Nerve growth factor and inducible nitric oxide synthase expression in the mesencephalon and diencephalon, as well as visual-and auditory-related nervous tissues, in a macaque model of type 2 diabetes

The present study detected distribution and expression of nerve growth factor and inducible nitric oxide synthase in the mesencephalon and diencephalon, as well as visual- and auditory-related nervous tissues, in a macaque model of type 2 diabetes using immunohistochemistry. Results showed that nerve growth factor expression decreased, but inducible nitric oxide synthase expression increased, in the mesencephalon and diencephalon, as well as visual- and auditory- related nervous tissues. These results suggested that nerve growth factor and inducible nitric oxide synthase play an important role in regulating the development of diabetic visual- and auditory-related diseases.

Biliverdin Reductase-A correlates with inducible nitric oxide synthasein in atorvastatin treated aged canine brain

Alzheimer’s disease is a neurodegenerative disorder characterized by progressive cognitive impairment and neuropathology. Recent preclinical and epidemiological studies proposed statins as a possible therapeutic drug for Alzheimer’s disease, but the exact mechanisms of action are still unknown. Biliverdin reductase-A is a pleiotropic enzyme involved in cellular stress responses. It not only transforms biliverdin-IX alpha into the antioxidant bilirubin-IX alpha but its serine/threonine/ tyrosine kinase activity is able to modulate cell signaling networks. We previously reported the beneficial effects of atorvastatin treatment on biliverdin reductase-A and heme oxygenase-1 in the brains of a well characterized pre-clinical model of Alzheimer’s disease, aged beagles, together with observed improvement in cognition. Here we extend our knowledge of the effects of atorvastatin on inducible nitric oxide synthase in parietal cortex, cerebellum and liver of the same animals. We demonstrated that atorvastatin treatment （80 mg/day for 14.5 months） to aged beagles selectively increased inducible nitric oxide synthase in the parietal cortex but not in the cerebellum. In contrast, inducible nitric oxide synthase protein levels were significantly decreased in the liver. Significant positive correlations were found between biliverdin reductase-A and inducible nitric oxide synthase as well as heme oxygenase-1 protein levels in the parietal cortex. The opposite was observed in the liver. Inducible nitric oxide synthase up-regulation in the parietal cortex was positively associated with improved biliverdin reductase-A functions, whereas the oxidative-induced impairment of biliverdin reductase-A in the liver negatively affected inducible nitric oxide synthase expression, thus suggesting a role for biliverdin reductase-A in atorvastatin-dependent inducible nitric oxide synthase changes. Interestingly, increased inducible nitric oxide synthase levels in the parietal cortex were not associated with higher oxidative/nitrosative stress levels. We hypothesize that biliverdin reductase-A-dependent inducible nitric oxide synthase regulation strongly contributes to the cognitive improvement observed following atorvastatin treatment.

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.

Effects of glucocorticoid dexamethasone on serum nitric oxide synthase activity and nitric oxide levels in a rat model of lung disease-induced brain injury

In this study, we investigated the effects of dexamethasone, pertussis toxin （a Gi protein inhibitor） and actinomycin （a transcription inhibitor） on serum nitric oxide synthase activity and nitric oxide content in a rat model of lung disease-induced brain injury. High-dose dexamethasone （13 mg/kg） and dexamethasone ＋ actinomycin reduced lung water content, increased serum nitric oxide synthase activity and nitric oxide content, diminished inflammatory cell infiltration in pulmonary alveolar interstitium, attenuated meningeal vascular hyperemia, reduced glial cell infiltration, and decreased cerebral edema. These results demonstrate that high-dose glucocorticoid treatment can reduce the severity of lung disease-induced brain injury by increasing nitric oxide synthase activity and nitric oxide levels.

