Hyperlipidemia affects neuronal nitric oxide synthase expression in brains of focal cerebral ischemia rat model

BACKGROUND： Hyperlipidemia, a risk factor for ischemic cerebrovascular disease, may mediate production of neuronal nitric oxide synthase （nNOS） to induce increased nitric oxide levels, resulting in brain neuronal injury. OBJECTIVE： To investigate effects of hyperlipidemia on brain nNOS expression, and to verify changes in infarct volume and pathology during reperfusion, as well as neuronal injury following ischemia/reperfusion in a rat model of focal cerebral ischemia. DESIGN, TIME AND SETTING： Complete, randomized grouping experiment was performed at the Laboratory of Physiology, Shanxi Medical University from March 2005 to March 2006. MATERIALS： A total of 144 eight-week-old, male, Wistar rats, weighing 160-180 g, were selected. A rat model of middle cerebral artery occlusion was established by suture method after 4 weeks of formulated diet. Nitric oxide kit and rabbit anti-rat nNOS kit were respectively purchased from Nanjing Jiancheng Bioengineering Institute, China and Wuhan Boster Biological Technology, Ltd., China. METHODS： The rats were equally and randomly divided into high-fat diet and a normal diet groups. Rats in the high-fat diet group were fed a high-fat diet, consisting of 10% egg yolk powder, 5% pork fat, and 0.5% pig bile salt combined with standard chow to create hyperlipidemia. Rats in the normal diet group were fed a standard rat chow. A total of 72 rats in both groups were randomly divided into 6 subgroups： sham-operated, 4-hour ischemia, 4-hour ischemia/2-hour reperfusion, 4-hour ischemia/4-hour reperfusion, 4-hour ischemia/6-hour reperfusion, and 4-hour ischemia/12-hour reperfusion, with 12 rats in each subgroup. MAIN OUTCOME MEASURES： nNOS expression was measured by immunohistochemistry, and pathomorphology changes were detected by hematoxylin-eosin staining. Infarct volume and nitric oxide levels were respectively measured using 2, 3, 5-triphenyltetrazolium chloride （TTC） and immunohistochemistry. RESULTS： In the ischemic region, pathology changes were significant in the 4-hour ischemia/4-hour, 4-hour ischemia/6-hour reperfusion, and 4-hour ischemia/12-hour reperfusion subgroups fed on a high-fat diet compared to the same groups fed on a normal diet. In each ischemia subgroup, nNOS expression in brain tissues was higher than in the sham-operated subgroups fed on either the high-fat diet or normal diet （P 〈 0.01）. At each ischemia/reperfusion time point, rats fed on a high-fat diet expressed higher levels of nNOS compared to rats fed on the normal diet （P 〈 0.05）. When tissue was stained with TTC, a white infarction area was detected in the ischemic hemisphere, demonstrating that the infarct volume gradually increased with prolonged reperfusion time in each ischemia subgroup. At each ischemia/reperfusion time point, the infarct volume was larger in rats fed on a high-fat diet compared to those fed on a normal diet. CONCLUSION： nNOS expression was greater in hyperlipidemia rats following ischemia/reperfusion. Cerebral ischemia/reperfusion injury is aggravated with prolonged reperfusion time.

The action mechanism by which C1q/tumor necrosis factor-related protein-6 alleviates cerebral ischemia/reperfusion injury in diabetic mice

