Identification and Pathogen Stimulation Patterns of Neuronal Nitric Oxide Synthase(nNOS)in Black Rockfish(Sebastes schlegelii)

Neuronal nitric oxide synthase(nNOS)was the producer of nitric oxide(NO)which played important gas messenger molecules in biological process.It also can take effect as immune regulation molecule in organism.Black rockfish(Sebastes schlegelii)is an important economic fish which were widely farmed in East Asia countries.Meanwhile,the pathogenic bacteria such as the Edwardsiella tarda and Vibrio anguillarum in seawater always brought serious obstacles to their healthy growth.In order to explore the expression pattern of n NOS gene under the pathogen stimulation and predict its immune function,the n NOS gene in black rockfish named Ssn NOS was identified.It was 3780 bp in length,located on chromosome 6,and contained 27 coding domain sequence(CDs).According to the phylogenetic analysis,the Ssn NOS showed closest relative to the counterpart gene of swamp eel(Monopterus albus).Meanwhile,analysis of Ssn NOS expression in various healthy tissues showed that Ssn NOS expression level was highest in healthy brain tissues,followed by intestinal tissues.In addition,Ssn NOS showed significant expression changes in response to stimulation by two pathogens.Particular in gill,the expression of Ssn NOS after pathogenic stimulation increased significantly.The Elisa analysis showed the Ssn NOS content in gills was much higher than that in other tissues at all time points.Moreover,the expression patterns of Ssn NOS in brain,intestine and kidney after stimulation by pathogens showed a distinct expression pattern which first down-regulated and then up-regulated.Therefore,the Ssn NOS may be an important signaling molecule for fish to respond rapidly in immune stimulation.

Role of brain-derived neurotrophic factor and neuronal nitric oxide synthase in stress-induced depression

BACKGROUND： Accumulated evidence indicates an important role for hippocampal dendrite atrophy in development of depression, while brain-derived neurotrophic factor （BDNF） participates in hippocampal dendrite growth. OBJECTIVE： To discuss the role of BDNF and neuronal nitric oxide synthase （nNOS） in chronic and unpredictable stress-induced depression and the pathogenesis of depression. DESIGN, TIME AND SETTING： Randomized, controlled animal experiment. The experiment was carded out from October 2006 to May 2007 at the Department of Animal Physiology, College of Life Science, Shaanxi Normal University. MATERIALS： Thirty-seven male Sprague-Dawley rats weighing 250-300 g at the beginning of the experiment were obtained from Shaanxi Provincial Institute of Traditional Chinese Medicine （Xi＇an, China）. BDNF antibody and nNOS antibody were provided by Santa Cruz （USA）. K252a （BDNF inhibitor） and 7-NI （nNOS inhibitor） were provided by Sigma （USA）. METHODS： Animals were randomly divided into five groups： Control group, chronic unpredicted mild stress （CUMS） group, K252a group, K252a＋7-NI group and 7-NI＋CUMS group. While the Control, K252a and K252a＋7-NI groups of rats not subjected to stress had free access to food and water, other groups of rats were subjected to nine stressors randomly applied for 21 days, with each stressor applied 2-3 times. On days 1, 7, 14 and 21 during CUMS, rats received microinjection of 1 μL of physiological saline in the Control and CUMS groups, 1 ~ L of K252a in the K252a group, 1 μL of K252a and 7-NI in the K252a＋7-NI group, and 1 μL of 7-NI in the 7-NI＋CUMS group. We observed a variety of alterations in sucrose preference, body weight change, open field test and forced swimming test, and observed the expression of BDNF and nNOS in rat hippocampus by immunohistochemistry; MAIN OUTCOME MEASURES： ① A variety.of behavioral alterations of rats; ② The expression of BDNF and nNOS in rat hippocampus. RESULTS： Compared with the Control group, the behavior of the CUMS rats was significantly depressed, the expression of BDNF decreased （P 〈 0.01） but the expression of nNOS increased （P 〈 0.01）. The behavior of rats given intra-hippocampal injection of BDNF inhibitor was significantly depressed and the expression of nNOS was significantly increased （P 〈 0.01）. Intra-hippocampal injections of an nNOS inhibitor reversed the depression-like behavioral changes induced by CUMS or intra-hippocampal injection of BDNF inhibitor. CONCLUSION： CUMS induced a decrease in expression of BDNF and an increase in expression of NO in the hippocampus, which may lead to depression.

