Distribution of nitric oxide synthase in stomach myenteric plexus of rats

AIM: To study the distribution of nitric oxide synthase (NOS) in rat stomach myenteric plexus. METHODS: The distribution of NOS in gastric wall was studied in quantity and location by the NADPH-diaphorase (NDP) histochemical staining method and whole mount preparation technique. RESULTS: NOS was distributed in whole stomach wall, most of them were located in myenteric plexus, and distributed in submucosal plexus.The shape of NOS positive neurons was basically similar, most of them being round and oval in shape. But their density, size and staining intensity varied greatly in the different parts of stomach. The density was 62+/-38 cells mm(2) (antrum), 43+/-32 cells/mm(2) (body), and 32+/-28 cells mm(2) (fundus), respectively. The size and staining intensity of NOS positive neurons in the fundus were basically the same, the neurons being large and dark stained, while they were obviously different in antrum. In the body of the stomach, the NOS positive neurons were in an intermediate state from fundus to antrum. There were some beadlike structures which were strung together by NOS positive varicosities in nerve fibers, some were closely adherent to the outer walls of blood vessels. CONCLUSION: Nitric oxide might be involved in the modulation of motility, secretion and blood circulation of the stomach, and the significant difference of NOS positive neurons in different parts of stomach myenteric plexus may be related to the physiologic function of stomach.

Role of chloride channels in nitric oxide-induced rat hippocampal neuronal apoptosis in vitro

BACKGROUND：Chloride channels participate in non-neuronal apoptosis.However,it remains unclear whether chloride channels are involved in ischemic neuronal apoptosis.OBJECTIVE：To explore the effects of 4-acetamido-4＇-isothiocyanatostilbene-2,2＇-disulfonic acid （SITS） and 4,4＇-diisothiocyanostilbene-2,2＇-disulfonic acid （DIDS）,two chloride channel blockers,on the hippocampal neuronal apoptosis induced by 3-morpholinosydnonimine （SIN-1） based on the nitric oxide toxicity theory of neuronal apoptosis following ischemic brain injury.DESIGN,TIME AND SETTING：Comparative observation and in vitro experiments were performed at the laboratory of Zhuhai Campus of Zunyi Medical College from January to May 2009.MATERIALS：SIN-1,SITS,and DIDS were purchased from Sigma,USA.METHODS：Hippocampal neurons from Sprague-Dawley rats,aged 1 day,were cultured In vitro for 12 days and randomly assigned to control,SIN-1,or chloride channel blocker groups.SIN-1 group neurons were induced by SIN-1 for 18 hours to establish a model of ischemic neuronal apoptosis.Neurons in chloride channel blocker groups were treated with SITS or DIDS plus SIN-1 for 18 hours.The controls were cultured in DMEM/Ham＇s F12 complete medium alone.MAIN OUTCOME MEASURES：The apoptotic neurons and nuclear appearance were detected by Hoechst 33258 fluorescence staining; neuronal viability was quantitatively determined by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide analysis.Caspase-3 activity was analyzed by Western blot.RESULTS：SIN-1 （1 mmol/L） dramatically induced apoptosis （50%-60%）.SITS and DIDS inhibited nitric oxide-induced neuronal injury in a dose-dependent manner,suppressed caspase-3 activation,reduced neuronal apoptosis,and improved neuronal survival.CONCLUSION：Chloride channel blockers can protect against neuronal injury induced by NO.Chloride channels might be involved in neuronal apoptosis following cerebral ischemia.

Protective effect of electroacupuncture and moxibustion on gastric mucosal damage and its relation with nitric oxide in rats

INTRODUCTION Gastric mucosal injury is one of the common disorders,there are many reports subjicted to its pathogenesis treatment and prevention.We investigated the protective effect

Effect of L-NAME on nitric oxide and gastrointestinal motility alterations in cirrhotic rats