Studies have shown that C1q/tumor necrosis factor-related protein-6 (CTRP6) can alleviate renal ischemia/reperfusion injury in mice. However, its role in the brain remains poorly understood. To investigate the role of CTRP6 in cerebral ischemia/reperfusion injury associated with diabetes mellitus, a diabetes mellitus mouse model of cerebral ischemia/reperfusion injury was established by occlusion of the middle cerebral artery. To overexpress CTRP6 in the brain, an adeno-associated virus carrying CTRP6 was injected into the lateral ventricle. The result was that oxygen injury and inflammation in brain tissue were clearly attenuated, and the number of neurons was greatly reduced. In vitro experiments showed that CTRP6 knockout exacerbated oxidative damage, inflammatory reaction, and apoptosis in cerebral cortical neurons in high glucose hypoxia-simulated diabetic cerebral ischemia/reperfusion injury. CTRP6 overexpression enhanced the sirtuin-1 signaling pathway in diabetic brains after ischemia/reperfusion injury. To investigate the mechanism underlying these effects, we examined mice with depletion of brain tissue-specific sirtuin-1. CTRP6-like protection was achieved by activating the sirtuin-1 signaling pathway. Taken together, these results indicate that CTRP6 likely attenuates cerebral ischemia/reperfusion injury through activation of the sirtuin-1 signaling pathway.

Effect of Magnesium on Nitric Oxide Synthase of Neurons in Cortex during Early Period of Cerebral Ischemia

Summary: To investigate the effect of magnesium on nitric oxide synthase (NOS) of neurons in cortex during early cerebral ischemic period, a rat model of middle cerebral artery occlusion (MCAO) was established. The results showed that the NOS activity of neurons in cortex was in- creased significantly at 15 min after MCAO, reached its peak at 30 min after MCAO and returned to normal levels at 60 min after MCAO. The NOS activity of neurons in the magnesium-treated group was decreased significantly as compared with that in the ischemic group at 15 min and 30 ruin after MCAO respectively. The results suggested that magnesium could inhibit the elevated NOS activity of neurons in cortex induced by cerebral ischemia.

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.

Agmatine reduces nitric oxide synthase expression and peroxynitrite formation in the cerebral cortex in a rat model of transient global cerebral ischemia

Agmatine, an analog of L-arginine, is an endogenous substance synthesized by arginine decarboxylase, which has been shown to possess neuroprotective effects following brain ischemia. Nitric oxide is generated by sequential oxidation of the guanidinium group in L-arginine, and agmatine might protect the brain from ischemic injury by interfering with nitric oxide signaling. This study investigated the effects of agmatine on cerebral cortex neuronal injury following transient global cerebral ischemia and also detected nitric oxide synthase expression and peroxynitrite formation. Results demonstrated that intraperitoneal injection of agmatine in global cerebral ischemia/reperfusion alleviated ischemia/reperfusion-induced cerebral cortical cortex neuronal injury and cellular apoptosis, decreased neuronal and inducible nitric oxide synthase expression at 24, 48, and 72 hours following global cerebral ischemia and reperfusion, and greatly inhibited nitrotyrosine levels, which reflect the amount of peroxynitrite formed. These findings indicated that agmatine alleviates cerebral cortex neuronal injury following global cerebral ischemia and decreases nitric oxide synthase expression and peroxynitrite formation following ischemia/repeffusion.

Cervical sympathetic trunk transection affects inducible nitric oxide synthase expression in rat hippocampus following focal cerebral ischemia/reperfusion injury