Spatiotemporal expression of inducible nitric oxide synthase and cyclooxygenase 2 in the spinal cord during early stage sciatic nerve crush injury

BACKGROUND： Previous studies have shown that inducible nitric oxide synthase （iNOS） and cyclooxygenase 2 （COX-2） participate in inflammatory immune responses and neuropathic pain following peripheral nerve injury. However, few reports have addressed time-dependent expression of iNOS and COX-2 following peripheral nerve injury. OBJECTIVE： To investigate spatiotemporal expression of iNOS and COX-2 during early stage sciatic nerve crush injury.DESIGN, TIME AND SETTING： The randomized, controlled, animal experiment was performed at the Laboratory of Applied Anatomy, Department of Human Anatomy and Neurobiology, Central South University, China from September 2006 to September 2007.MATERIALS： Mouse anti-rat iNOS monoclonal antibody and goat anti-rat COX-2 monoclonal antibody （Transduction Laboratory, USA）, as well as biotinylated rabbit anti-mouse lgG and biotinylated rabbit anti-goat IgG （Santa Cruz Biotechnology, USA） were used in the present study.METHODS： A total of 48 healthy, adult, Sprague Dawley rats were randomly assigned to three groups. In the model group （n = 32）, crush injury to the right sciatic nerve was established using an artery clamp. The model group was further assigned to four subgroups according to survival time （6,12, 24, and 72 hours）, respectively （n = 8）. Sham surgery （n = 8） and normal control （n = 8） groups were also established.MAIN OUTCOME MEASURES： iNOS and COX-2 expression was detected in the L4-6 spinal cord with immunohistochemistry. Gray values of iNOS- and COX-2-postive cells in the anterior horn and posterior horn of spinal cord, as well as quantification of iNOS- and COX-2-positive cells in the anterior horn of spinal cord, were measured.RESULTS： iNOS and COX-2 expression gradually increased in the anterior horn and posterior horn of the spinal cord on the damaged side over time from 6 hours following sciatic nerve injury （P〈0.05） and peaked at 72 hours. Simultaneously, the number of iNOS- and COX-2-positive cells similarly increased in the anterior horn of spinal cord on the damaged side （P〈 0.05）.CONCLUSION： iNOS and COX-2 expression increased in the spinal cord during early stage sciatic nerve crush, which suggested that iNOS and COX-2 participate in occurrence and development of inflammatory immune responses following peripheral nerve injury.

Effects of melatonin on learning abilities, cholinergic fibers and nitric oxide synthase expression in rat cerebral cortex

