Glutamate Excitotoxicity Leads To Selective Loss Of NNOS Containing Neurons And Gastroparesis In Diabetic Rats

Background and AimsGastroparesis is a clinical disorder manifested by delayed gastric emptying andsymptoms such as nausea, vomiting, pain and flatulence, without mechanical obstruction.Diabetes is one of the leading causes of gastroparesis. With the increase of diabeticpatients, there are more and more studies focus on diabetic gastroparesis. Whilegastroparesis may not increase mortality, it can adversely affect the quality of life andlead to nutritional insufficiency, electrolyte imbalance, impaired glycemic control, andfrequent hospitalizations.Gastric emptying is a highly complex function which needs the coordination ofsmooth muscles, enteric nervous system and extrinsic nerves, and interstitial cells ofCajal. Therefore, many researchers found the changes of vagus, enteric nervous system,interstitial cells of Cajal and smooth muscles in diabetic gastroparesis. Among thesechanges, the selective loss of nNOS neurons is noticeable.Glutamate is one of the classic excitatory neurotransmitter, which also plays a keyrole in mediating excitotoxicity. Excitotoxicity is a process of neurodegenerationtriggering through the over activation of glutamate receptor NR2B expressed on nNOSneurons, the over influx of Calcium and the over production of NO. While the antagonistof NR2B could prevent the neurons from excitotoxicity, it would block the vitalfunctions of cells and lead to adverse effect and mortality of stoke.There are some literatures have mentioned that the glutamate and its receptors existin the myenteric plexus of stomach, but there is no article have reported the functions ofglutamate system in the process of diabetic gastroparesis and selective loss of nNOSneurons. In this study, we determined:(1) glutamate system exists in the myenteric plexus of stomach,(2) excitotoxicity leads to diabetic gastroparesis and selective loss ofnNOS neurons, and (3) perturbing NR2B receptor–PSD-95–protein interactionsprevents selective loss of nNOS neurons and improve gastric emptying in diabetic rats.Methods1. Constructing diabetic gastroparesis animal model in Sprague-Dawley rats.1) Intraperitoneal injecting of streptozocin (STZ,55mg/kg), and measuring theblood glucose and body weight after48hours.2) Evaluating gastric delayed emptying by measuring gastric solid emptying in4hours after6weeks.3) Evaluating the changes of nNOS and Choline acetyltransferase (ChAT) byimmunofluorescence and Western blot.2. Mimicking glutamate excitotoxicity by suppressing the re-uptake of glutamate.After the release of glutamate into the synapse, the termination of neurotransmitterdepends on the function of re-uptake. Excitatory amino acid transporter2(EAAT2),which distributed on glial cells, accounts for more than90%of re-uptake of glutamate.Small interfering RNA (siRNA) could combine with the complementary mRNA andsilence its expression.1) Confirming if all the components of glutamate system, such as vGLUT1, NR2B,PSD-95, GS, EAAT2, exist in the myenteric plexus of stomach.2) Injecting the siRNA target EAAT2into subserosa and transferring them into cellsby electroporation.3) Evaluating the expression of mRNA and protein by qPCR and Western blotafter siRNA injection and electroporation.4) Evaluating gastric delayed emptying by measuring gastric solid emptying in4hours after5days. 5) Evaluating the changes of nNOS and ChAT by immunofluorescence and Westernblot.3. Synthesizing the peptide named Tat-NR2B9c（Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg--Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val）to perturb NMDAR-PSD-95-nNOS protein interactions, and evaluating itsprevention effects on nNOS and gastroparesis both in vitro and in vivo.1) In the cultured gastric whole mount (muscularis propria with enteric nerveswithout mucosa and submucosa), treating with100uM Tat-NR2B9c attached5-FAMand evaluating if the peptide could enter into neurons or not by immunofluorescence.2) In the cultured gastric whole mount, pretreating with50nM Tat-NR2B9c