BACKGROUND： The stellate ganglion block （SGB） plays a protective role in focal cerebral ischemia/reperfusion injury. The human SGB can be simulated by transection of the cervical sympathetic trunk （TCST） in rats. OBJECTIVE： To observe the effects of TCST on inducible nitric oxide synthase （iNOS） levels and cerebral infarct volume in the hippocampus of rats with cerebral ischemia/reperfusion injury, and to analyze the mechanism of action. DESIGN, TIME AND SETTING： A completely randomized, controlled, neuropathological experiment was performed at the Institute of Neurological Disease, Taihe Hospital, Yunyang Medical College between March and September 2006. MATERIALS： A total of 93 Wistar rats, aged 1718 weeks, of either gender, were used for this study. 2, 3, 5-triphenyl tetrazolium chloride was purchased from Changsha Hongyuan Biological Reagent Company China. Rabbit iNOS antibody and goat anti-rabbit IgG antibody were the products of Wuhan Boster Biological Reagent Co., Ltd., China. METHODS： Ten rats were randomly selected for the sham-operated group. Cerebral ischemia/reperfusion injury was induced by middle cerebral artery occlusion （MCAO） using the suture method in the remaining rats. Forty successful rat models were randomly and equally divided into the following two groups： （1） TCST group： subsequent to TCST, MCAO was performed for 2 hours, followed by 24 hours reperfusion; （2） model group： rats underwent experimental procedures similar to the TCST group, with the exception of TCST. Rats in the sham-operated group were subjected to experimental procedures similar to the model group; however, the thread was only introduced to a depth of 10 mm. MAIN OUTCOME MEASURES： Following 24 hours of reperfusion, functional neurological deficits were scored. Brain tissue sections from ten rats of each group were used to measure cerebral infarct volume by TTC staining. Hippocampal tissue sections of an additional ten rats from each group were used to detect iNOS levels using the streptavidin-peroxidase immunohistocbemistry method. Experimental data were expressed as absorbance values. RESULTS： Of the 93 selected rats, 50 were included in the final analysis. After MCAO for 2 hours, followed by 24 hours reperfusion, the absorbance values of iNOS-immunoreactive product were significantly greater in the model group compared to the TCST and sham-operated groups （P 〈 0.05）. However, there was no difference between the TCST and sham-operated groups （P 〉 0.05）. In addition, cerebral infarct volume in the model group was significantly greater compared to the TCST group （P 〈 0.05）. The TCST group performed with lower total functional neurological scores compared to the model group （P 〈 0.05）. CONCLUSION： TCST protects the brain against focal cerebral ischemia/reperfusion injury by possibly down-regulating hippocampal iNOS expression.

Effects of low molecular weight heparin-superoxide dismutase conjugate on serum levels of nitric oxide,glutathione peroxidase,and myeloperoxidase in a gerbil model of cerebral ischemia/reperfusion injury

BACKGROUND： Several studies have demonstrated that low molecular weight heparin-superoxide dismutase （LMWH-SOD） conjugate may exhibit good neuroprotective effects on cerebral ischemia/reperfusion injury though anticoagulation, decreasing blood viscosity, having anti-inflammatory activity, and scavenging oxygen free radicals. OBJECTIVE： To investigate the intervention effects of LMWH-SOD conjugate on serum levels of nitric oxide （NO）, glutathione peroxidase （GSH-Px）, and myeloperoxidase （MPO） following cerebral ischemia/reperfusion injury. DESIGN, TIME AND SETTING： A randomized, controlled, and neurobiochemical experiment was performed at the Institute of Biochemical Pharmacy, School of Pharmaceutical Sciences, Shandong University between April and July 2004. MATERIALS： A total of 60 Mongolian gerbils of either gender were included in this study. Total cerebral ischemia/reperfusion injury was induced in 50 gerbils by occluding bilateral common carotid arteries. The remaining 10 gerbils received a sham-operation （sham-operated group）. Kits of SOD, NO, and MPO were sourced from Nanjing Jiancheng Bioengineering Institute, China. LMWH, SOD, and LMWH-SOD conjugates were provided by Institute of Biochemistry and Biotechnique, Shandong University, China. METHODS： Fifty successful gerbil models of total cerebral ischemia/reperfusion injury were evenly randomized to five groups： physiological saline, LMWH-SOD, SOD, LMWH ＋ SOD, and LMWH. At 2 minutes prior to ischemia, 0.5 mL/65 g physiological saline, 20 000 U/kg LMWH-SOD conjugate, 20 000 U/kg SOD, a mixture of SOD （20 000 U/kg） and LMWH （LMWH dose calculated according to weight ratio, LMWH： SOD = 23.6：51）, and LMWH （dose as in the LMWH ＋ SOD group） were administered through the femoral artery in each above-mentioned group, respectively. MAIN OUTCOME MEASURES： Serum levels of NO, MPO, and GSH-Px. RESULTS： Compared with 10 sham-operated gerbils, the cerebral ischemia/reperfusion injury gerbils exhibited decreased serum levels of GSH-Px and increased serum levels of NO and MPO （P 〈 0.01）. The serum level of GSH-Px was significantly upregulated in all groups, in particular in the LMWH-SOD group （P 〈 0.01）, compared with the physiological saline group （P 〈 0.05-0.01）. Following medical treatment, serum levels of NO and MPO were significantly downregulated in all groups, in particular in the LMWH-SOD group （P 〈 0.01）. Serum levels of GSH-Px, NO, and MPO in the LMWH-SOD group were close to those in the sham-operated group （P 〉 0.05）. CONCLUSION： In cerebral ischemia/reperfusion injury, LMWH-SOD conjugate exhibits stronger neuroprotective effects on free radical scavenging, inflammation inhibition, and cytotoxicity inhibition than simple or combined application of LMWH and SOD by downregulating NO and MPO levels and upregulating the GSH-Px level.