Targeting neuronal nitric oxide synthase as a valuable strategy for the therapy of neurological disorders

The management of neurological disorders have huge and increasing human and economic costs. Despite this, there is a scarcity of effective therapeutics, and there is an extreme urgency for new and real treatments. In this short review we analyze some promising advancements in the search of new bioactive molecules targeting neuronal nitric oxide synthase （nNOS）, an enzyme deputed to the biosynthesis of nitric oxide （NO）. In different conditions of neuronal damages, this molecule is overproduced, contributing to the pathogenesis and progression of neuronal diseases. Two main approaches to modulate nNOS are discussed： a first one consisting in the direct inhibition of the enzyme by means of small organic molecules, which can be also active against other different targets involved in such diseases. A second section is dedicated to molecules able to prevent the formation of the ternary complex N-methyl-D-aspartate （NMDA）type glutamate receptors, postsynaptic density-95 （PSD95） protein-nNOS, which is necessary to activate the latter for the biosynthesis of NO.

Role of neuronal nitric oxide synthase and inducible nitric oxide synthase in intestinal injury in neonatal rats

瞄准：与肠的损害在新生的老鼠调查神经元的氮的氧化物 synthase ( nNOS )和可诱导的氮的氧化物 synthase ( i NOS )的动态变化和角色并且定义引起坏死小肠结肠炎( NEC )是否在粘膜与氮的氧化物 synthase ( NOS )的层次被联系影响的肠织物。方法：Wistar 老鼠在年龄的不到 24 h 与 5 mg/kg 收到了腹膜内注射脂肪的多糖(LPS ) 。回肠纸巾为 NEC 的组织学的评估并且为 nNOS 和 i NOS 的大小在 1， 3， 6， 12 和 24 h 追随者 LPS 挑战被收集。在肠的损害的度和 NOS 的层次之间的关联是坚定的。结果：注射 LPS 的小狗在损害分数对控制显示出重要增加。nNOS 蛋白质和 mRNA 的表示在 LPS 注射以后被减少。在 nNOS 蛋白质和在 24 h 以内的中部的肠的损害的等级之间有否定重要关联。i NOS 蛋白质和 mRNA 的表示显著地在肠的损害的山峰被增加。结论：nNOS 和 i NOS 在导致 LPS 的肠的损害起不同作用。小心应该在 NEC 有关 NOS 禁止者的潜在的治疗学的使用被施加。

Changes of learning and memory ability associated with neuronal nitric oxide synthase in brain tissues of rats with acute alcoholism

BACKGROUD： Ethanol can influence neural development and the ability of leaming and memory, but its mechanism of the neural toxicity is not clear till now. Endogenous nitric oxide （NO） as a gaseous messenger is proved to play an important role in the formation of synaptic plasticity, transference of neuronal information and the neural development, but excessive nitro oxide can result in neurotoxicity. OBJECTIVE ： To observe the effects of acute alcoholism on the learning and memory ability and the content of neuronal nitric oxide synthase （nNOS） in brain tissue of rats. DESIGN ： A randomized controlled animal experiment. SETTING ： Department of Physiology, Xinxiang Medical College MATERIALS： Eighteen male clean-degree SD rats of 18-22 weeks were raised adaptively for 2 days, and then randomly divided into control group （n = 8） and experimental group （n = 10）. The nNOS immunohistochemical reagent was provided by Beijing Zhongshan Golden Bridge Biotechnology Co.,Ltd. Y-maze was produced by Suixi Zhenghua Apparatus Plant. METHODS ： The experiment was carded out in the laboratory of the Department of Physiology, Xinxiang Medical College from June to October in 2005. ① Rats in the experimental group were intraperitoneally injected with ethanol （2.5 g/kg） which was dissolved in normal saline （20%）. The loss of righting reflex and ataxia within 5 minutes indicated the successful model. Whereas rats in the control group were given saline of the same volume. ② Examinations of learning and memory ability： The Y-maze tests for learning and memory ability were performed at 6 hours after the models establishment. The rats were put into the Y-maze separately. The test was performed in a quiet and dark room. There was a lamp at the end of each of three pathways in Y-maze and the base of maze had electric net. All the lamps of the three pathways were turned on for 3 minutes and then turned off. One lamp was turned on randomly, and the other two delayed automatically. In 5 seconds after alternation, pulsating electric current presented in the base of unsafe area to stimulate rat＇s feet to run to the safe area. The lighting lasted for 15 seconds as one test. Running from unsafe area to safe area at one time in 10 seconds was justified as successful. Such test was repeated for 10 times for each rat and the successful frequency was recorded. The qualified standard of maze test was that the rat ardved in the safe area g times during 10 experiments. The number of trainings for the qualified standard was used to represent the result of spatial learning. ③ Determination of the content of nNOS in brain tissue： After the Y-maze test, the rats were anaesthetized, and blood was let from the incision on right auricle, transcardially perfused via the left ventricle with about 200 mL saline, then fixed by perfusion of 40 g/L paraformaldehyde. Hippocampal CA1 region, corpus striatum and cerebellum were taken to prepare serial freezing coronal sections. The nNOS contents in the brain regions were determined with the immunohistochemical methods to reflect the changes of nitdc oxide in brain tissue. MAIN OUTCOME MEASURES ： The changes of learning and memory ability and the changes of the nNOS contents in the brain tissue of rats with acute alcoholism were observed. RESULTS ： One rat in the experimental group was excluded due to its slow reaction to electdc stimulation in the Y-maze test, and the other 17 rats were involved in the analysis of results. ① The training times to reach qualifying standards of Y-maze in the expedmental group was more than that in the control group [（34.33 ±13.04）, （27.50±8.79） times, P〈 0.05]. ② Forms and numbers of nNOS positive neurons in brain tissue： It could be observed under light microscope that in the hippocampal CA1 region, there were fewer nNOS positive neurons, which were lightly stained, and the processes were not clear enough; But the numbers of the positive neurons which were deeply stained as huffy were obviously increased in the experimental group, the cell body and cyloplasm of process were evenly stained, but the nucleus was not stained. The nNOS positive neurons in corpus stdatum had similar forms and size in the experimental group and control group. The form of the nNOS positive neurons in cerebellum were similar between the two groups. The numbers of nNOS positive neurons in hippocampal CA1 region and corpus striatum in the expedmental group [（18.22±7.47）, （11.38±5.00） cells/high power field] were obviously higher than those in the control group [（10.15±4.24）, （6.15±3.69） cells/high power field. The number of nNOS positive neurons in cerebellum had no significant difference between the two groups [（49.56±18.84）, （44.43±15.42） cells/high power field, P〉 0.05]. CONCLUSION ： Acute alcoholism may impair learning and memory ability, and nitric oxide may be involved in mediating the neurotoxic role of ethanol.

