A feasible strategy for focal cerebral ischemiareperfusion injury: remote ischemic postconditioning

It is difficult to control the degree of ischemic postconditioning in the brain and other isch- emia-sensitive organs. Remote ischemic postconditioning could protect some ischemia-sensitive organs through measures on terminal organs. In this study, a focal cerebral ischemia-reperftlsion injury model was established using three cycles of remote ischernic postconditioning, each cycle consisted of 10-minute occlusion of the femoral artery and 10-minute opening. The results showed that, remote ischemic postconditioning significantly decreased the percentage of the in- farct area and attenuated brain edema. In addition, inflammatory nuclear factor-KB expression was significantly lower, while anti-apoptotic Bcl-2 expression was significantly elevated in the ce- rebral cortex on the ischemic side. Our findings indicate that remote ischemic postconditioning attenuates focal cerebral ischemia/reperfusion injury, and that the neuroprotective mechanism is mediated by an anti-apoptotic effect and reduction of the inflammatory response.

Electroacupuncture preconditioning protects against focal cerebral ischemia/reperfusion injury via suppression of dynamin-related protein 1

Electroacupuncture preconditioning at acupoint Baihui （GV20） can reduce focal cerebral ischemia/reperfusion injury. However, the precise protective mechanism remains unknown. Mitochondrial fission mediated by dynamin-related protein 1 （Drp1） can trigger neuronal apoptosis following cerebral ischemia/reperfusion injury. Herein, we examined the hypothesis that electroacupuncture pretreatment can regulate Drp1, and thus inhibit mitochondrial fission to provide cerebral protection. Rat models of focal cerebral ischemia/reperfusion injury were established by middle cerebral artery occlusion at 24 hours after 5 consecutive days of preconditioning with electroacupuncture at GV20 （depth 2 mm, intensity 1 mA, frequency 2/15 Hz, for 30 minutes, once a day）. Neurological function was assessed using the Longa neurological deficit score. Pathological changes in the ischemic penumbra on the injury side were assessed by hematoxylin-eosin staining. Cellular apoptosis in the ischemic penumbra on the injury side was assessed by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling staining. Mitochondrial ultrastructure in the ischemic penumbra on the injury side was assessed by transmission electron microscopy. Drp1 and cytochrome c expression in the ischemic penumbra on the injury side were assessed by western blot assay. Results showed that electroacupuncture preconditioning decreased expression of total and mitochondrial Drp1, decreased expression of total and cytosolic cytochrome c, maintained mitochondrial morphology and reduced the proportion of apoptotic cells in the ischemic penumbra on the injury side, with associated improvements in neurological function. These data suggest that electroacupuncture preconditioning-induced neuronal protection involves inhibition of the expression and translocation of Drp1.

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.

Vascular endothelial growth factor induced angiogenesis following focal cerebral ischemia/reperfusion injury in rabbits