Changes of Nitric Oxide Synthase Activity in Penumbral and Core Area during Focal Cerebral Ischemia and Reperfusion in Rats

Objective:To study the changes of nitric oxide synthase(NOS)activity in penumbral and core area during focal cerebral ischemia and reperfusion,and to explore the therapeutic window of focal cerebral ischemia. Methods:The middle cerebral artery of rats was occluded for 15,30,60,90 and 120 min by an intraluminal filament respectively,and recirculation was instituted for 24 h.The changes of NOS activity in ischemic core area(parietal cortex and caudoputamen)and penumbral area (frontal cortex)were examined after focal cerebral ischemia and reperfusion using NADPH-d histochemistry technique.Results:The NOS activity of the ischemic penumbral area peaked at 60 min while the ischemic core area peaked at 30 min then declined at 90-120 min sharply.Conclusion:NOS takes part in cerebral ischemic damage during focal cerebral ischemia and reperfusion.The NOS activity of the ischemic penumbral area is different from the ischemic core area.The peak time of the penumbral area is delayed comparing with the core area.The data suggest that the best time to apply NOS inhibitor is within 30 min in ischemic core area, and 60 min in penumbral area.

Effect of nitric oxide synthase inhibitor N^G-nitro-L-arginine on the content of amino acid in ischemic brain tissues of rats