Role of hippocampal neuronal nitric oxide synthase in epilepsy

OBJECTIVE To study the function of neuronal nitric oxide synthase(nNOS) in the dentate gyrus(DG) in the pathology of epilepsy.METHODS The expression of nNOS in the DG was measured by qPCR and Western blotting in mice 3 and 12 h,1,7,14,and 60 d after treatment with pilocarpine(280 mg·kg-1,ip,one time).We constructed a type of lentiovirus encoding the full length cDNA of nNOS(LV-nNOS-GFP) and injected it and LV-GFP(1 μL) into the DG of the hippocampus 7 d after pilocarpine-induced seizure.The occurrence of epileptic spikes and spontaneous seizure(SRS)were monitored through electroencephalo-graph(EEG) and the protein expression was confirmed by Western blotting.We also constructed a lentioviral vehicle to interfere the expression of nNOS mRNA,which was named as LV-n NOSRNAi-GFP.A volume of 1 μL of LV-nNOS-RNAiGFP or LV-GFP was injected into the DG of the hippocampus 7 d before pilocarpine-induced seizure followed by EEG record and protein detection 2 months later.By EEG,we compared the susceptibility of nNOS knockout and wild-type mice to seizure induction and the development of epilepsy.In addition,we measured the influence of nNOS knockout on the excitability of dentate cells including mEPSC and mIPSC by using patch clamp technique.RESULTS Western blotting and qPCR measurement showed that the mRNA and protein expression of nNOS in the DG was not significantly changed in pilocarpinetreated mice compared with control mice.But the both m RNA and protein expression of nNOS decreased 7,14 and 60 d after treatment with pilocarpine(280 mg·kg-1,ip,one time).With infection of LV-nNOS-GFP in the DG,the decreased level of nNOS was recovered 7 d after seizure induction and the frequency of epileptic spikes and SRS were reversed by nNOS overexpression.We found that nNOS knockout caused a higher susceptive level to seizure induction by pilocarpine.Re-expression of nNOS in the DG of nNOS knockout mice relived the severity of epilepsy.By patch clamp recording,we found that there was no significant difference in the amplitude of mEPSC and mIPSC between nNOS knockout and wild-type mice,but the frequency of mEPSC was increased in nN OS knockout mice.Consistently,knockdown of nNOS by injection of LV-nNOS-RNAi-GFP into the DG caused higher frequency of epileptic spikes and SRS 2 months after pilocarpine-induced seizure.CONCLUSION Neurons expressing nNOS in the DG play an important role in the development of epilepsy.