BACKGROUND： Therapeutic angiogenesis has opened up new pathway for the treatment of ischemic cerebrovascular disease in recent years. The exploration of the effect of vascular endothelial growth factor （VEGF） on inducing angiogenesis following ischemia/reperfusion injury can provide better help for the long-term treatment of cerebrovascular disease in clinic. OBJECTIVE： To observe the effect of VEGF on inducing angiogenesis following focal cerebral ischemia /reperfusion injury in rabbits through the angiogenesis of microvessels reflected by the expression of the factors of vascular pseudohemophilia. DESIGN： A randomized controlled animal tria SETTNG： Department of Medical Imaging, Second Hospital of Hebei Medical University MATERIALS： Sixty-five healthy male New Zealand rabbits of clean degree, weighing （2.6±0.2） kg, aged 4.5-5 months, were used. The polyclonal antibody against vascular pseudohemophilia （Beijing Zhongshan Company）, recombinant VEGF165 （Peprotech Company, USA）, biotinylated second antibody and ABC compound （Wuhan Boster Company） were applied. METHODS： The experiments were carried out in the Laboratory of Neuromolecular Imaging and Neuropathy, Second Hospital of Hebei Medical University from May to August in 2005. （1） The rabbits were randomly divided into three groups： sham-operated group （n=15）, control group （n=25） and VEGF-treated group （n=-25）. In the control group and VEGF-treated group, models were established by middle cerebral artery occlusion （MCAO） induced focal cerebral ischemia/reperfusion. In the VEGF-treated group, VEGF165 （2.5 mg/L） was stereotactically injected into the surrounding regions of the infarcted sites immediately after the 2-hour ischemia/reperfusion; Saline of the same dosage was injected in the control group. But the rabbits in the sham-operated group were only drilled but not administrated. （2） The experimental indexes were observed on the 3^rd 7^th, 14^th, 28^th and 70^th days of the experiment respectively, 3 rabbits in the sham-operated group and 5 in the control group and VEGF-treated group were observed at each time point. The brain tissues in the surrounding regions of the infarcted sites were collected. The positive expressions of the factors of vascular pseudohemophilia in vascular endothelial cells were analyzed with immunohistochemical method. The microvessels in unit statistical field were counted with the imaging analytical software. MAIN OUTCOME MEASURES： The changes of microvascular density in the brain tissue and the positive expressions of the factors of vascular pseudohemophilia in the surrounding regions of the infarcted sites were observed on the 3^rd 7^th, 14^th, 28^th and 70^th days of the experiment. RESULTS： All the 65 New Zealand rabbits were involved in the analysis of results without deletion. Changes of the number of microvessels at different time points in each group： There were no obvious changes at different time points in the sham-operated group. The numbers of microvessels at 7 and 14 days were obviously more in the control group than in the sham-operated group [（6.0±1.1）, （9.0±0.9） microvessels; （3.0±1.1）, （3.0±1.1） microvessels; P〈 0.05-0.01], and those at 3, 7, 14 and 28 days were obviously more in the VEGF-treated group than in the control group [（8.3±2.0）, （13.4±1.4）, （15.5±2.3）, （6.8± 1.0） microvessels; （3.4±0.6）, （6.0±1.1）, （9.0±0.9）, （3.2±0.8） microvessels; P 〈 0.01]. （2） Positive expressions of the factors of vascular pseudohemophilia in the surrounding regions of infarcted sites： There were no obvious changes at different time points in the sham-operated group. In the control group, the changing law of the expressions was the same as that for the number of microvessels that the expression began to mildly increase at 7 days, reached the peak value at 14 days, and began to reduce at 28 days. In the VEGF-treated group, the expression was obviously increased at 3 days, also reached the peak value at 14 days, and reduced to the normal level at 70 days, but the expressions were obviously stronger than those in the control group at the same time points. CONCLUSION： Angiogenesis can be obviously induced in rabbits after the focal cerebral ischemia/reperfusion injury is treated with VEGF for 18 days.

Electroacupuncture-attenuated ischemic brain injury increases insulin-like growth factor-1 expression in a rat model of focal cerebral ischemia

Acupuncture has recently gained popularity in many countries as an alternative and complementary therapeutic intervention. Previous studies have shown that changes in genes, proteins, and their metabolites were measureable during acupuncture for treatment of cerebral ischemia. Through the use of in situ hybridization and immunohistochemistry, the present study confirmed that electroacupuncture increased insulin-like growth factor-1 mRNA and protein expression in the corpus stfiatum following cerebral ischemia, reduced brain edema following middle cerebral artery occlusion reperfusion, and decreased infarct volume. Results suggested that electroacupuncture is effective in the relief of cerebral ischemia by increasing endogenous insulin-like growth factor-1 expression.