andevaluating if the peptide could suppress the production of cGMP induced by100uMNMDA by enzyme linked immunosorbent assay (ELISA).3) In the cultured gastric whole mount, pretreating with50nM Tat-NR2B9c andevaluating if the peptide could prevent apoptosis of nNOS by immunofluorescence.4) After the injection and electroporation of siRNA, treating the rats withTat-NR2B9c（2.6mg/kg）on D0,2,4by intraperitoneal injection, and evaluating theexpression of EAAT2, nNOS, ChAT and gastric solid emptying.5) After the intraperitoneal injection of STZ, treating the rats with Tat-NR2B9c（2.6mg/kg）from week4to week6everyday by intraperitoneal injection, and evaluatingthe expression of nNOS, ChAT and gastric solid emptying.Results1. STZ successfully induced diabetic animal model in rats1) After the intraperitoneal injection of streptozocin (55mg/kg), the blood glucosesof rats increased to300~450mg/dl, while control rats were70~110mg/dl.2) Gastric solid emptying of STZ-induced diabetic rats decreased to46.8+3.3%,compared to71.7+4.4%of control. 3) The expression of nNOS of STZ-induced diabetic rats decreased to54.1±3.55%of control, while there was no significance changes of the expression of ChAT.2. The gastric solid emptying and the expression of nNOS decreased after theinjection of siRNA targeted EAAT2.1) The expression of vGLUT1、NR2B、PSD-95、GS、EAAT2exist in the myentericplexus of stomach by immunofluorescence.2) After the injection of siRNA targeted EAAT2into subserosa, the expression ofmRNA of EAAT2decreased to54.03±3.54%of control, and the expression of protein ofEAAT2decreased to67.47±2.92%of control.3) After the injection of siRNA targeted EAAT2into subserosa, the expression ofnNOS decreased to40.9±2.73%of control, while there was no significance changes ofthe expression of ChAT.4) After the injection of siRNA targeted EAAT2into subserosa, gastric solidemptying decreased to43.4±4.04%%, compared to72.6±2.93of control.3. Tat-NR2B9c prevented nNOS and improved gastric solid emptying both in vitroand in vivo.1) According to immunofluorescence, Tat-NR2B9c could enter into neurons incultured gastric whole mounts in vitro.2) The level of cGMP of gastric whole mounts increased with the stimulation ofNMDA, while50nM Tat-NR2B9c suppressed26.8±3.12%production of cGMP inducedby100uM NMDA.3) In vitro cultured gastric whole mounts, with the stimulation of100uM NMDA,the expression of Caspase-3in nNOS neurons increased to59.17±3.43%, compared to0of control, while treated with50nM Tat-NR2B9c prevented the increased expression ofCaspase-3in nNOS neurons (24.58±3.14%VS59.17±3.43%).4) In vitro cultured gastric whole mounts, with the stimulation of100uM NMDA, the number of nNOS neurons in ganglions decreased to4.0±0.40/per ganglion, comparedto7.0±0.40/per ganglion of control, and while treated with50nM Tat-NR2B9c preventedthe decreased number of nNOS neurons (6.25±0.63/per ganglion VS4.0±0.40/perganglion).5) After the injection of siRNA targeted EAAT2into subserosa, the expression ofnNOS increased to78.4±4.36%while treated with50nM Tat-NR2B9c, as well as thenumber of nNOS neurons (14.8±1.15%VS24.4±2.25%) and gastric solid emptying（65.9±5.38%VS43.4±4.04%）.6) After the intraperitoneal injection of streptozocin, the expression of nNOSincreased to77.8±3.38%while treated with50nM Tat-NR2B9c, as well as the number ofnNOS neurons (26.3±0.95%VS16.8±0.85%) and gastric solid emptying （64.6±2.61%VS46.8+3.3%）.Conclusions1. The intraperitoneal injection of streptozocin could induced diabetic gastroparesisand selective loss of nNOS neurons.2. Glutamate system exists in myenteric plexus of stomach. The injection andelectroporation of siRNA targeted EAAT2into subserosa could induce excitotoxicity,leading to the selective loss of nNOS and gastroparesis.3. Tat-NR2B9c could enter into neuronal cells and decrease the production ofcGMP by suppressing the activity of nNOS.4. Tat-NR2B9c could not only prevent nNOS neurons from excitotoxicity in vitro,but also prevent nNOS neurons selective loss and improve gastric solid emptying in bothmodels of siRNA targeted EAAT2and STZ induced diabetes.