AIM: To investigate the effect of L-NAME on nitric oxide and gastrointestinal motility alterations in cirrhotic rats. METHODS: Rats with cirrhosis induced by carbon tetrachloride were randomly divided into two groups, one n =13 receiving 0.5mg.kg(-1) per day of N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, for 10 days, whereas the other group (n =13) and control (n =10) rats were administrated the same volume of 9g.L(-1) saline. Half gastric emptying time and 2h residual rate were measured by SPECT, using (99m)Tc-DTPA-labeled barium sulfate as test meal. Gastrointestinal transition time was recorded simultaneously. Serum concentration of nitric oxide (NO) was determined by the kinetic cadmium reduction and colorimetric methods. Immunohistochemical SABC method was used to observe the expression and distribution of three types of nitric oxide synthase (NOS) isoforms in the rat gastrointestinal tract. Western blot was used to detect expression of gastrointestinal NOS isoforms. RESULTS: Half gastric emptying time and trans-gastrointestinal time were significantly prolonged(124.0 +/- 26.4 min; 33.7 +/- 8.9 min; 72.1 +/- 15.3 min; P【0.01), (12.4 +/- 0.5h; 9.5 +/- 0.3h; 8.2 +/- 0.8h; P【0.01), 2h residual rate was raised in cirrhotic rats than in controls and cirrhotic rats treated with L-NAME (54.9 +/- 7.6%,13.7 +/- 3.2%, 34.9 +/- 10.3%, P【0.01). Serum concentration of NO was significantly increased in cirrhotic rats than in the other groups (8.20 +/- 2.48) micromol.L(-1), (5.94 +/-1.07) micromol.L(-1) and control (5.66 +/- 1.60 micromol.L(-1), P【0.01. NOS staining intensities which were mainly located in the gastrointestinal tissues were markedly lower in cirrhotic rats than in the controls and cirrhotic rats after treated with L-NAME. CONCLUSION: Gastrointestinal motility was remarkably inhibited in cirrhotic rats, which could be alleviated by L-NAME. Nitric oxide may play an important role in the inhibition of gastrointestinal motility in cirrhotic rats.

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

AIM： TO investigate the dynamic change and role of neuronal nitric oxide synthase （nNOS） and inducible nitric oxide synthase （iNOS） in neonatal rat with intestinal injury and to define whether necrotizing enterocolitis （NEC） is associated with the levels of nitric oxide synthase （NOS） in the mucosa of the affected intestine tissue. METHODS： Wistar rats less than 24 h in age received an intraperitoneal injection with 5 mg/kg lipopolysaccharide （LPS）. Ileum tissues were collected at 1, 3, 6, 12 and 24 h following LPS challenge for histological evaluation of NEC and for measurements of nNOS and iNOS. The correlation between the degree of intestinal injury and levels of NOS was determined. RESULTS： The LPS-injected increase in injury scores pups showed a significant versus the control. The expression of nNOS protein and mRNA was diminished after LPS injection. There was a negative significant correlation between the nNOS protein and the grade of median intestinal injury within 24 h. The expression of iNOS protein and mRNA was significantly increased in the peak of intestinal injury. CONCLUSION： nNOS and iNOS play different roles in LPS-induced intestinal injury. Caution should be exerted concerning potential therapeutic uses of NOS inhibitors in NEC.

Effects of aminoguanidine on nitric oxide production induced by inflammatory cytokines and endotoxin in cultured rat hepatocytes

AIM: To study the effects of aminoguanidine (AG) and two L-arginine analogues N(omega)-nitro-L-arginine methyl ester (L-NAME) and N(omega)-nitro-L-arginine (L-NNA) on nitric oxide (NO) production induced by cytokines (TNF-alpha, IL-1 beta, and IFN-gamma) and bacterial lipopolysaccharide (LPS) mixture (CM) in the cultured rat hepatocytes, and examine their mechanisms action. METHODS: Rat hepatocytes were incubated with AG, L-NAME, L-NNA, Actinomycin D (ActD) and dexamethasone in a medium containing CM (LPS plus TNF-alpha, IL-1 beta, and IFN-gamma) for 24h. NO production in the cultured supernatant was measured with the Griess reaction. Intracellular cGMP level was detected with radioimmunoassy. RESULTS: NO production was markedly blocked by AG and L-NAME in a dose-dependent manner under inflammatory stimuli condition triggered by CM in vitro. The rate of the maximum inhibitory effects of L-NAME (38.9%) was less potent than that obtained with AG(53.7%, P 【 0.05). There was no significant difference between the inhibitory effects of AG and two L-arginine analogues on intracellular cGMP accumulation in rat cultured hepatocytes. Non-specific NOS expression inhibitor dexamethasone (DEX)and iNOS mRNA transcriptional inhibitor ActD also significantly inhibited CM-induced NO production. AG(0.1 mmol x L(-1)) and ActD (0.2 ng x L(-1)) were equipotent in decreasing NO production induced by inflammatory stimuli in vitro, and both effects were more potent than that induced by non-selectivity NOS activity inhibitor L-NAME (0.1 mmol x L(-1)) under similar stimuli conditions (P【0.01). CONCLUSION: AG is a potent selective inhibitor of inducible isoform of NOS,and the mechanism of action may be not only competitive inhibition in the substrate level, but also the gene expression level in rat hepatocytes.