Effects of NG-nitro-L-arginine on focal cerebral ischemic injury in rats

BACKGROUND： Previous researches have proved that aminoguanidin can cure cerebral ischemic injury remarkably as a selective induced nitricoxide synthase （iNOS） inhibitor. However, whether nonselective NOS inhibitor could protect cerebral ischemic injury or not is unclear. OBJECTIVE： To investigate the effects of NG-nitro-L-arginine （L-NA）, a nonselective nitricoxide synthase （NOS） inhibitor, on cerebral ischemic injury of rats and the possible mechanism.DESIGN： Randomized controlled study.SETTING ： Pharmacological Department of Medical Academy of Science of Hebei Province.MATERIALS： A total of 56 male healthy SD rats, of grade Ⅱ, weighting 250-290 g, were provided by the Experimental Animal Center of Hebei Province （certification： 04036）. METHODS： The experiment was completed in the Pharmacological Department of Medical Academy of Science of Hebei Province from March 2005 to January 2006.① Grouping： Rats were randomly divided into 3 groups： sham operation group （n=8）, model group （n=24） and L-NA group （n=24）.② Modeling： Middle cerebral artery （MCA） was established on rats in model group and L-NA group with intreluminal line occlusion methods, and rats in sham operation group were separated their external carotid arteries without occlusion of internal carotid artery. ③ Intervention study： Rats in model group and L-NA group were injected intreperitoneally with 10 mL/kg and 20 mg/kg L-NA at 2, 6 and 12 hours respectively after ischemia twice a day for 3 consecutive days. ④ Rats were sacrificed on the third day for measuring volume of cerebral infarction with image analysis and swelling degrees and activities of mitochondria with electron microscope. Effect of L-NA on ultrastructural changes of neurons in cortex was observed after ischemia. MAIN OUTCOME MEASURES：① Volume of cerebral infarction; ②Swelling degrees, contents of nitric oxide （NO） and malondialdehyde （MDA） and activities of adenosine triphosphatase （ATPase）, superoxide dismutase （SOD） and glutathione peroxidase （GSH-Px） in mitochondria;③ Ultrastructural changes of mitochondria in brain tissue after cerebral ischemia. RESULTS： ① At 12 hour after ischemia, volume of cerebral infarction in L-NA group was lower than that in model group （P 〈 0.01）. ② Content of NO in mitochondria in L-NA group was decreased as compared with that in model group at 2, 6 and 12 hours after ischemia （P 〈 0.05）; swelling degree of mitochondria in brain tissue was relieved in L-NA group at 12 hour after ischemia, and content of MDA was decreased （P 〈 0.05）; mitochondrial activity in L-NA group was increased at 12 hour after ischemia, and activities of ATPase, SOD and GSH-Px in mitochondria were increased （P 〈 0.05）.③ Degrees of mitochondrial injury in brain tissue were relieved in L-NA group at 12 hour after ischemia as compared with those in model group and L-NA group at 2 and 6 hours after ischemia. CONCLUSION ： ①L-NA can beneficially inhibit NO production, but not protect brain against damage in ischemia acute stage. ②L-NA might have protective effects on cerebral injury through inhibiting the production of oxygen free radical, increasing antioxidation, ameliorating energy metabolism, beneficially improving the integrity of form and function of mitochondria in brain tissue during postischemia in rats.

IMM-H004 therapy for permanent focal ischemic cerebral injury and cardiopulmonary complications via CKLF1/CCR4-mediated inflammation pathway

OBJECTIVE To investigate the effects of IMM-H004 on permanent focal cerebral ischemia injury and associated cardiopulmonary complications,further elucidating the molecular mechanisms.METHODS The effects of IMM-H004 were investigated in wild-type(WT) and CKLF1-/-rats.The effects of IMM-H004 on ischemic stroke injury and its cardiopulmonary complications were determined using 2,3,5-triphenyltetrazolium chloride(TTC) staining,behavior tests,magnetic resonance imaging(MRI)scans,enzyme-linked immunosorbent assay(ELISA),Nissl staining,and histo-pathological examination.Multiple molecular experiments including immunohistological staining,immunofluorescence staining,quantitative RT-PCR,Western blotting,and co-immunoprecipitation assays were used to elucidate the underlying mechanisms.RESULTS IMM-H004 treatment provided significant protection against ischemic stroke-induced brain injury and associated cardiopulmonary complications,through CKLF1-depedent-anti-inflammation pathway in rats.IMM-H004 downregulated the amount of CKLF1 and disturbed the combination between CKLF1 and C-C chemokine receptor type 4,suppressing the inflammatory response and protecting the damaged organs in ischemic setting.CONCLUSION This preclinical study established efficacy of IMM-H004 as a potential therapeutic medicine for ischemic stroke and associated cardiopulmonary complications.The protective effects of IMM-H004 may due to its specific mechanism through CKLF1.These results support further efforts to develop IMM-H004 for human clinical trials in acute cerebral ischemia,especially for patients who are not suitable for reperfusion therapy.