COLOCALIZATION OF NITRIC OXIDE SYNTHASE IN GNRH NEURONS OF RAT FOREBRAIN AND HYPOTHALAMUS

In order to gain more information about the effect of nitric oxide (NO) on GnRH release, double NADPH diaphorase histochemistry GnRH immunohistochemistry staining was used to investigate the morphological relationship between NO synthase (NOS) containing cells and GnRH neurons in the forebrain and hypothalamus of rats. The results showed that some of the GnRH neurons in the diagonal band of Broca, olfactory tubercle and deeper part of temporal cortex had the NOS activity, suggesting GnRH secretion can be rapidly regulated by NO derived from GnRH neurons themselves in an autocrine manner.

Subcellular distribution of nitric oxide synthase isoforms in the rat duodenum

AIM:To study the cell-type specific subcellular distribution of the three isoforms of nitric oxide synthase(NOS) in the rat duodenum.METHODS:Postembedding immunoelectronmicroscopy was performed,in which primary antibodies for neuronal NOS(nNOS),endothelial NOS(eNOS),and inducible NOS(iNOS),were visualized with protein A-gold-conjugated secondary antibodies.Stained ultrathin sections were examined and photographed with a Philips CM10 electron microscope equipped with a MEGAVIEW II camera.The specificity of the immunoreaction in all cases was assessed by omitting the primary antibodies in the labeling protocol and incubating the sections only in the protein A-gold conjugated secondary antibodies.RESULTS:Postembedding immunoelectronmicroscopy revealed the presence of nNOS,eNOS,and iNOS immunoreactivity in the myenteric neurons,the enteric smooth muscle cells,and the endothelium of capillariesrunning in the vicinity of the myenteric plexus of the rat duodenum.The cell type-specific distributions of the immunogold particles labeling the three different NOS isozymes were revealed.In the control experiments,in which the primary antiserum was omitted,virtually no postembedding gold particles were observed.CONCLUSION:This postembedding immunoelectronmicroscopic study provided the first evidence of celltype-specific differences in the subcellular distributions of NOS isoforms.

Dose-dependent and combined effects of N-methyl-D-aspartate receptor antagonist MK-801 and nitric oxide synthase inhibitor nitro-L-arginine on the survival of retinal ganglion cells in adult hamsters

This study investigated the effects of daily intraperitoneal injections of N-methyl-D-aspartate receptor antagonist MK-801 and nitric oxide synthase inhibitor nitro-L-arginine （L-NA） on the survival of retinal ganglion cells （RGCs） at 1 and 2 weeks after unilateral optic nerve transection in adult hamsters. The left optic nerves of all animals were transected intraorbitally 1 mm from the optic disc and RGCs were retrogradely labeled with Fluorogold before they received different daily dosages of single MK-801 or L-NA as well as daily combinational treatments of these two chemicals. All experimental and control animals survived for 1 or 2 weeks after optic nerve transection. Our results revealed that the mean numbers of surviving RGCs increased and then decreased when the dosage of MK-801 （1.0, 3.0 and 4.5 mg/kg） and L-NA （1.5, 3.0, 4.5 and 6.0 mg/kg） increased at both 1 and 2 weeks survival time points. Daily combinational use of 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA lead to a highest RGC number that was even higher than the sum of the RGC numbers in 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA subgroups at 2 weeks. These findings indicated that both MK-801 and L-NA can protect axotomized RGCs in a dose-dependent manner and combinational treatment of these chemicals possesses a potentiative and protective effect.