Distribution of constitutive nitric oxide synthase in the jejunum of adult rat

AIM: To study the distribution of the constitutive nitric oxide synthase (NOS) in the jejunum of adult rat. METHODS: The distribution of endothelial NOS (eNOS) was detected by immunohistochemistry. Immunofluorescence histochemical dual staining technique were used for studying the distribution of neuronal NOS (nNOS) and eNOS. The dual stained slides were observed under a confocal laser scanning microscope. RESULTS: Positive neuronal NOS (nNOS) and endothelial NOS (eNOS) cells were found to be distributed in lamina propria of villi, and the epithelial cell was not stained. eNOS was mainly located in submucosal vascular endothelia, while nNOS was mainly situated in myenteric plexus. Some cells in the villi had both nNOS and eNOS. More than 80% of the cells were positive for both nNOS and eNOS, the rest cells were positive either for nNOS or for eNOS. CONCLUSION: The two constitutive nitric oxide synthases are distributed differently in the jejunum of rat. nNOS distributed in myenteric plexus is a neurotransmitter in the non-adrenergic non-cholinergic (NANC) inhibitory nerves. eNOS distributed in endothelial and smooth muscle cells of blood vessels plays vasodilator role. eNOS and nNOS are coexpressed in some cells of lamina propria of villi. NO generated by those NOS is very important in the physiological and pathological process of small intestine.

Attenuation of Nitric Oxide Synthase Gene Expression in Rat Lung Induced by Hypoxia

In order to investigate the role of nitric oxide(NO) in pathogenesis of hypoxic pulmonary hypertension(HPH), the mean pulmonary arterial pressure(mPAP), mRNA expression of NO synthase(NOS) in lung tissues, cGMP levels, and their relationships were studied in rats exposed to hypoxia from 8 h to 28days. The results showed that mPAP began to increase in animals exposed to 10 % O2 for 8 h. Moreover, the longer the exposure, the higher the mPAP.Northern blot analysis and dot blot hybridization indicated that mRNA expression of NO in lung tissues of hypoxic rats tended to decrease with exposure days, but that of β-actin which acted as a control did not alter. The cGMP levels of plasma and lung tissues in hypoxic rats also inclined to be lower with exposure days. A marked negative correlations between the changes of cGMP levels and those of mPAP were found. It was suggested that mRNA expression of NOS gene was attenuated in hypoxic lung tissues, which may be one of important pathogenetic mechanisms of HPH.

Nitric oxide synthase distribution in esophageal mucosa and hemodynamic changes in rats with cirrhosis

AIM To observe the nitric oxide synthase (NOS) distribution in the esophageal mucosa andhemodynamic changes in cirrhotic rats.METHODS NOS distribution in the loweresophagus of rats with carbon tetrachlorideinduced cirrhosis was assessed by using NADPHdiaphorase (NADPH-d ) histochemical method.Concentration of NO in serum were measured byfluorometric assay. Mean arterial pressure(MAP), cardiac output (CO), cardiac index (CI),splanchnic vascular resistance (SVR ), andsplanchnic blood flow (SBF ) were alsodetermined using 5’CO-labeled microspheretechnique.RESULTS Intensity of NOS staining in theesophageal epithelium of cirrhotic rats wassignificantly stronger than that in controls.There was a NOS--positive staining area in theendothelia of esophageal submucosal vessels ofcirrhotic rats, but the NOS staining was negativein normal rats. NO concentration of serum incirrhotic rats were significantly higher incomparison with that of controls. Cirrhotic ratshad significantly lower MAP, SVR and higherSBF than those of the controls.CONCLUSION SPlanchnic hyperdynamiccirculatory state was observed in rats withcirrhosis. The endogenous NO may play animportant role in development of esophagealvarices and in changes of hemodynamics incirrhosis.