Neuroprotective effect of ischemic preconditioning in focal cerebral infarction: relationship with upregulation of vascular endothelial growth factor

Neuroprotection by ischemic preconditioning has been confirmed by many studies, but the precise mechanism remains unclear. In the present study, we performed cerebral ischemic pre- conditioning in rats by simulating a transient ischemic attack twice （each a 20-minute occlusion of the middle cerebral artery） before inducing focal cerebral infarction （2 hour occlusion-reper- fusion in the same artery）. We also explored the mechanism underlying the neuroprotective effect of ischemic preconditioning. Seven days after ocdusion-reperfusion, tetrazolium chloride staining and immunohistochemistry revealed that the infarct volume was significantly smaller in the group that underwent preconditioning than in the model group. Furthermore, vascular endothelial growth factor immunoreactivity was considerably greater in the hippocampal CA3 region of preconditioned rats than model rats. Our results suggest that the protective effects of ischemic preconditioning on focal cerebral infarction are associated with upregulation of vascu- lar endothelial growth factor.

Normobaric oxygen for cerebral ischemic injury

Oxygen inhalation has been shown to increase oxygen supply to tissues after cerebral ischemia/ reperfusion injury, protecting injured neural cells. However, hyperbaric oxygen may aggravate oxi- dative stress. By contrast, normobaric oxygen has the rapid and non-invasive characteristics and may have therapeutic effects on ischemic/hypoxic disease. Rats inhaled normobaric oxygen （95% 02） for 6 consecutive days, and then a rat model of focal cerebral ischemia was established. Nisst and 2,3,5-triphenyltetrazolium chloride （TTC） staining revealed that normobaric oxygen pretreat- ment improved neurological deficits and reduced infarct volume. Immunohistochemical staining and western blot assay revealed that the expression of hypoxia-inducible factor-la, Notch-l, vascular endothelial growth factor and erythropoietin were increased. Behavioral studies also verified that neurological deficit scores increased. The hypoxia-inducible factor inhibitor 2-methoxyestradiol treatment at 1 hour before administration of normobaric oxygen could suppress the protective effect of normobaric oxygen. Given these observations, normobaric oxygen pretreatment may alleviate cerebral ischemic injury via the hypoxia-inducible factor signal pathway.

Acupuncture effects on serum myelin basic protein and remyelination following 30 minutes and 2 hours of ischemia in a rat model of cerebral ischemia-reperfusion injury

BACKGROUND： Acupuncture treatment on injured cerebral axons has shown to provide efficacy in clinical practice. It is unknown whether acupuncture produces therapeutic effects by protecting injured cerebral myelin in ischemic stroke. OBJECTIVE： To test whether acupuncture provides protection for injured cerebral myelin, based on quantitative data from cerebral ischemia-reperfusion rats, and to compare the effects of early and late acupuncture on serum myelin basic protein （MBP） content and remyelination of the ischemic internal capsule.DESIGN, TIME AND SETTING： A randomized, controlled experiment was performed at the Neurobiological Laboratory, Sichuan University from March 2005 to March 2006. MATERIALS： ＂Hua Tuo＂ Brand filiform needles were produced by the Medical Instrument Factory of Suzhou, China.METHODS： A total of 52 adult, healthy, male, Sprague Dawley rats were randomly assigned to four groups： control （n = 4）, model （n = 16）, early acupuncture （n = 16）, and late acupuncture （n = 16）. The focal cerebral ischemia-reperfusion model was established by middle cerebral artery occlusion in the right hemisphere using the modified thread embolism method in the latter three groups. Early and late acupuncture groups underwent acupuncture after ischemia for 30 minutes and 2 hours using the Xingnaokaiqiao needling method, respectively. Acupoints were ＂Neiguarf＇ （PC 6） and ＂Sanyinjiao＂ （SP 6） on the bilateral sides, as well as ＂Shuigou＇ （DU 26） and ＂Baihui＂ （DU 20） with stimulation for 1 minute at each acupoint. Acupuncture at all acupoints was performed two or three times while the needle was retained, once per day. No special handling was administered to the control clroup.MAIN OUTCOME MEASURES： For each group, remyelination of the internal capsule was observed by Pal-Weigert＇s myelin staining and serum MBP content was detected using enzyme-linked immunosorbent assay method on days 1,3, 5, and 7 following ischemia-reperfusion injury.RESULTS： Compared with the control group, massive demyelination of the internal capsule occurred, and serum MBP content increased in the model group （P 〈 0.05）. Compared with the model group, the extent of demyelination in the internal capsule was less distinct and serum MBP content was significantly less in the early and late acupuncture group （P 〈 0.01 ）. Compared with the late acupuncture group, serum MBP content reached a peak later and the peak value was less in the early acupuncture group. CONCLUSION： Results suggest that acupuncture exerts a protective effect on injured cerebral myelin in ischemia-reperfusion rats by reducing serum MBP content and promoting remyelination. The study also suggests that the effect of early acupuncture is superior to late acupuncture.

