| ObjectiveThis study aims to observe the projection of nerve fiber between the ventromedial hypothalamic nucleus(VMH)and the hippocampus;To explore the effects of Nesfatin-1 on gastric distention(GD)sensitive neurons and the potential mechanism for Nesfatin-1 to regulate gastric motility in VMH;To discuss the regulation of hippocampus CA1 area on the effect of Nesfatin-1 in the VMH.Methods782 adult male wistar rats were used in this study.The body weights of the rats were 250-300 g.The rats were fed with standard pellet food,animal housing temperature maintained 22±3℃,relative humidity maintained 60±5%.The light of day and night was 12h:12h.Adaptive feeding was about one week before experiment.The main experiments methods include preparation of diabetic rats model,single neurons discharge records,fluorogold retrograde tracing experiment,immunohistochemistry staining,electrical stimulation and electrical damage,gastric motility research in vivo.400 rats were selected to prepare diabetic rat model.Rats were fasted for 12 hours before modeling.The rats in the model group were injected intraperitoneally with 35mg/kg 1%(STZ),and the rats were fed for one week after the first injection.In the same way,the rats were given intraperitoneal injection with the same dose for the second time in eighth day.The tail blood of the rats was used to measure fasting blood glucose.While the fasting blood glucose was more than 7.8mmol/L or postprandial blood glucose more than 11.1mmol/L,the rats were selected as diabetic model.Single neurons discharge recording was used to observe the effects of Nesfatin-1 on gastric distention(GD)sensitive neurons in VMH;Fluorogold retrograde tracing combined with immunohistochemistry staining were used to Nesfatin-1 neuron fiber link between VMH and hippocampus CA1;Electrical stimulation and electrical damage were used to observe the hippocampus CA1 regulating on the effect of Nesfatin-1 in the VMH;Gastric motility in vivo was used to observe the effects of Nesfatin-1 on gastric motility in the VMH.Results1.In the experiment of single unit discharge recording,176 neurons were recorded in the extracellular neurons of the VMH nucleus in the 55 normal rats,and 118 neurons(118/176,67%)responded to gastric distension stimulation,identified as GD sensitive neurons.Among the 118 GD sensitive neurons,there were 61 GD excitatory(GD-E)neurons and 57 GD inhibitory(GD-I)neurons.Out of 61 GD-E neurons in the VMH,16(16/61,26%)were inhibited by the VMH microinjection of Nesfatin-1,8 GD-E neurons showed excitatory effect,and there was no significant change in the 37 neurons.The VMH nucleus microinjection of Nesfatin-1,12 of 57(12/57,21%)GD-I neurons were excited by Nesfatin-1,7 neurons showed inhibitory effect,and the 38 neurons showed no significant change.After pretreatment wit h corticotropin-releasing factor antagonist astressin-B,the effect of Nesfatin-1 on the discharge of GD-E or GD-I neurons was significantly decreased(P<0.05).Compared with normal rats,the proportion of GD-E or GD-I neurons and its discharge frequency recorded in the VMH nucleus of diabetic rats was no significantly difference(P>0.05)after gastric distension stimulation.But in the diabetic rat,the inhibition of GD-E neurons were significantly increased(26.2% VS.38.5%,P<0.05),the discharge frequency change rate increased significantly(34.1 ± 5.31% VS.57.2 ± 9.4%,P<0.05)while nesfatin-1 was injected into the VMH;The activation of GD-I neurons discharge ratio increased significantly(21.1% VS.33.9%,P<0.05),as well as the rate of discharge frequency change was significantly increased(26.8 ± 7.2% VS.38.2 ± 6.5%,P<0.05)compared with the normal rats.In diabetic rats,regulation of Nesfatin-1 on the rate of discharge frequency of GD-E or GD-I neurons was more effective than in normal rats after the injection of astressin-B into the VMH nucleus.2.Gastric motility research in vivo showed that different concentrations of nesfatin-1 could significantly decrease the amplitude and frequency of gastric motility and it showed a dose-dependent manner(p<0.05~0.01).After injection of nesfatin-1 into the ventromedial hypothalamic area about 5min,the