Rosuvastatin reduces rat intestinal ischemia-reperfusion injury associated with the preservation of endothelial nitric oxide synthase protein

AIM： To investigate the protective effect of rosuvastatin on ischemia-reperfusion （I-R）-induced small intestinal injury and inflammation in rats, and to determine the effect of this agent on the expression of endothelial nitric oxide synthase （eNOS） protein. METHODS： Intestinal damage was induced in male Sprague-Dawley rats by clamping both the superior mesenteric artery and the celiac trunk for 30 min, followed by reperfusion for 60 min. Rosuvastatin dissolved in physiological saline was administered intraperitoneally 60 min before ischemia. The severity of the intestinal mucosal injury and inflammation were evaluated by several biochemical markers, as well as by histological findings. The protein levels of eNOS were determined by Western blot. RESULTS： The levels of both intraluminal hemoglobin and protein, as indices of mucosal damage, were significantly increased in the I-R group compared with those in the sham-operated group. These increases, however, were significantly inhibited by treatment with rosuvastatin in a dose-dependent manner. The protective effects of rosuvastatin were also confirmed by histological findings. Exposure of the small intestine to I-R resulted in mucosal inflammation characterized by significant increases in thiobarbituric acid-reactive substances, tissueassociated myeloperoxidase activity, and the mucosal contents of rat cytokine-induced neutrophil chemoattracrant-1 （CINC-1） and tumor necrosis factor-α（TNF-α）. These increases in inflammatory parameters after I-R were significantly inhibited by pretreatment with rosuvo astatin at a dose of 10 mg/kg. Furthermore, mRNA expression of CINC-1 and TNF-α was increased after I-R, and this increase was also inhibited by rosuvastatin. The mucosal protein levels of eNOS decreased during I-R, but were preserved in rats treated with rosuvastatin. CONCLUSION： Rosuvastatin inhibits rat intestinal injury and inflammation induced by I-R, and its protection is associated with the preservation of eNOS protein.

Effects of nitric oxide on the prefrontal cortex in stressed rats

BACKGROUND： Nitric oxide （NO） exhibits both protective and detrimental effects in the central nervous system. OBJECTIVE： To investigate the effect of NO on the prefrontal cortex in neonatal stressed rats. DESIGN, TIME AND SETTING： A randomized, controlled, animal study was performed at the Anatomical Department of Iran University of Medical Sciences from May 2007 to August 2008. MATERIALS; Forty-eight male, Wistar rats were obtained from Pasteur＇s Institute, Tehran, Iran. METHODS： Rat stress models were established by immobilization and randomly received intraperitoneal injection of 2 mL physiological saline, L-arginine （200 mg/kg） as a NO precursor, N（G）-nitro-L-arginine methyl ester （20 mg/kg）, or subcutaneous injection of 7-nitroindazole （25 mg/kg） as a NO synthase inhibitor. MAIN OUTCOME MEASURES： After the rats were treated for 4 weeks, the frontal cortex was harvested for histological observation and NO detection. RESULTS： Subcutaneous administration of N（G）-nitro-L-arginine methyl ester or 7-nitroindazole resulted in significantly lower prefrontal cortex thickness and NO production compared with subcutaneous administration of L-arginine （P 〈 0.05）. Prefrontal cortex thickness significantly increased in rats following L-arginine treatment, compared with physiological saline intervention （P 〈 0.05）. CONCLUSION： NO exhibited protective effects on the prefrontal cortex of stressed rats.

Expression of nitric oxide synthase in the colon of diabetic rats

Objective: To investigate the differen t expression of three isozymes of nitric oxide synthase (NOS) in diabetic rat colo ns and the contribution to the colonic dysfunction. Methods: Sprague-Dawley (SD) rats were used in this experiment and diabetes were induc ed by streptozotocin (65 mg/kg, i.v.). Three isozymes of NOS (nNOS, iNOS and eNO S) expression in proximal and distal colon were measured in two weeks after diab etes induction using the methods of immunocytochemistry and semi-quantitative r everse transcription-polymerase chain reaction (RT-PCR). Results: Positive immunoreactivity for nNOS was found in intermuscular and submucous plexus neuronal cells, neither eNOS nor iNOS had been found in any layers of col on in the two groups. The expression of nNOS mRNA was significantly increased in diabetic colon than that in control rats as determined by RT-PCR. The eNOS mRN A level of diabetic colon was lower compared to thecontrol rats, while no expres sion of iNOS mRNA was found in the normal or diabetic rats. Conclusion: This report has demonstrated that nNOS increased and eNOS decreased in rat colon in the early stages of diabetes. NO production by the nNOS might play a key role in colonic dysfunction, as supported by raised nNOS mRNA and enzyme e xpression in the diabetic colon. Reduced eNOS activity might also contribute to colonic dysfunction in experimental diabetes.