BACKGROUND: Melatonin is a kind of hormones derived from pineal gland. Recent researches demonstrate that melatonin is characterized by anti-oxidation, anti-senility and destroying free radicals. While, effect and pathogenesis of pineal gland on learning ability should be further studied. OBJECTIVE: To investigate the effects of pinealectomy on learning abiliy, distribution of cholinesterase and expression of neuronal nitric oxide synthase (nNOS) in cerebral cortex of rats and probe into the effect of melatonin on learning ability, central cholinergic system and nNOS expression. DESIGN: Randomized grouping design and animal study. SETTING: Department of Neurology, the 187 Hospital of Chinese PLA. MATERIALS: A total of 12 male SD rats, of normal learning ability testing with Y-tape maze, of clean grade, weighing 190-210 g, aged 6 weeks, were selected in this study. METHODS: The experiment was carried out in the Department of Neurology, Zhujiang Hospital from July 1997 to June 2000. All SD rats were divided into experimental group (n =6, pinealectomy) and control group (n =6, sham operation). Seven days later, rats in both two groups were continuously fed for 33 days. ① Learning ability test: The learning ability of rats was tested by trisection Y-type maze and figured as attempting times. ② Expression of acetylcholinesterase (AchE) was detected by enzyme histochemistry and nNOS was measured by SABC method. ③ Quantitative analysis of AchE fibers: AchE fibers density in unit area (surface density) was surveyed with Leica Diaplan microscope and Leica Quantimet 500+ image analytic apparatus and quantitative parameter was set up for AchE fibers covering density (μm2) per 374 693.656 μm2, moreover, the AchE fibers density was measured in Ⅱ-Ⅳ layers of motor and somatosensory cortex (showing three layers per field of vision at one time), in radiative, lacunaria and molecular layers of CA1, CA2 and CA3 areas, and in lamina multiforms of dentate gyrus. Three tissue slices were picked up randomly in the same part of each rat, together six tissue slices for nNOS expression and four near view (× 400) were selected in the parts of right neocortex, medial septal nucleus-diagonal band nucleus (SM-DB), corpus striatus and hippocampus to count nNOS-positive cells. MAIN OUTCOME MEASURES: Learning ability; distribution and quantitative analysis of AchE fibers; expression of nNOS in various cerebral areas. RESULTS: The twelve rats were all involved in the final analysis. ① Learning ability test: The learning abilities before operation in the experimental group [(14.67±4.97) times] were consistent with those in the control group [(14.33±4.32) times, P > 0.05], the learning abilities in the experimental group at 40 days after pinealectomy [(28.67±2.42) times] were obviously more than those before pinealectomy and those in the control group after operation [(13.83±8.33) times, P < 0.01]. ② Results of AchE-positive fibers density in cerebral cortex of rats: The AChE-positive fibers densities in motor and somatosensory cortex, CA1, CA2 and CA3 areas of hippocampus and in lamina multiforms of dentate gyrus in the experimental group were obviously lower than those in the control group [experimental group: (15 244±1 339), (14 764±1 391), (12 991±970), (15 077±1 020), (19 546±1 489), (19 337±1 378) μm2; control group: (21 001±1 021), (17 930±2 225), (17 260±1 342), (18 911±1 048), (24 108±1 671), (22 917±1 909) μm2, P < 0.01]. ③ Expression of nNOS in various cerebral areas: nNOS-positive cells in cerebral cortex of rats of the experimental group were more, furthermore the ones in somatosensory cortex were slightly more in motor cortex and the number (5.90±0.68) was more than that in the control group (3.68±0.39,P < 0.05). The nNOS-positive cells in SM-DB (16.21±2.03) were markedly more than those in the control group (9.32±1.05,P < 0.01). The nNOS-positive cells in hippocampus (4.27±0.75) and in corpus striatus (9.35±2.58) were not different with those in the control group (3.94±0.53, 8.96±2.31, P > 0.05). CONCLUSION: Decrease of melatonin due to pinealectomy of rats can result in learning disorder, which may be related to trauma of cholinergic neuron in cerebral cortex which were caused by nitric oxide neurotoxicity arose from the overexpression of nNOS in cerebral neocortex and SM-DB.

Effect of extracellular signal-regulated kinase and nitric oxide on compressive neuralgia formation and maintenance