BACKGROUND： Nitric oxide synthase （NOS） inhibrtors have been widely used to investigate the role of NO on cerebral ischemic injury, but the results are controversial. Moreover, it has been considered to aggravate the ischemic neuronal damage with the release of excessively excitatory amino acids （EAA） during cerebral ischemia. On the other hand, some inhibitory amino acid is suggested to be important for the neuronal protection against ischemic brain damage. Our study has recently showed that treatment with the NOS inhibitor NG-nitro-L-arginine （L-NA） reduced focal cerebral ischemic damage. The effect of L-NA on the contents of excitatory and inhibitory amino acid in the rat brain following cerebral ischemia is still unclear. OBJECTIVE： By evaluating the effect of NOS inhibitor, L-NA on the contents of aspartate, glutamate, glycine and γ-aminobutyric acid （GABA） in striatum, hippocampus and cortex in the rat brain following cerebral ischemia respectively, to investigate the beneficial effect of L-NA on cerebral ischemic injury and the possible mechanism. DESIGN： A randomized and controlled experiment SETTING ： Department of Pharmacology, Hebei Academy of Medical Sciences MATERIALS： A total of 42 male healthy SD rats （grade Ⅱ, weighting 250-300 g） were provided by the Experimental Animal Center of Hebei Province （Certification： 04036）. Aspartate, glutamate, glycine, GABA, L-NA and 2,3,5-triphenyltetrazolium chloride （TTC） were obtained from Sigma Chemicals Co, St Louis, MO, USA. HPLC-ultraviolet detector system consisted of Agilent 1100 HPLC. METHODS： The experiment was carried out in Department of Pharmrcology, Hebei Academy of Medical Sciences from June 2005 to June 2006. Rats were randomly divided into three groups： sham-operated group （n = 6）, ischemic group （n = 18）, L-NA group （n = 18）. The model of focal cerebral ischemia in rat was prepared with intraluminal line occlusion methods. In sham-operated rats, the external carotid artery was surgically prepared, but the filament was not inserted. Each group was further divided into 3 subgroups （n = 6 for each）： drugs were administrated at 2, 6 and 12 hours after the middle cerebral artery occlusion （MCAO） respectively. L-NA （20 mg/kg, ip） was administrated, twice a day, for 3 consecutive days. Same volume of normal saline was administrated in ischemic and sham operation groups. The changes of infarcted volume and the contents of amino acids were respectively assayed. Image analysis software was used for the measurement of the infarcted area. The results were expressed as a percentage of the infarcted volume of cerebral/volume of whole brain （IV%） in order to control for edema formation. The contents of aspartate, glutamate, glycine and GABA in striatum, hippocampus and cortex in the rat brain following cerebral ischemia were respectively measured by HPLC method. All data were analyzed with one-way ANOVA and Dunnett＇s test. MAIN OUTCOME MEASURES： （1） The volume of cerebral infarction; （2） The contents of aspartate, glutamate glycine and GABA in brain tissue after cerebral ischemia. RESULTS ： All 42 rats were involved in the final analysis. （1） Infarcted volume： Volume was 0 in sham-operated group. When L-NA was administrated at 2 and 6 hours after MCAO, the infarcted volume was （20.13±3.59）% and （23.12±5.84）% in L-NA group, which was not similar to that in ischemic group [（22.10±3.98）%, （25.38± 5.37）%, P〉 0.05]. However, the infarcted volume was markedly decreased compared with that of ischemic group when L-NA was administrated at 12 hours after MCAO [（26.11±3.55）% and （37.15±3.58）%, P 〈 0.01]. Changes of amino acid content： At 2 and 6 hours after ischemia, the contents of aspartate, glutamate, glycine and GABA in striatum, hippocampus and cortex in ischemic group were significantly increased compared with those in sham-operated group （ P〈 0.05-0.01）. However, contents in L-NA group were similar to those in ischemic group （P 〉 0.05）. At 12 hours after ischemia, the contents of aspartate [（0.21 ±0.06）, （0.36±0.05）, （0.29±0.12） mg/g] and glutamate [（0.55±0.06）, （0.78±0.10）, （0.52±0.10） mg/g] in striatum, hippocampus and cortex in L-NA group were significantly decreased compared with those in ischemic group [（0.49±0.17）, （0.63± 0.03）, （0.51±0.15） mg/g; （0.98±0.30）, （1.15±0.15）, （0.93±0.15） mg/g, P〈 0.05-0.01]. Glycine in hippocampus was （0.40±0.07） mg/g, which was higher than that in ischemic group [（0.21±0.07） mg/g, P 〈 0.05]. GABA in striatum, hippocampus and cortex was （0.93±0.10）, （0.62±0.12） and （0.81 ±0.10） mg/g, respectively, which was higher than that in ischemic group [（0.60±0.08）, （0.37±0.17）, （0.59±0.10） mg/g, P 〈 0.05-0.01]. CONCLUSION ： It may be concluded that L-NA have beneficial effect on ischemic cerebral injury in ischemic later stage in rats. The possible mechanism is that L-NA can decrease the contents of aspartate and glutamate, increase the contents of glycine and GABA.

EFFECT OF RADIX SALVIAE MILTIORRHIZAE ON THE GENE EXPRESSION OF NITRIC OXIDE SYNTHASE IN ISCHEMIC RAT BRAINS

The effect of Radix Salviae Miltiorrhizae (RSM) on the gene expression of nitric oxide synthase (NOS) in rat brains during ischemia was studied with in situ hybridization and the results were analyzed with IBAS 2000 Image Analysis System. It was found that NOS gene expression of cerebral cortex and caudate-putamen was markedly increased in 24 hours in ischemia group (P

Blood-letting punctures at twelve Jing-Well points of the hand can treat cerebral ischemia in a similar manner to mannitol