生姜提取物对染铝毒大鼠学习记忆功能和神经细胞结构的影响及其可能作用机制

目的分析生姜提取物(GRE)对铝染毒大鼠学习记忆功能及神经细胞结构的影响,并基于钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)、神经元型一氧化氮合酶(nNOS)、蛋白激酶C(PKC)探讨其可能的作用机制。方法将36只SD雄性大鼠随机分为空白对照组、铝染毒组、临床药物组、低剂量GRE(L⁃GRE)组、中剂量GRE(M⁃GRE)组、高剂量GRE(H⁃GRE)组,每组6只大鼠。空白对照组大鼠自由饮用不含氯化铝的饮用水,其余组大鼠饮用含10 mg/mL结晶氯化铝的饮用水。3个月后给予空白对照组和铝染毒组大鼠灌胃生理盐水,临床药物组大鼠灌胃盐酸多奈哌齐溶液,分别给予L⁃GRE组、M⁃GRE组、H⁃GRE组大鼠灌胃100 mg/kg、200 mg/kg、400 mg/kg GRE溶液。持续干预4周后,采用Morris水迷宫实验评估大鼠的学习记忆功能,通过HE染色观察大鼠海马组织形态变化,分别采用实时荧光定量PCR和Western blot检测大鼠海马组织CaMKⅡ、nNOS、PKC mRNA和蛋白表达水平。结果(1)与空白对照组相比,铝染毒组大鼠的逃避潜伏期延长且穿越平台次数减少(P<0.05),海马组织的神经细胞皱缩,神经细胞数量明显减少,CaMKⅡ、nNOS、PKC的mRNA表达水平及nNOS、PKC的蛋白表达水平降低(P<0.05)。(2)与铝染毒组比,L⁃GRE组、M⁃GRE组和H⁃GRE组大鼠的逃避潜伏期缩短,临床药物组、M⁃GRE组、H⁃GRE组大鼠的穿越平台次数增加(P<0.05);各给药组大鼠海马组织病理形态有不同程度的改善;临床药物组大鼠的CaMKⅡ、nNOS和PKC mRNA表达水平升高,各剂量GRE组大鼠nNOS和PKC mRNA和蛋白表达水平升高(P<0.05)。(3)与临床药物组相比,M⁃GRE组和H⁃GRE组大鼠的逃避潜伏期,以及各剂量GRE组的穿越平台次数和目标象限停留时间差异无统计学意义(P>0.05);各剂量GRE组大鼠海马组织病理形态改善程度更为明显;M⁃GRE组、H⁃GRE组大鼠的nNOS、PKC蛋白表达水平升高,L⁃GRE组大鼠的nNOS蛋白表达水平升高(P<0.05)。(4)各剂量GRE组组间大鼠逃避潜伏期、穿越平台次数及目标象限停留时间差异无统计意义(P>0.05);随GRE干预剂量增加,大鼠海马组织病理形态的改善效果越明显;H⁃GRE组大鼠的CaMKⅡmRNA表达水平高于L⁃GRE组(P<0.05)。结论GRE能够改善铝染毒大鼠的神经细胞结构和学习记忆功能,其中对神经细胞结构的改善效果优于盐酸多奈哌齐,对学习功能的改善效果与盐酸多奈哌齐相当。GRE的作用机制可能与拮抗铝所致CaMKⅡ、nNOS和PKC表达水平下降有关。

人脐带间充质干细胞恢复糖尿病勃起功能障碍大鼠海绵体神经功能的实验研究

目的探索人脐带间充质干细胞通过再生修复海绵体神经对糖尿病大鼠勃起功能的改善作用。方法收集健康足月产新生儿脐带,提取分离人脐带间充质干细胞(hUCMSC)并鉴定表面标志物。通过高脂饮食联合腹腔注射链脲佐菌素(STZ)的方法诱导大鼠糖尿病,10周后选取高血糖大鼠进行阿扑吗啡(APO)试验,筛查、建立糖尿病引起的勃起功能障碍(DIED)大鼠模型。hUCMSC移植8周后,通过APO实验及最大海绵体内压/平均动脉压(ICP/MAP)评估大鼠勃起功能;通过免疫组化法分析大鼠海绵体组织中神经元型一氧化氮合酶(nNOS)水平;通过ELISA法检测各组大鼠阴茎组织中BDNF、NGF、VEGF和IGF-1神经营养生长因子的表达水平。结果第4代hUCMSC表面高表达CD73、CD90、CD105,低表达CD11b、CD19、CD34、CD45、HLA-DR。糖尿病引起的勃起功能障碍大鼠模型制备成功。海绵体内移植hUCMSC 8周后,APO实验表明hUCMSC可增加正常勃起的大鼠数量;中剂量和高剂量hUCMSC治疗后,相对于阴性对照组,ICP/MAP值升高,阴茎内血流灌注明显改善;免疫组化实验显示中剂量和高剂量hUCMSC治疗后,大鼠阴茎海绵体中nNOS水平明显增加,启动大鼠阴茎勃起;ELISA检测结果证实中剂量和高剂量hUCMSC治疗后,均能不同程度恢复DIED大鼠阴茎组织中BDNF、NGF、VEGF和IGF-1表达水平。结论hUCMSC是一种多功能的成体干细胞,经海绵体移植治疗高脂饮食联合STZ诱导的DIED大鼠是有效的,且有剂量依赖性。hUCMSC治疗DIED的机制与上调BDNF、NGF、VEGF和IGF-1表达水平提供的神经保护和再生修复作用有关。