BACKGROUND： Previous studies have shown that extracellular signal-regulated kinase 1/2 （ERK1/2） and nitric oxide activation play a pivotal role in central sensitization and long-term neuronal plasticity induced by noxious stimulation. However, their effects on compressive neuralgia formation and maintenance remain poorly understood.OBJECTIVE： To investigate effects of the specific inhibitor of ERK1/2 signal pathway U0126 on neuronal nitric oxide synthase （nNOS） expression in the dorsal horn of the spinal cord in a compressive neuralgia rat model.DESIGN, TIME AND SETTING： A randomized, controlled experiment was performed at the Institute of Otolaryngology, Head and Neck Surgery, First Hospital of Jilin University from July 2008 to March 2009.MATERIALS： U0126 （Bio-Mol, USA） was used in this study.METHODS： A total of 84 rats were randomly assigned to two groups. In the first part of the experiment, 24 rats were used for behavioral testing, and they were randomly assigned to three sub-groups （n =8）： U0126, dimethyl sulfoxide （DMSO） and model control. In the second part of the experiment, 60 rats were used for immunofluorescence and Western blot analysis, and they were randomly assigned to six sub-groups （n = 10）： sham surgery, model control, U0126 post-injection at 0.5, 2, 12 and 24 hours. Neuropathic pain was produced by chronic compression to the dorsal root ganglion in rats from each sub-group. Rats in the U0126 group were administered a 5-ug U0126 intrathecal injection, and rats in the DMSO group were administered a 10-μL 5% DMSO intrathecal injection.MAIN OUTCOME MEASURES： Changes in mechanical and thermal hyperalgesia were observed using von Frey filaments and thermalqia stimular. Thermal and mechanical hyperalgesia were stimulated at different time points following intrathecal injection of U0126. nNOS activation and expression in the spinal cord dorsal horn were determined by immunofluorescence and Western blot analysis.RESULTS： Intrathecal injection of U0126 significantly attenuated chronic compression of dorsal root ganglion-induced mechanical and thermal hyperalgesia. Immunofluorescence staining results demonstrated that, compared to the sham surgery group, the number of nNOS-positive neurons was significantly increased in the injured spinal dorsal horn in the model control group （P〈0.01）. However, compared to the model control group, there were significantly decreasing numbers of nNOS-positive neurons in the U0126 post-injection groups at 0.5-hour, 2-hour, and 12-hour （P〈0.05）. Western blot analysis revealed similar results. CONCLUSION： Decreased activity in the ERK signal pathway resulted in down regulated nNOS expression in the dorsal horn of the spinal cord. These results suggested that ERK is involved in nitric oxide reaction to neuropathic pain.

Effects of resveratrol on hydrogen peroxide-induced oxidative stress in embryonic neural stem cells

Resveratrol, a natural phenolic compound, has been shown to prevent cardiovascular diseases and cancer and exhibit neuroprotective effects. In this study, we examined the neuroprotective and antJoxJdant effects of resveratrol against hydrogen peroxide in embryonic neural stem cells. Hydrogen peroxide treatment alone increased catalase and glutathione peroxidase activities but did not change superoxide dismutase levels compared with hydrogen peroxide ＋ resveratrol treatment. Nitric oxide synthase activity and concomitant nitric oxide levels increased in response to hydrogen peroxide treatment. Conversely, resveratrol treatment decreased nitric oxide synthase activity and nitric oxide levels. Resveratrol also attenuated hydrogen peroxide-induced nuclear or mitochondrial DNA damage. We propose that resveratrol may be a promising agent for protecting embryonic neural stem cells because of its potential to decrease oxidative stress by inducing higher activity of antioxidant enzymes, decreasing nitric oxide production and nitric oxide synthase activity, and alleviating both nuclear and mitochondrial DNA damage.

鞘内注射氯胺酮对甲醛炎性疼痛大鼠脊髓背角nNOS表达的影响

目的：观察甲醛炎性疼痛大鼠脊髓背角神经元型一氧化氮合酶（nNOS）的表达，以及鞘内注射氯胺酮对甲醛炎性痛大鼠脊髓背角nNOS表达的影响。方法：32只SD大鼠采用改良Yaksh法进行鞘内置管，随机分为4组（n=8）：对照组（C组）、鞘内注射生理盐水组（NS组）、氯胺酮50μg组（K1组）和氯胺酮100μg组（K2组）。NS，K1和K2组于置管5d后，复制甲醛炎性疼痛模型。采用疼痛加权评分法（PIS）评估大鼠甲醛致痛后1h内的疼痛行为，24h后用免疫组织化学法观察大鼠腰5节段水平脊髓背角nNOS的表达。结果：与NS组比较，K1组和K2组在甲醛炎性痛第2时相（15～60min）的PIS值明显降低（P〈0．01）；NS组大鼠脊髓背角nNOS免疫反应阳性细胞数量及免疫组织化学评分均较C组明显增加（P〈0．01），而K1和K2组则明显低于Ns组，差异有统计学意义（P〈0．01）。结论：鞘内注射氯胺酮对甲醛炎性痛大鼠具有明显的抗伤害作用；鞘内注射氯胺酮可明显抑制甲醛炎性痛引起的脊髓背角nNOS表达的增加，表明nNOS在脊髓水平伤害性信息的传递和调制中可能发挥重要作用。