A rat model of middle cerebral artery permanent occlusion was established using the modified Longa method. Successfully established model animals were treated by blood-letting puncture at twelve Jing-Well points of the hand, and/or by injecting mannitol into the caudal vein twice daily. Brain tissue was collected at 24, 48 and 72 hours after modeling, and blood was collected through the retinal vein before Evans blue was injected, approximately 1 hour prior to harvesting of brain tissue. Results showed that Evans blue leakage into brain tissue and serum nitric oxide synthase activity were significantly increased in model rats. Treatment with blood-letting punctures at twelve Jing-Well points of the hand and/or injection of mannitol into the caudal vein reduced the amount of Evans blue leakage into the brain tissue and serum nitric oxide synthase activity to varying degrees. There was no significant difference between single treatment and combined treatment. Experimental findings indicate that blood-letting punctures at twelve Jing-Well points of the hand can decrease blood-brain barrier permeability and serum nitric oxide synthase activity in rats following middle cerebral artery occlusion, and its effect is similar to that of mannitol injection alone and Jing-Well points plus mannitol injection.

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.

Anti-oxidant effect of picroside II in a rat model of cerebral ischemia/reperfusion injury

Picroside II,the major active component of picroside,has been shown to induce PC12 cell axonal growth and relieve free radical damage.In vivo experiments have demonstrated that picroside II can improve neurological function in rats with cerebral ischemia/reperfusion injuries.In the present in vivo study,enzyme-linked immunosorbent assay and immunohistochemistry revealed that picroside II increased superoxide dismutase content and reduced inducible nitric oxide synthase content in the ischemic hemisphere.The effects of picroside II were similar to those of salvianic acid A sodium,an active control drug.These results indicate that picroside II exerts a neuroprotective effect,possibly by downregulating inducible nitric oxide synthase expression,increasing superoxide dismutase activity,and inhibiting neuronal apoptosis.

Neuronal apoptosis and inflammatory reaction in rat models of focal cerebral ischemia following 40-minute suspended moxibustion

The treatment duration of heat-sensitive moxibustion（approximately 40 minutes on average） is longer than that of traditional suspended moxibustion.The present study investigated expression changes of three inflammatory and apoptosis-associated proteins（inducible nitric oxide synthase,cyclooxygenase-2 and caspase-3） in transient middle cerebral artery occlusion model rats following suspended moxibustion for 40 minutes,to explore the mechanisms underlying neuroprotective action of suspended moxibustion.The results indicated that suspended moxibustion at acupoint Dazhui（DU 14） for 40 minutes reduced the cortical expression of caspase-3,cyclooxygenase-2 and inducible nitric oxide synthase proteins of transient middle cerebral artery occlusion model rats,as well as decreasing infarct volume and ameliorating the neurological deficit score.Outcomes with 40 minutes of moxibustion were superior to the outcomes after suspended moxibustion for 15 minutes.

Protective effect of ginkgo proanthocyanidins against cerebral ischemia/reperfusion injury associated with its antioxidant effects

Proanthocyanidins have been shown to effectively protect ischemic neurons, but its mechanism remains poorly understood. Ginkgo proan-thocyanidins （20, 40, 80 mg/kg） were intraperitoneally administered 1, 24, 48 and 72 hours before reperfusion. Results showed that ginkgo proanthocyanidins could effectively mitigate neurological disorders, shorten infarct volume, increase superoxide dismutase activity, and de-crease malondialdehyde and nitric oxide contents. Simultaneously, the study on grape seed proanthocyanidins （40 mg/kg） confirmed that different sources of proanthocyanidins have a similar effect. The neurological outcomes of ginkgo proanthocyanidins were similar to that of nimodipine in the treatmen＇t of cerebral ischemia/reperfusion injury. （Sur results suggestthat-ginkgo proanthocyanidins can effectively lessen cerebral ischemia/reperfusion injury and protect ischemic brain tissue and these effects are associated with antioxidant properties.