The Role of Nitric Oxide in Hyperoxic Lung Injury in Premature Rats

To investigate the role of nitric oxide (NO) in hyperoxic lung injury, the 3 day old preterm rats were randomly assigned to four groups: group I (hyperoxia group), group Ⅱ (hyperoxia+N w nitro L arginine methyl ester (L NAME) group), group Ⅲ (air group), and group Ⅳ (air+L NAME) group. Group Ⅰ and Ⅱ were exposed to ≥90 % O 2 for 3 or 7 days. Group Ⅱ and Ⅳ received subcutaneous L NAMEy on daily basis (20 mg/kg). After 3 day or 7 day exposure, the lung wet weight/dry weight ratio (W/D), total protein and malondialdehyde (MDA) in bronchoalveolar lavage fluid (BALF) and lung pathology were examined in all groups. NO content, expression of endothelial NOS (eNOS) and inducible NOS (iNOS) in lungs were measured in group Ⅰ and Ⅲ. Our results showed that after 3 day exposure, group Ⅰ appeared acute lung injury characterized by the increase of MDA content ( P <0.01) and the presence of hyperaemia, red cell extravasation and inflammatory infiltration; after 7 day exposure, except MDA, total protein and W/D were also increased in comparison with group Ⅲ ( P <0.01, 0.05), pathological changes were more severe than those after 3 day exposure. After 3 and 7 day exposure, total protein in group Ⅱ was significantly increased as compared with group Ⅰ ( P <0.01 for both). The pulmonary acute inflammatory changes were more obvious in group Ⅱ than in group Ⅰ. Occasionally, mild hemorrhage was detected in the lungs of group Ⅳ. BALF protein content in group IV was higher than that in group Ⅲ after 7 day exposure ( P <0.01). After 3 and 7 day exposure, NO content in BALF were all significantly elevated in group Ⅰ as compared with group Ⅲ ( P <0.01 for all). In the lungs of group Ⅰ, strong immunostaining for iNOS was observed in airway and alveolar epithelia, inflammatory cells, which were stronger than those in group Ⅲ. Expression of iNOS in rats after 7 day hyperoxic exposure was stronger than that after 3 day exposure. Shortly after 7 day exposure, stronger immunostaining for eNOS in airway epithelia in group Ⅰ than that in group Ⅲ was seen. Our study suggested that treatment with L NAME worsened acute hyperoxic lung injury in preterm rats and also had a deleterious effect on the rats exposed to air, indicating that endogenous nitric oxide may play a protective role in rats under both physiological and hyperoxic status. Hyperoxia can significantly upregulate the expression of iNOS and eNOS in inflammatory cells, epithelia in the lungs of preterm rats, promote NO generation, which suggests that endogenous NO may mediate the hyperoxic pulmonary damage. Over stimulation of iNOS may contribute to the pathogenesis of hyperoxic lung injury. NO may have dual roles in pulmonary oxygen toxicity.

Expression of inducible nitric oxide synthase in transplanted rat liver and its inhibitory effect

It is reported that inducible nitric oxide synthase (iNOS) plays an important role in rejection. This study was designed to observe the expression of iNOS in the transplanted liver tissues and the effect of aminoguanidine (an inhibitor of iNOS) and FK506 (immunosuppression) on acute rejection after liver transplantation. METHODS:All rats were divided into four groups:homogeneous (LEW-LEW), acute rejection (BN-LEW), aminoguanidine (BN-LEW), and FK506 (BN-LEW). In the last two groups the drugs were used in heterogeneous rats after liver transplantation.The expression of iNOS in liver tissues of all groups was investigated immunohistochemically. RESULTS:In the acute rejection group, the expression of iNOS was extremely positive compared to the other three groups. The difference was statistically significant. CONCLUSIONS:During the process of acute rejection in rat orthotopic liver transplantation, the enhanced expression of iNOS correlates to the severity of rejection. Aminoguanidine and FK506 can inhibit the expression of iNOS and palliate acute rejection.