运动性疲劳对大鼠乳头体nNOS表达的影响

目的:探讨乳头体中神经元型一氧化氮合酶(nNOS)与运动性疲劳的关系。方法:建立运动性疲劳动物模型,用免疫组织化学方法检测乳头体内侧核内侧部(mmm)、乳头体内侧核外侧部(mml)、乳头体内侧核后部(mmp)和乳头体外侧核(lm)等部位nNOS的表达。结果:疲劳组大鼠mmm处nNOS阳性神经元数量为(4.50±2.84)cells/U,面积为(179.81±130.15)μm2,均大于对照组(P<0.05,P<0.01);疲劳组大鼠mml处nNOS阳性神经元数量为(43.7±6.93)cells/U,面积为(5208.63±1253.20)μm2,均大于对照组(P<0.01);疲劳组大鼠mmp处nNOS阳性神经元数量为(8.88±4.26)cells/U,面积为(202.75±109.67)μm2,均大于对照组(P<0.05);疲劳组大鼠lm处nNOS阳性神经元数量为(27.2±6.94)cells/U,面积为(2763.23±1107.35)μm2,均大于对照组(P<0.01);疲劳组大鼠mmp处和lm处nNOS阳性神经元灰度值分别为54.27±14.86和72.45±28.07,均大于对照组(P<0.01,P<0.05)。结论:乳头体nNOS神经元与运动性疲劳密切相关,NO可能在乳头体对疲劳应激反应的调节中发挥重要作用。

兔脑nNOS阳性神经元的形态、结构和分布规律

利用SABC免疫组织化学方法,对兔脑神经型一氧化氮合酶(nNOS)阳性神经元的形态、结构及分布规律进行了系统研究,结果表明:①nNOS阳性神经元呈深棕色,着色主要在胞质内,细胞核处较为浅淡;神经元胞体形态多种多样,有三角形、圆形、椭圆形、梭形等,神经元突起有一个或者数个;nNOS阳性神经纤维大多呈棕色串珠样,有些区域的阳性纤维交错分布,相互交织成网状。②家兔的各个脑区均有nNOS阳性神经元和神经纤维出现,其中在大脑皮质、小脑、丘脑下部、中脑和脑桥广泛分布,延髓分布极少。③nNOS阳性神经元,在仔兔时就已基本发育到成年兔水平,以后随着年龄增长逐渐发育成熟至衰老,到2岁龄(老年兔)时就出现了显著的衰老变化,即密度减小,表达强度减弱,第一级突起数减少,最长突起长度变短等。结果提示:nNOS阳性神经元及其催化产生的NO在家兔中枢神经系统的发育和神经调控中起重要作用。

捕食应激致大鼠海马NO释放增多及nNOS表达增强

目的 探讨一氧化氮 (NO)在创伤后应激障碍 (PTSD)样行为异常大鼠的变化规律 ,以进一步揭示PTSD的神经生物学机制。方法 在大鼠捕食应激PTSD动物模型基础上 ,动态检测了大鼠海马、额叶皮层组织匀浆NO、一氧化氮合酶 (NOS)含量及神经元型NOS(nNOS)表达。结果 PTSD样行为异常大鼠海马NO含量于捕食应激后即刻明显升高 ,1 2h达高峰 ,2 4h时仍明显增多 ;海马nNOS表达亦同步增高 ,而额叶皮层则无明显改变。结论 严重心理 /生理应激所致海马nNOS持续性高表达与NO释放明显增多 。