Changes in secretory pathway Ca^(2+)-ATPase 2 following focal cerebral ischemia/reperfusion injury

This study aimed to investigate changes in secretory pathway Ca2＋-ATPase 2 expression following cerebral ischemia/reperfusion injury, and to define the role of Ca2＋-ATPases in oxidative stress. A rat model of cerebral ischemia/reperfusion injury was established using the unilateral middle cerebral artery occlusion method. Immunohistochemistry and reverse transcription-PCR assay results showed that compared with the control group, the expression of secretory pathway Ca2＋-ATPase 2 protein and mRNA in the cerebral cortex and hippocampus of male rats did not significantly change during the ischemic period. However, secretory pathway Ca2＋-ATPase 2 protein and mRNA expression reduced gradually at 1, 3, and 24 hours during the reperfusion period. Our experimental findings indicate that levels of secretory pathway Ca2＋-ATPase 2 protein and mRNA expression in brain tissue change in response to cerebral ischemia/reperfusion injury.

3′-Daidzein sulfonate sodium improves mitochondrial functions after cerebral ischemia/reperfusion injury

3′-Daidzein sulfonate sodium is a new synthetic water-soluble compound derived from daidzein（an active ingredient of the kudzu vine root）. It has been shown to have a protective effect on cerebral ischemia/reperfusion injury in rats. We plan to study the mechanism of its protective effect. 3′-Daidzein sulfonate sodium was injected in rats after cerebral ischemia/reperfusion injury. Results showed that 3′-daidzein sulfonate sodium significantly reduced mitochondrial swelling, significantly elevated the mitochondrial membrane potential, increased mitochondrial superoxide dismutase and glutathione peroxidase activities, and decreased mitochondrial malondialdehyde levels. 3′-Daidzein sulfonate sodium improved the structural integrity of the blood-brain barrier and reduced blood-brain barrier permeability. These findings confirmed that 3′-daidzein sulfonate sodium has a protective effect on mitochondrial functions after cerebral ischemia/reperfusion injury, improves brain energy metabolism, and provides protection against blood-brain barrier damage.

红毛五加叶水提取物对大鼠局灶性脑缺血损伤的预防作用

目的:研究红毛五加叶水提取物(acanthopanax giraldii leaves extract,AGLE)对大鼠局灶性脑缺血损伤的预防作用。方法 :将138只SPF级雄性SD大鼠随机分为假手术组、模型组、银杏叶片组(0.04 g/kg)及AGLE高、中、低剂量组(20、10、5 g/kg),每组23只,采用插线法阻塞大鼠大脑中动脉(middle cerebral artery,MCA)制备局灶性脑缺血模型;采用氯化三苯基四氮唑(2.3.5-Triphenytetrya-zolium chloride,TTC)染色法测定脑梗死范围;Longa法观测大鼠神经功能缺失评分;光镜下观察缺血侧大脑皮层病理学改变;分光光度法和硝酸还原酶法分别测定脑组织一氧化氮合酶(nitric oxide synthase,NOS)活性及一氧化氮(nitric oxide,NO)含量。结果:模型组大鼠行为指标评分、脑梗死范围、NOS活性及NO含量均高于假手术组(P<0.01);缺血侧大脑皮层出现神经元结构不清,神经细胞肿胀,核固缩、溶解、破裂或消失等病理改变。与模型组比较,AGLE各剂量组大鼠行为指标评分、脑梗死范围、NOS活性及NO含量均降低(P<0.01)。与银杏叶片组比较,AGLE低剂量组改善大鼠行为指标评分更明显(P<0.01)。AGLE各剂量组脑梗死范围、NOS活性及NO含量与银杏叶片组比较,差异无统计学意义(P>0.05)。结论:AGLE对大鼠局灶性脑缺血损伤具有预防作用,其作用机制可能与降低脑组织中NOS活性及NO含量有关。