活动性肺结核患者血清中SAMD9L水平变化及与巨噬细胞相关细胞因子的关系

目的 探讨血清中无菌α基序域9样蛋白(SAMD9L)和巨噬细胞相关细胞因子在活动性肺结核(APTB)患者诊断中的应用价值。方法 回顾性选取2019年5月至2022年5月就诊的APTB患者84例为APTB组,另选取同期84例潜伏期结核感染(LTBI)患者作为LTBI组,并选取同期84例健康志愿者作为对照组。检测3组患者血清SAMD9L、iNOS和Arg-1表达量。Pearson法分析APTB组血清SAMD9L与iNOS、Arg-1表达水平间的相关性;多因素Logistic回归模型分析发生APTB的影响因素。ROC曲线分析SAMD9L、iNOS和Arg-1表达量对APTB的预测价值。结果 APTB组血清中SAMD9L和Arg-1水平显著高于LTBI组和对照组(P<0.05);iNOS水平显著低于LTBI组和对照组(P<0.05)。iNOS水平分别与SAMD9L和Arg-1水平呈负相关(r=-0.405、-0.585,P<0.05),SAMD9L与Arg-1水平呈正相关(r=0.416,P<0.05)。Logistic回归显示iNOS含量是APTB患者的保护因子(P<0.05),SAMD9L和Arg-1含量是APTB患者危险因子(P<0.05)。SAMD9L、iNOS和Arg-1三者联合诊断APTB的结果优于任何一项单独分析(Z三项联合-SAMD9L=1.730,Z三项联合-iNOS=2.839,Z三项联合-Arg-1=2.086;P<0.05);治疗半年后血清中SAMD9L和Arg-1表达水平下降,iNOS表达量上升(P<0.05)。结论 APTB患者血清中SAMD9L与Arg-1表达水平上调,二者与巨噬细胞相关细胞因子关系密切,且三者联合检测对APTB的诊断具有临床意义。

Oxidative damage of primary cultured hippocampal neurons Does androgen have an antagonistic effect?

BACKGROUND： Evidence illustrates that androgen has a neuroprotective role. However, whether androgen also has the protective effect on hippocampal neurons during free radical mediated injury remains unclear. OBJECTIVE： To investigate the neuroprotective effect of androgen on hippocampal neurons during free radical damage. DESIGN, TIME AND SETTING： A controlled in vitro experiment was performed at the Department of Human Anatomy, Cell Culture Lab, and Neuroendocrinology Lab, Basic Medical School, Hebei Medical University from February to June 2009. MATERIALS： Testosterone was provided by Tianjin Jinyao Amino Acid Company, China. METHODS： Primary cultured neurons from 24 Sprague Dawley rats were randomly assigned into four groups： control, H202, testosterone, and testosterone （pre-added） plus H2O2 groups. MAIN OUTCOME MEASURES： The positive cell ratio of microtubule associated protein-Ⅱ and neuron specific enolase was determined by immunocytochemistry. Neuronal morphology was observed by hematoxylin-eosin staining and Nissl staining. Cell vitality and viability were determined using an inverted phase contrast microscope. The content of nitric oxide synthase, malondialdehyde, and superoxide dismutase were measured with a spectrophotometer. RESULTS： As compared with the control group, cell vitality and viability, and superoxide dismutase level were significantly decreased in the H202 group （P 〈 0.05）, while nitric oxide synthase and malondialdehyde levels were significantly increased （P 〈 0.05）. Neuronal vitality and viability as well as superoxide dismutase level in the testosterone plus H2O2 group were significantly greater than in the H2O2 group （P 〈 0.05）, and nitric oxide synthase and malondialdehyde levels were significantly less than in the H2O2 group （P〈 0.05）. CONCLUSION： Androgen partially reversed H2O2-induced neuronal damage and protected neurons.

Effects of Activated ACM on Expression of Signal Transducers in Cerebral Cortical Neurons of Rats

To explore the roles of astrocytes in the epileptogenesis, astrocytes and neurons were isolated, purified and cultured in vitro from cerebral cortex of rats. The astrocytes were activated by ciliary neurotrophic factor (CNTF) and astrocytic conditioned medium (ACM) was collected to treat neurons for 4, 8 and 12 h. By using Western blot, the expression of calmodulin dependent protein kinase Ⅱ (CaMKⅡ), inducible nitric oxide synthase (iNOS) and adenylate cyclase (AC) was de- tected in neurons. The results showed that the expression of CaMKⅡ, iNOS and AC was increased significantly in the neurons treated with ACM from 4 h to 12 h (P<0.05), and that of iNOS and AC peaked at 8 h and 12 h respectively. It was suggested that there might be some epileptogenic factors in the ACM and such signal pathways as NOS-NO-cGMP, Ca2+?CaM-CaMKⅡ and AC-cAMP-PKA might take part in the signal transduction of epileptogenesis.