不同补锌剂量对大鼠海马nNOS蛋白表达影响

目的探讨补锌对临床亚健康干预的合理剂量以及高锌对脑功能的影响及机制。方法采用Y-型迷宫行为学测试结合免疫组化卵白素-生物素-过氧化酶复合物法(ABC)法,分别观察大鼠海马结构不同亚区内神经元型一氧化氮合酶(nNOS)蛋白表达的变化。结果适量补锌〔200 mg/(kg.bw)饲料锌水平〕可使大鼠海马结构CA1、CA3、齿状回的nNOS表达增加(P<0.05),促进动物的学习记忆功能。而超高剂量补锌〔≥600 mg/(kg.bw)饲料锌水平〕,可使大鼠海马结构CA1、CA3、齿状回的nNOS表达减少(P<0.05),损害动物的学习记忆能力。结论适量补锌对大鼠学习记忆功能有促进作用,其机制可能与海马nNOS蛋白表达水平上调有关。

成年猕猴背根神经节nNOS免疫阳性神经元的分布

目的:观察正常成年猕猴背根神经节神经元型一氧化氮合酶(nNOS)免疫阳性神经元的分布。方法:ABC免疫细胞化学方法显示nNOS免疫阳性神经元,并用体视方法进行定量分析。结果:猕猴颈、胸、腰各段背根神经节nNOS免疫阳性神经元的分布相似,数量较多,阳性神经元的大小不等,多呈圆形或椭圆形;胞浆着色较深,胞核位于细胞中央,不着色,细胞被神经纤维束分隔成群。nNOS免疫阳性神经元以中型神经元为主,其次为小型神经元,其胞浆呈强阳性染色,细胞直径<50μm,大型神经元较少。颈、胸、腰各段背根神经节nNOS免疫阳性神经元的密度以及阳性细胞与总细胞数的比值均无明显差异。结论:猕猴背根神经节nNOS主要表达在中、小型神经元,提示NO可能主要参与痛觉等浅感觉的传导和调制。

电针对血管性痴呆大鼠学习记忆与皮层nNOS表达的影响

目的:探讨电针对血管性痴呆幼龄大鼠学习记忆与大脑皮层神经性一氧化氮合酶(nNOS)表达的影响。方法:采用改良的Pusinelli(4-VO)4-血管阻断法,建立全脑缺血再灌注大鼠痴呆模型,随机分为假手术组、模型组、电针组、脑复康组,分别给予相应措施处理后,同时处死检测各组大鼠脑皮质nNOS的表达变化,结合图像分析统计皮质等处阳性细胞数,用Y型电迷宫进行行为学测试并定量测定学习记忆成绩。经统计学处理,比较组间差异。结果:模型组大鼠Y电迷宫成绩显著低于假手术组(P<0.05),而大脑皮层nNOS蛋白表达和神经元丢失较假手术组为多(P<0.05);各治疗组Y电迷宫学习成绩优于模型组(P<0.05),而其皮层nNOS蛋白表达及神经元丢失较模型组明显减少(P<0.05),且与假手术组无显著性差异(P>0.05)。结论:电针治疗可明显改善血管性痴呆大鼠的Y迷宫学习记忆成绩,推测可能与电针抑制脑缺血后nNOS的过量表达和其介导的细胞毒性作用有关。

人胎儿和新生儿脊髓和DRG内nNOS阳性神经元的表达和分布

目的 :观察 2 4～ 2 7w人胎儿和新生儿的脊髓和背根神经节 (DRG)内神经元型一氧化氮合酶 (nNOS)阳性神经元的表达和分布。方法 :ABC免疫细胞化学方法。结果 :( 1 ) 2 4～ 2 7w人胎儿胸髓和腰 1～ 3节段的中间带外侧核和前角Ⅷ层和Ⅸ层内可观察到nNOS阳性神经元。颈、胸、腰各段DRG内nNOS免疫阳性神经元占DRG细胞总数的 84%～ 88%。 ( 2 )新生儿脊髓和DRG内nNOS免疫阳性神经元的分布情况与上述胎儿相似。但DRG内nNOS免疫阳性神经元的体积有所增加 ,数量明显减少 ,约占DRG细胞总数的 70 %～ 73 %。结论 :人胎儿脊髓和DRG在发育的 2 4～ 2 7w至出生时 ,nNOS阳性神经元的表达在定位分布上无差异 。