电针预处理丰隆穴对脑血栓大鼠神经生长因子、一氧化氮水平的影响

目的 观察电针预处理丰隆穴(ST40)对三氯化铁(FeCl_(3))诱导的远端大脑中动脉血栓(distal middle cerebral artery thrombus,dMCAT)模型大鼠行为学、脑梗死体积及梗死侧海马神经生长因子(nerve growth factor,NGF)、缺血区皮层一氧化氮(nitric oxide,NO)水平的影响。方法 成年雄性SD大鼠52只,随机分为假手术组、模型组、电针组、阿司匹林组,假手术组10只,其余每组14只。对电针组予电针双侧丰隆穴(2/100 Hz,1~3 mA),每天1次,每次30分钟,对阿司匹林组予阿司匹林50 mg/(kg·d)灌胃,均持续7天。第8天,模型组、电针组、阿司匹林组以50%FeCl_(3)溶液贴敷左侧大脑中动脉,建立dMCAT模型。术后24小时和7天对大鼠进行行为学评价(Longa评分、网屏实验、平衡木实验);术后24小时以TTC染色法测定脑梗死体积百分比;术后24小时和7天取大鼠梗死侧脑组织,以蛋白免疫印迹法检测海马NGF水平,硝酸还原酶法检测缺血区皮层NO含量。结果 与假手术组比较,术后24小时,模型组大鼠Longa评分、网屏实验评分、平衡木实验评分和脑梗死体积百分比显著升高(P<0.01);与模型组比较,术后24小时,电针组大鼠Longa评分降低(P<0.05),电针组和阿司匹林组网屏实验评分和脑梗死体积百分比降低(P<0.05,P<0.01),平衡木实验评分降低(P<0.05)。术后7天电针组与阿司匹林组海马NGF表达水平升高(P<0.01,P<0.05)。各组皮层NO水平比较无统计学差异(P>0.05)。结论 电针和阿司匹林预处理均能一定程度减轻脑血栓大鼠早期脑损伤,改善运动功能,并提高术后7天梗死侧海马NGF的表达水平,可能有利于后期的神经恢复。

cGMP通路激活对小鼠脑缺血后神经发生的作用

目的观察小鼠脑缺血后海马体神经发生的变化及其与环磷酸鸟苷(cGMP)相关调节机制。方法76只C57BL/6雄性小鼠按照随机数字表法分为假手术组和模型组,每组38只,采用双侧颈总动脉夹闭法建立脑缺血模型。采用Morris水迷宫检测其学习记忆功能,采用苏木精-伊红(HE)染色法检测海马体CA1区病理变化,采用免疫荧光法检测海马体齿状回神经发生标志物5′-溴脱氧尿嘧啶核苷(BrdU)、双肾上腺皮质激素(DCX)、BrdU/神经元核抗原(NeuN)阳性细胞数的表达,采用比色法、硝酸还原法分别检测海马体一氧化氮合酶(NOS)活性和一氧化氮(NO)水平,采用酶联免疫吸附试验(ELISA)检测cGMP水平和磷酸二酯酶(PDE)9活性,采用Western bolt法检测cGMP依赖性蛋白激酶G(PKG)、脑源性神经营养因子(BDNF)蛋白表达。结果多变量方差分析结果显示,在训练2~5 d,在相同训练天数模型组小鼠逃避潜伏期的时间同假手术组相比明显延长,差异均有统计学意义(F=13.683、8.625、73.266、90.327,P<0.05)。在第6天的空间探索试验中,假手术组、模型组小鼠穿越平台次数分别为(6.67±1.37)、(1.67±0.51)次,模型组小鼠穿越平台次数明显少于假手术组,差异有统计学意义(t=8.300,P<0.05)。模型组海马体CA1区锥体神经元数目明显减少,差异有统计学意义(P<0.05),提示脑缺血模型建立成功;与假手术组比较,模型组小鼠海马体马齿状回神经发生标志物BrdU、DCX、BrdU/NeuN阳性细胞数明显增加,同时NOS活性下降,NO生成减少,PDE9活性降低,而cGMP水平增加,PKG和BDNF蛋白表达上调,差异均有统计学意义(P<0.05)。结论PDE9活性降低,cGMP-PKG信号通路激活可能参与促进小鼠脑缺血后海马体神经发生过程。