression of the P2X 2 R with neuronal nitric oxide synthase (nNOS), choline acetyltrans-ferase (ChAT), and calretinin (CalR) in neurons of the small intestine myenteric plexus in ob/ob and control female mice In these sections, we used scanning confocal microscopy to analyze the co-localization of these markers as well as the neuronal density (cm 2 ) and area profile (μm2) of P2X 2 R-positive neurons In addition, enteric neurons were labeled using the nicotinamide adenine dinucleotide (NA H) diaphorase method and analyzed with light microscopy as an alternate means by which to analyze neuronal density and areaRESULTS:In the present study, we observed a 29 6% increase in the body weight of the ob/ob animals (OG) compared to the control group (CG) In addition, the average small intestine area was increased by approxi-mately 29 6% in the OG compared to the CG Immu-noreactivity (IR) for the P2X 2 R, nNOS, ChAT and CalR was detectable in the myenteric plexus, as well as in the smooth muscle, in both groups This IR appeared to be mainly cytoplasmic and was also associated with the cell membrane of the myenteric plexus neurons, where it outlined the neuronal cell bodies and their processes P2X 2 R-IR was observed to co-localize 100% with that for nNOS, ChAT and CalR in neurons of both groups In the ob/ob group, however, we observed that the neuronal density (neuron/cm 2 ) of P2X 2 R-IR cells was in-creased by 62% compared to CG, while that of NOS-IR and ChAT-IR neurons was reduced by 49% and 57%, respectively, compared to control mice The neuronal density of CalR-IR neurons was not different between the groups Morphometric studies further demonstrated that the cell body profile area (μm2) of nNOS-IR, ChAT-IR and CalR-IR neurons was increased by 34%, 20% and 55%, respectively, in the OG compared to controls Staining for NA H diaphorase activity is widely used to detect alterations in the enteric nervous system; however, our qualitative examination of NA H-diaphorase positive neurons in the myenteric ganglia revealed an overall similarity between the two groups CONCLUSION:We demonstrate increases in P2X2R expression and alterations in nNOS, ChAT and CalR IR in ileal myenteric neurons of female ob/ob mice compared to wild-type controls.

Morphological effects of autoclaved diet on the myenteric neurons of rats

AIM:To evaluate the effect of autoclaved diet on the jejunum neurons of the myenteric plexus of rats during their growth.METHODS:The experimental groups were made up of rats going through weaning whose mothers received either an autoclaved or a non-autoclaved diet during gestation and lactation,and rats that were fed the same diet as their mothers during the post-weaning period.In order to measure the neurons'body profile and to quantify the number of neurons per area,preparations were stained by the nicotinamide adenine dinucleotide-diaphorase method.RESULTS:No significant changes were observed in rats'body weight or in the number of neurons regardless of the diet used(P>0.05) .There was a decrease in the jejunum-ileum length in rats treated with an autoclaved diet(P<0.05) .An increase in the neuronal cross-sectional area was seen in rats that had received the autoclaved diet,an effect that was significant for animals undergoing weaning.In addition,all observed factors showed significant differences when related to the age of the animals.CONCLUSION:The autoclaved diet did not alter the quantity of neurons,but increased their cell body area,suggesting changes similar to those observed in protein deficiency.

Effect of motilin and erythromycin on intracellular Ca2+ mobilization in rat myenteric neurons in culture

Objective: To investigate the effects of motilin and erythromycin on intracellular Ca2+ mobi-lization in cultured myenteric neurons of rats. Methods: The cultured myenteric neurons were identified with immunofluorescence staining technique. Motilin-induced and erythromycin-induced intracellular Ca2+ mobilization was studied in primary cultures of myenteric neurons using the ratiometric Ca2+ indicator Furo3/AM, with a laser confocal microscope. Results: The effects of motilin and erythromycin on intracellular Ca2+ mobilization were as follows: (1)In Hank's solution, 10 -8, 10-7, 10-6 mol/L motilin could elevate intracellular Ca2+ concentration ([Ca2+]i)in a dose-dependent manner. (2) In Hank's solution, 10μg/ ml erythromycin also could induce the elevation of [Ca2+]i. (3) After pretreatment with antibody against the motilin receptor in Hank's solution, the Ca2+ response to erythromycin was almost restricted. Conclusion: It is suggested that motilin could increase [Ca2+]i in myenteric neurons in a dose-dependent manner, and erythromycin may also have this effectivenesss by binding to the motilin receptor.