Involvement of nitrergic neurons in colonic motility in a rat model of ulcerative colitis

BACKGROUND The mechanisms underlying gastrointestinal(GI)dysmotility with ulcerative colitis(UC)have not been fully elucidated.The enteric nervous system(ENS)plays an essential role in the GI motility.As a vital neurotransmitter in the ENS,the gas neurotransmitter nitric oxide(NO)may impact the colonic motility.In this study,dextran sulfate sodium(DSS)-induced UC rat model was used for investigating the effects of NO by examining the effects of rate-limiting enzyme nitric oxide synthase(NOS)changes on the colonic motility as well as the role of the ENS in the colonic motility during UC.AIM To reveal the relationship between the effects of NOS expression changes in NOS-containing nitrergic neurons and the colonic motility in a rat UC model.METHODS Male rats(n=8/each group)were randomly divided into a control(CG),a UC group(EG1),a UC+thrombin derived polypeptide 508 trifluoroacetic acid(TP508TFA;an NOS agonist)group(EG2),and a UC+NG-monomethyl-L-arginine monoacetate(L-NMMA;an NOS inhibitor)group(EG3).UC was induced by administering 5.5%DSS in drinking water without any other treatment(EG1),while the EG2 and EG3 were gavaged with TP508 TFA and L-NMMA,respectively.The disease activity index(DAI)and histological assessment were recorded for each group,whereas the changes in the proportion of colonic nitrergic neurons were counted using immunofluorescence histochemical staining,Western blot,and enzyme linked immunosorbent assay,respectively.In addition,the contractile tension changes in the circular and longitudinal muscles of the rat colon were investigated in vitro using an organ bath system.RESULTS The proportion of NOS-positive neurons within the colonic myenteric plexus(MP),the relative expression of NOS,and the NOS concentration in serum and colonic tissues were significantly elevated in EG1,EG2,and EG3 compared with CG rats.In UC rats,stimulation with agonists and inhibitors led to variable degrees of increase or decrease for each indicator in the EG2 and EG3.When the rats in EGs developed UC,the mean contraction tension of the colonic smooth muscle detected in vitro was higher in the EG1,EG2,and EG3 than in the CG group.Compared with the EG1,the contraction amplitude and mean contraction tension of the circular and longitudinal muscles of the colon in the EG2 and EG3 were enhanced and attenuated,respectively.Thus,during UC,regulation of the expression of NOS within the MP improved the intestinal motility,thereby favoring the recovery of intestinal functions.CONCLUSION In UC rats,an increased number of nitrergic neurons in the colonic MP leads to the attenuation of colonic motor function.To intervene NOS activity might modulate the function of nitrergic neurons in the colonic MP and prevent colonic motor dysfunction.These results might provide clues for a novel approach to alleviate diarrhea symptoms of UC patients.

Effects of adrenocorticotropic hormone and electroacupuncture on formalin-induced NOS-positiTe neurons in the spinal cord of rats

AIM:To study the effects of adrenocorticotropic hormone(ACTH) and electroacupuncture (EA) on formalin-induced nitric oxide synthetase (NOS)-positive neurons increases in the spinal cord or rats.METHODS:ACTH was administered by intrathecal injection (i.t.)and EA stimulation on “jiaji” point was performed by 1 mA 50Hz,5mA 5Hz and 1mA 5Hz respectively.The NOS-positive neurons were assayed by NADPH-diaphorase histochemistry.RESULTS:The results showed that both ACTH(0.5u,i.t.)and EA stimulation (1mA 50Hz,5mA 5Hz,1mA 5Hz) on “jiaji” point 30min significantly reduced the formalin-induced NOS-ositive neurons in the rat dorsal horn.The combinative use of ACTH (0.5u,i.t.) and EA(1mA 5Hz) caused a more marked reduction of the numbers of NOS positive neurons than that of the single ACTH or EA.Those effects were partially reversed by pretreatment with either the substrate of NOS,L-arginine (10nmol,i.t.)or opioid antagonist naloxone(10g,i.t.).CONCLUSION:These results suggests that both ACTH and EA might inhibit the formalin-induced NOS-positive neurons increases and have a synergic effect acting via a different pathway.