Japanese herbal medicine, Saiko-keishi-to, prevents gut ischemia/reperfusion-induced liver injury in rats via nitric oxide

AIM:To determine whether Saiko-keishi-to(TJ-10),a Japanese herbal medicine,could protect liver injury induced by gut ischemia/reperfusion(I/R),and to investigate the role of NO. METHODS:Male Wistar rats were exposed to 30-min gut isohemia followed by 60 min of reperfusion.Intravital microscopy was used to monitor leukocyte recruitment.Plasma tumor necrosis factor(TNF)levels and alanine aminotransferase (ALT)activities were measured.TJ-10 1 g/(kg.d)was intragastrically administered to rats for 7 d.A NO synthase inhibitor was administered. RESULTS:In control rats,gut I/R elicited increases in the number of stationary leukocytes,and plasma TNF levels and ALT activities were mitigated by pretreatment with TJ-10.Pretreatment with the NO synthase inhibitor diminished the protective effects of TJ-10 on leukostasis in the liver,and the increase of plasma TNF levels and ALT activities.Pretreatment with TJ-10 increased plasma nitrite/nitrate levels. CONCLUSION:TJ-10 attenuates the gut I/R-induced hepatic microvascular dysfunction and sequential hepatocellular injury via enhancement of NO production.

Effects of Infrasound on Gastric Motility, Gastric Morphology and Expression of Nitric Oxide Synthase in Rat

Infrasound widely condition, productive exists in nature, our living and traffic environment. Gastrointestinal tract is relatively sensitive to infrasound. However, the effect of infrasound on gastrointestinal function is unclear. Therefore, the purpose of our study was to observe the effects of infrasound on gastric motiliW and gastric morphology and to assess the expression of nitric oxide synthase （NOS） in gastric antrum after exposure to infrasound of 8 Hz - 130 dB for 2 hours per day for 14 consecutive days. Gastric motility was assessed by gastric fluid-emptying rate. Gastric morphology was evaluated by HE. The expression of NOS was measured by tissue microarray technology. The results would contribute to understand the role of infrasound in gastroenterology, and help to explain the mechanism of infrasound on gastroenterology.

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.

Sevoflurane and nitric oxide synthase expression in rat cochlea

Sevoflurane exhibits anesthetic action by inhibiting the auditory cortex, brain stem nitric oxide synthase activity, and reducing nitric oxide （NO）, thereby interfering with the hearing process. However, the influence of sevoflurane on peripheric receptor （cochlea） NO remains poorly understood. Results from the present study showed that sevoflurane downregulated cochlear inducible NO synthase, endothelial NO synthase and neuronal NO synthase expression in a dose dependent manner. This suggests that sevoflurane can decrease cochlear NO synthase expression in a dose dependent manner.

Spatio-temporal expression of inducible nitric oxide synthase in and surrounding a region of rat frontal lobe damaged with a sharp instrument