Distribution of the P2X2 receptor and chemical coding in ileal enteric neurons of obese male mice(ob/ob)

AIM:To investigate the colocalization,density and profile of neuronal areas of enteric neurons in the ileum of male obese mice.METHODS:The small intestinal samples of male mice in an obese group(OG)(C57BL/6J ob/ob)and a control group(CG)(+/+)were used.The tissues were analyzed using a double immunostaining technique for immunoreactivity(ir)of the P2X2 receptor,nitric oxide synthase(NOS),choline acetyl transferase(ChAT)and calretinin(Calr).Also,we investigated the density and profile of neuronal areas of the NOS-,ChAT-and Calrir neurons in the myenteric plexus.Myenteric neurons were labeled using an NADH-diaphorase histochemical staining method.RESULTS:The analysis demonstrated that the P2X2receptor was expressed in the cytoplasm and in the nuclear and cytoplasmic membranes only in the CG.Neuronal density values(neuron/cm2)decreased 31%(CG:6579±837;OG:4556±407)and 16.5%(CG:7796±528;OG:6513±610)in the NOS-ir and calretininir neurons in the OG,respectively(P<0.05).Density of ChAT-ir(CG:6200±310;OG:8125±749)neurons significantly increased 31%in the OG(P<0.05).Neuron size studies demonstrated that NOS,ChAT,and Calr-ir neurons did not differ significantly between the CG and OG groups.The examination of NADH-diaphorase-positive myenteric neurons revealed an overall similarity between the OG and CG.CONCLUSION:Obesity may exert its effects by promoting a decrease in P2X2 receptor expression and modifications in the density of the NOS-ir,ChAT-ir and CalR-ir myenteric neurons.

Effects of protein deprivation and re-feeding on P2X_2 receptors in enteric neurons

AIM:To investigate the effects of malnutrition and refeeding on the P2X2 receptor,nitric oxide synthase(NOS),calretinin,calbindin and choline acetyltransferase(ChAT) in neurons of the rat ileum.METHODS:We analyzed the co-localization,numbers and sizes of P2X2-expressing neurons in relation to NOS-immunoreactive(IR),calbindin-IR,ChAT-IR,and calretinin-IR neurons of the myenteric and submucosal plexus.The experimental groups consisted of:(1) rats maintained on normal feed throughout pregnancy until 42 d post-parturition(N);(2) rats deprived of protein throughout pregnancy and 42 d post-parturition(D);and(3) rats undernourished for 21 d post-parturition and then given a protein diet from days 22 to 42(DR).The myenteric and submucosal plexuses were evaluated by double labeling by immunohistochemical methods for P2X2 receptor,NOS,ChAT,calbindin and calretinin.RESULTS:We found similar P2X2 receptor immunoreactivity in the cytoplasm and surface membranes of myenteric and submucosal neurons from the N,D and DR groups.Double labeling of the myenteric plexus demonstrated that approximately 100% of NOS-IR,calbindin-IR,calretinin-IR and ChAT-IR neurons in all groups also expressed the P2X2 receptor.In the submucosal plexus,the calretinin-IR,ChAT-IR and calbindin-IR neurons were nearly all immunoreactive for the P2X2 receptor.In the myenteric plexus,there was a 19% increase in numbers per cm2 for P2X2 receptor-IR neurons,64% for NOS-IR,84% for calretinin-IR and 26% for ChAT-IR neurons in the D group.The spatial density of calbindin-IR neurons,however,did not differ among the three groups.The submucosal neuronal density increased for calbindin-IR,calretinin-IR and ChAT-IR neurons.The average size of neurons in the myenteric plexus neurons in the D group was less than that in the controls and,in the re-fed rats;there was a 34% reduction in size only for the calretinin-IR neurons.CONCLUSION:This work demonstrates that expression of the P2X2 receptor is present in inhibitory,intrinsic primary afferent,cholinergic secretomotor and vasomotor neurons.Undernutrition affected P2X2 receptor expression in the submucosal plexus,and neuronal and size.These changes were rescued in the re-fed rats.

Alleviated mucosal and neuronal damage in a rat model of Crohn's disease

AIM:To establish a rat model suitable to investigate the repetitive relapsing inflammations(RRI)characteristic to Crohn’s disease.METHODS:Colitis was induced by 2,4,6-trinitrobenzenesulfonic acid(TNBS).RRI were mimicked by repeating administrations of TNBS.Tissue samples were taken from control,once,twice and three times treated rats from the inflamed and adjacent non-inflamed colonic segments at different timepoints during the acute intestinal inflammation.The means of the ulcerated area were measured to evaluate the macroscopic mu-cosal damage.The density of myenteric neurons was determined on whole mounts by Hu C/Hu D immunohistochemistry.Heme oxygenase-1(HO-1)expression was evaluated by molecular biological techniques.RESULTS:TNBS-treated rats displayed severe colitis,but the mortality was negligible,and an increase of body weight was characteristic throughout the experimental period.The widespread loss of myenteric neurons,and marked but transient HO-1 up-regulation were demonstrated after the first TNBS administration.After repeated doses the length of the recovery time and extent of the ulcerous colonic segments were markedly decreased,and the neuronal loss was on a smaller scale and was limited to the inflamed area.HO-1 m RNA level was notably greater than after a single dose and overexpression was sustained throughout the timepoints examined.Nevertheless,the HO-1protein up-regulation after the second TNBS treatment proved to be transient.Following the third treatment HO-1 protein expression could not be detected.CONCLUSION:Experimentally provoked RRI may exert a protective preconditioning effect against the mucosal and neuronal damage.The persistent up-regulation of HO-1 m RNA expression may correlate with this.