地高辛标记的大鼠nNOS mRNA探针的制备和应用

目的 制备大鼠神经元型一氧化氮合酶 (neuronalnitricoxidesynthase,nNOS)地高辛 (digoxigenin)标记的RNA探针 ,探讨nNOS在脊髓中的表达定位。方法 采用RT PCR方法 ,从大鼠脑组织中扩增nNOS基因mRNA部分片段 ,并经序列测定。以dig nNOSmRNA为探针 ,采用原位杂交观察成年大鼠脊髓组织中nNOSmRNA表达。结果 RT PCR法扩增出一特异产物与预期长度 2 4 0bp相符 ,T载体克隆测序与nNOS基因 10 0 %同源。原位杂交结果显示阳性信号出现在成年大鼠脊髓组织中。结论 采用RT PCR和T载体技术获得了大鼠脑组织nNOS基因克隆 ,dig nNOSmRNA探针原位杂交显示正常SD大鼠腰段脊髓组织中表达nNOSmRNA。

nNOS参与OGD介导的促神经干细胞增殖效应

目的:体外研究脑缺血促神经发生的机制。方法:体外培养神经干细胞(neural stem cells,NSCs)与海马神经元,并采用氧糖剥夺模型(oxygen and glucose deprivation,OGD)模拟在体缺血,通过RT-PCR、Western blot、免疫细胞化学、酶活性测定、亚硝酸盐/硝酸盐(nitrite/nitrate,NOx)含量测定等多种方法研究神经干细胞的生物学行为以及介导这种效应的分子机制。结果:(1)OGD通过直接和间接(神经元)作用增加神经干细胞中BrdU+细胞比例。(2)OGD上调神经干细胞中神经元型一氧化氮合酶(neuronal nitric oxide synthase,nNOS)并下调神经元中nNOS,且都有利于神经干细胞增殖。(3)采用nNOS基因敲除小鼠来源的神经干细胞以及神经元(用于共培养)进行实验,发现OGD并不能升高神经干细胞中BrdU+细胞比例。结论:神经干细胞和神经元中nNOS水平变化共同参与了OGD诱导的促神经干细胞增殖效应。

血管性痴呆小鼠大脑皮质NOS活性及nNOS蛋白表达的改变

目的观察血管性痴呆小鼠大脑皮质一氧化氮合酶(NOS)和神经元型一氧化氮合酶(nNOS)阳性神经元的变化,探讨血管性痴呆的发生机制。方法复制小鼠血管性痴呆模型,利用Y-迷宫检测血管性痴呆模型小鼠学习记忆能力,采用NADPH-d组织化学和ABC免疫组织化学方法,研究血管性痴呆小鼠与正常小鼠大脑皮质NOS和nNOS阳性神经元数量的变化。结果血管性痴呆小鼠比正常小鼠Y-迷宫学习记忆训练次数明显增多,差异有统计学意义(P<0.01);大脑皮质NOS和nNOS阳性神经元的数量明显增多,差异有统计学意义(P<0.05)。结论血管性痴呆的发生可能与大脑皮质NOS和nNOS阳性神经元的数量明显增多有关。

大鼠臂旁核内nNOS阳性传入纤维的脑内来源

目的探讨大鼠臂旁核(PBN)内神经元型一氧化氮合酶(nNOS)阳性传入纤维的脑内来源。方法在大鼠PBN内微量注射荧光金(FG),结合nNOS免疫荧光双标技术,观察FG/nNOS双标神经元在脑内的分布情况。结果FG/nNOS双标神经元主要分布在孤束核(NST)、延髓嘴侧腹外侧区(RVL)、中缝背核(DR)、导水管周围灰质(PAG)和下丘脑室旁核(PVN)。结论NST、RVL、DR、PAG和PVN内的nNOS阳性投射神经元是PBN内nNOS阳性纤维终末的主要来源。