BACKGROUND： Inducible nitric oxide synthase （iNOS） cannot be detected in the neurons and glial cells of normal rats, but iNOS can be found in some neurons and glial cells of rats following ischemic, traumatic, neurotoxic or inflammatory damage. OBJECTIVE： To investigate iNOS expression and iNOS-positive cell types at various time points following damage to the rat frontal lobe using a sharp instrument. DESIGN： A nerve molecular biology, randomized, controlled study. TIME AND SETTING： This experiment was performed at the Department of Human Anatomy, Institute of Neurobiology, Medical School of Nantong University, between April 2006 and December 2007. MATERIALS： Rabbit anti-iNOS antibody （Santa Cruz, USA）, biotin labeled goat anti-rabbit antibody （Sigma, USA）, reverse transcription kit （Biouniquer, Hong Kong, China） and horseradish peroxidase labeled goat anti-rabbit antibody （Pierce, USA） were used for this study. METHODS： A total of 112 healthy rats aged 3 months were randomly assigned into a sham operation group （n = 28） and a damage group （n = 84）. Rat models of frontal lobe damage were induced in the damage group using a sharp instrument to make an incision in the frontal lobe cortex. In the sham operation group, the rat bone window was opened but brain tissues were left intact. MAIN OUTCOME MEASURES： Parameters were measured at 3, 6, 12, 24, 72, 120 and 168 hours following damage in both groups. Pathological changes were observed using Nissl staining and hematoxylin-eosin staining. Expression of iNOS mRNA, iNOS protein and iNOS-positive cells were examined by RT-PCR, Western blot analysis and immunohistochemistry, respectively. RESULTS： A large number of inflammatory cells infiltrated the damaged region 12 and 24 hours following damage, iNOS mRNA and iNOS protein expression increased in and around the damaged region 3 hours following damage, reached a peak at 24 hours, and then gradually decreased. The changes in iNOS-positive cell number reflected the changes in iNOS mRNA and iNOS protein expression after damage, iNOS was mainly found in neural cells at 3 and 6 hours, in macrophages at 12 and 24 hours, and in glial cells at 72 and 120 hours after damage. iNOS-positive cells were few in and surrounding the damaged region at 168 hours. There were a few iNOS-positive neural cells in the rat frontal lobe cortex in the sham operation group. CONCLUSION： Neurons, macrophages and glial cells can express iNOS following rat frontal lobe damage caused by a sharp instrument. The levels of iNOS expression, and the cell types expressing iNOS, change with time.

Distribution of nitric oxide synthase positive neurons in the substantia nigra of rats with liver cirrhosis

BACKGROUND：Nitrogen monoxide plays an important role in the physiological activity and pathological process of striatum in substantia nigra, and the nitric oxide synthase in substantia nigra may have characteristic changes after liver cirrhosis.OBJECTIYE： To observe the distribution and forms of nitric oxide synthase （NOS） positive neurons and fibers in substantia nigra of rats with liver cirrhosis.DESIGN： A comparative observational experiment.SETTINGS： Beijing Friendship Hospital; Capital Medical University.MATERIALS： Twenty 4-month-old male Wistar rats （120 - 150 g） of clean grade, were maintained in a 12-hour light/dark cycle at a constant temperature with free access to standard diet and water. Cryostat microtome （LEICA, Germany）; All the reagents were purchased from Sigma Company.METHODS： The experiment was carried out in the Department of Anatomy （key laboratory of Beijing city）,Capital Medical University from July 2000 to March 2002. The rats were randomly divided into normal group （n=10） and liver fibrosis group （n=10）. Rats in the liver fibrosis group were subcutaneously injected with 60% CCl4 oil at a dose of 5 mL/kg for the first time, and 3 mL/kg for the next 14 times, twice a week,totally 15 times. Liver fibrosis of grades 5 - 6 was taken as successful models. Whereas rats in the normal group were not given any treatment. Four months after CCl4 treatment, all the rats were anesthetized to remove brain, and frontal frozen serial sections were prepared. The expressions of nitric oxide synthase positive neurons in substantia nigra of rats were observed under inverted microscope. The number and gray scale of cell body of nitric oxide synthase positive neurons in substantia nigra were detected with NADPH-diaphorase staining.MAIN OUTCOME MEASURES： ①Number and gray scale of cell body of nitric oxide synthase positive neurons in substantia nigra; ②Expressions of nitric oxide synthase positive neurons in substantia nigra.RESULTS： All the 20 rats were involved in the analysis of results. ①The nitric oxide synthase positive neurons in substantia nigra were obviously fewer in the liver cirrhosis group than in the normal group （P〈0.01）, and the gray scale of the positive cell body was higher in the liver cirrhosis group than in the normal group （P〈0.05）. ② Abundant nitric oxide synthase positive neurons were observed in substantia nigra of neurons in substantia nigra were obviously fewer in the liver cirrhosis group than in the normal group （P〈0.01）, and the gray scale of the positive cell body was higher in the liver cirrhosis group than in the normal normal rats, the cell body of nitric oxide synthase positive neurons was clear and transparent, with short own cloudy processes. In substantia nigra of rats with liver cirrhosis, the body of nitric oxide synthase positive neurons were observed shrink obviously, less fibrin than normal.CONCLUSION： Rats with liver cirrhosis may suffer from the physiological dysfunction of neurons due to lack of fibers. The nitric oxide synthase positive neurons in substantia nigra can shrink and reduce.