Effects of Extracellular ATP on Survival of Sensory Neurons in the Dorsal Root Ganglia of Rats

ATP was added to the cultured sensory neurons obtained from the dorsal root ganglia of the neonatal rats and PBS was added to serve as control. MTT assays were conducted to evaluate the survival and activity of the cultured neurons. And the silicone regenerative chamber was used after the sciatic nerve incision of the mature SD rat. 1 mmol/L ATP was injected into the left chamber and 0.09 % natrium chloride was injected into the right chamber as controls. The changes of nitric oxide synthase (NOS) activity in the corresponding dorsal root ganglia were measured histochemically and image analysis was also performed 4 days after the sciatic nerve injury. The results showed that extracellular ATP could enhance the survival of the neurons and the number of NOS positive neurons were significantly different between the ATP and control groups ( P <0.05). It was suggested that extracellular ATP had neurotrophic effect on neurons survival and could inhibit the NOS activity of the sensory neurons after the peripheral nerve incision, hence exerting the protective effect on the neurons, which was valuable for nerve regeneration after nerve injury.

Preconditioning crush increases the survival rate of motor neurons after spinal root avulsion

In a previous study, heat shock protein 27 was persistently upregulated in ventral motor neurons following nerve root avulsion or crush. Here, we examined whether the upregulation of heat shock protein 27 would increase the survival rate of motor neurons. Rats were divided into two groups： an avulsion-only group （avtflsion of the L4 lumbar nerve root only） and a crush-avulsion group （the L4 lumbar nerve root was crushed 1 week prior to the avulsion）. Immunofluores- cent staining revealed that the survival rate of motor neurons was significantly greater in the crush-avulsion group than in the avulsion-only group, and this difference remained for at least 5 weeks after avulsion. The higher neuronal survival rate may be explained by the upregulation of heat shock protein 27 expression in motor neurons in the crush-avulsion group. Further- more, preconditioning crush greatly attenuated the expression of nitric oxide synthase in the motor neurons. Our findings indicate that the neuroprotective action of preconditioning crush is mediated through the upregulation of heat shock protein 27 expression and the attenuation of neuronal nitric oxide synthase upregulation following avulsion.

Gut region-dependent alterations of nitrergic myenteric neurons after chronic alcohol consumption

Chronic alcohol abuse damages nearly every organ in the body. The harmful effects of ethanol on thebrain, the liver and the pancreas are well documented. Although chronic alcohol consumption causes serious impairments also in the gastrointestinal tract like altered motility, mucosal damage, impaired absorption of nu-trients and inflammation, the effects of chronically consumed ethanol on the enteric nervous system are less detailed. While the nitrergic myenteric neurons play an essential role in the regulation of gastrointestinal peristalsis, it was hypothesised, that these neurons are the first targets of consumed ethanol or its metabolites generated in the different gastrointestinal segments. To reinforce this hypothesis the effects of ethanol on the gastrointestinal tract was investigated in different rodent models with quantitative immunohistochemistry, in vivo and in vitro motility measurements, western blot analysis, evaluation of nitric oxide synthase enzyme activity and bio-imaging of nitric oxide synthesis. These results suggest that chronic alcohol consumption did not result significant neural loss, but primarily impaired the nitrergic pathways in gut region-dependent way leading to disturbed gastrointestinal motility. The gut segment-specific differences in the effects of chronic alcohol consumption highlight the significance the ethanol-induced neuronal microenvironment involving oxidative stress and intestinal microbiota.

The role of nitric oxide and neuronal nitric oxide synthase in zebrafish(Danio rerio)shoaling

Nitric oxide(NO)-the product of arginine metabolism catalyzed by nitric oxide synthases(NOS)-is a well-known neurotransmitter which plays an important role in metabolism and amino acid transportation in the nervous system.In particular,it can inhibit monoamine neurotransmitter transportation which affects animal behavior,especially social behavior.Shoaling-is a one kind of social behavior.It is a behavior that individual fish choose to join with their group within two factors;food and predation risk.Shoaling fish has quickly responded to predator and increased the change in feeding competition.In addition,shoaling also effect to stress response on stock density of aquaculture system.The effect of NO molecular signaling on the dopamine pathway was investigated using zebrafish(Danio rerio)as a model organism.Our aim was to understand the role of NOS and NO in shoaling behavior,which is typical of zebrafish.The concentration of NO in the zebrafish brain was modulated using a knockout for the neuronal NOS gene,and NO production was induced through treatment with L-arginine.The existence of NO in the zebrafish brain was confirmed by using a fluorescent probe.Dopamine concentration in the brain was measured by UPLC tandem mass spectrometer.We measured shoaling cohesion of all individual fish of D.rerio,using average distance between all pairs of fish(nearest neighbor distance)and analyzed tracking by Zebralab ViewPoint software.Collectively,our results suggest that a lower level of NO was associated with a higher level of dopamine,which in turn leads to the shoaling behavior.