amplitude of gastric contraction began to decrease,decline reached the peak at about 10-15 min.Astressin-B could partly abolish the effect of Nesfatin-1(P<0.05).The VMH injection of different concentrations of Nesfatin-1 could significantly decrease the amplitude and frequency of gastric motility,and it showed a dose-dependent manner in diabetic rats(P<0.05~0.01).Compared with the normal rats,while the d iabetic rats were given the same dose of nesfatin-1 in the VMH nucleus,the gastric motility index was significantly reduced,and the difference was statistically significant(P<0.05).Further studies showed that after VMH nucleus injection of different doses of Nesfatin-1,the EC50Nesfatin-1 of the gastric motility index in DM rats was significantly lower than that of EC50Nesfatin-1(P<0.05)in normal rats.3.Fluorogold retrograde tracing combined with immunohistochemistry staining showed that the injection of fluorescent gold into the VMH nucleus after 7 days,the fluorescent gold labeled neurons were detected in the hippocampal CA1 region.And nesfatin-1 immunofluorescence staining show that the expression of nesfatin-1 immunoreactivity in the cytoplasm that partially labeled fluorescent gold in hippocampal CA1 region.It is suggested the projection of nesfatin-1 neurons in the hippocampal C A1 region to the VMH.4.Electrical stimulating the hippocampal CA1 region could excite the GD-E neurons and GD-I neurons in normal rat;Pre-administration of anti-nesfatin-1 antibody in VMH,then stimulated the hippocampus CA1 area,the excitatory effect of GD-E neurons was further enhanced.Electrical stimulating the hippocampal CA1 region could also excite the GD-I neurons.Compared with normal rats,pre-administration of anti-nesfatin-1 antibody in VMH,then stimulated the hippocampus C A1 area,the excitatory effect of Nesfatin-1 inhibitory GD-E neurons was significantly Nhigher in diabetic rats than that in normal rats(P<0.05),and the excitatory effect of nesfatin-1 on GD-I neurons was significantly lower than that in the normal rats(P<0.05).5.After electrical stimulation of the hippocampus CA1 area,gastric contraction amplitude and contraction frequency increased significantly in normal rat(P<0.05),peaked at about 13 min.Administration of anti-NUCB2/Nesfatin-1 antibody into the VMH in normal rat,the excitation effect induced by electrical stimulation of the hippocampal CA1 was slightly enhanced(P>0.05).After ventromedial hypothalamic injection of anti-NUCB2/Nesfatin-1 antibody,electrical stimulating the hippocampal CA1 area,the gastric contraction amplitude and frequency was significantly increased(P<0.05).Compared with normal rats,the gastric motility index of diabetic rats increased,but no statistically significant(P>0.05).6.The effects of the nesfatin-1 on GD responsive neurons in the VMH were no significant change in the hippocampus lesion rats(P>0.05).Compared with the false lesioned group,nesfatin-1 could inhibit the GD-E neurons and excite the GD-I neurons in hippocampal CA1 by nesfatin-1 administration to VMH.But the gastric motility had no significantly difference before or after electrical lesion of the hippocampal C A1 region(P<0.05).Compared with the normal rats,electrical injury of hippocampus CA1 and VMH injection of Nesfatin-1,there was no significant change in gastric motility index in diabetic rats(P>0.05).ConclusionsThe nesfatin-1 nerve fibers in the hippocampal CA1 area may projected into the VMH;The Nesfatin-1 signaling pathway in the hippocampus is involved in the regulation of gastric afferent information and gastric motility,which maybe relate to the activity of the CRF system;Hypothalamic nesfatin-1 maybe play an important ro le in the regulation of gastric afferent information and motility disorders in the patients with diabetes;The hippocampus may be involved in the regulation of nesfatin-1 on gastric afferent neurons excitability and gastric motility which were regulated by the hypothalamus feeding related central nervous system;This study provides a theoretical basis and therapeutic targets for the diagnosis and treatment of gastrointestinal disorders. |
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