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.

Effects of reduction of Sheng-Nao-Kang decoction in focal cerebral ischemia/reperfusion model rats

Aim Reduction of Sheng-Nao-Kang decoction （RSNK）, is a modified traditional Chinese medicinal formula of Sheng-Nao-Kang pill preparation, which is protective in rats against focal cerebral ischemia/reperfusion （I/R） injury. In the current study, we investigate the protective effect of RSNK against apoptosis and oxidative damage induced by cerebral I/R and explore the underlying mechanisms. Cerebral I/R injury was induced by in- traluminal middle cerebral artery occlusion （MCAO） for 2 h followed by reperfusion for 24 h in adult male Sprague- Dawley rats. Rats were randomized into seven groups （n- 8）： Sham group, I/R group, RSNK-treated groups （ 0.7 g · kg ^- 1, 1 . 4 g · kg ^- 1 and 2. 8 g · kg^ - 1 ） , nimodipine （NMP） -treated group and Whitmania pigra Whitman （WW）-treated group. Neurological deficit scores, cerebral humidity content and cerebral infarction volume were measured after the 24 h reperfusion. Malondialdehyde （ MDA）, superoxide dismutase （ SOD）, catalase （ CAT）, inducible nitric oxide synthase （iNOS） and total nitric oxide synthase （TNOS） in serum were measured by assay kits for biochemical analysis. Histological structures of the cortex of the ipsilateral ischemic cerebral hemisphere in rats were observed by Nissl staining. The caspase-3 protein content in the hippocampus and cortex was detected by immunohistochemistry. Additionally, Bax and Bcl-2 protein expressions in the injured brain were evaluated by Western blot. RSNK administration not only markedly improved neurological deficit scores, but also reduced cere- bral humidity content and cerebral infarction volume, lowered MDA content, up-regulated SOD and CAT levels, down-regulated iNOS and TNOS levels, restrained the expression of caspase-3 positive protein and alleviated the Bax and Bcl-2 protein expressions.

Effects of Salvia miltiorrhiza on cerebral infarction volume,nerve behavior and SOD,GSH-px,MDA,BDNF and GDNF in rats

Objective:To investigate the effects of Salvia miltiorrhiza on cerebral infarction volume,nerve behavior and brain tissue SOD,GSH-Px,MDA,GDNF,BDNF in rats with focal cerebral ischemia reperfusion injury.Methods:Healthy adult male SD rats(clean grade)80,were randomly divided into sham operation group(saline),model group(saline),low dose group(10 mg/kg salvianolate),high dose group(30 mg/kg salvianolate)and edaravone group(6 mg/kg)with 16 rats in each groups were compared after operation in rats,48 h neural behavior of 24 h and 72 h scores,cerebral infarction volume 24 h after operation;SOD,GSH-Px and other indicators of brain tissue of rats after the determination of 72 h.Results:After 24 h,low dose group,high dose group and edaravone group,the cerebral infarction volume were less than the model group,high dose group and edaravone group,the infarct volume was less than the low dose group;after 24 h,48 h,72 h,scores of neurological behavior in the low dose group and high dose group and Yidala in the study group were lower than model group,neural behavior in high dose group and edaravone group scores were lower than the low dose group;low dose group,high dose group and edaravone group in the brain tissue of SOD,GSH-Px,BDNF,GDNF were higher than that of model group,and MDA was lower than that of model group;high dose group and edaravone group in the brain tissue of SOD,GSH-Px,BDNF,GDNF were higher than those in the low dose group,and MDA was lower than that of low dose group.Conclusion:The treatment of focal cerebral ischemia reperfusion injury in rats by inhibiting lipid peroxidation,protecting Magnesium Oxide's activity,thereby reaching the therapeutic effect.

基于环磷酸腺苷/蛋白激酶A/环磷酸腺苷反应元件结合蛋白通路探究丙泊酚对局灶性脑缺血再灌注大鼠神经功能改善机制

目的:基于环磷酸腺苷/蛋白激酶A/环磷酸腺苷反应元件结合蛋白(cAMP/PKA/CREB)通路探究丙泊酚对局灶性脑缺血再灌注大鼠神经功能的改善机制。方法:采用改良线栓法缺血2 h,再灌注24 h建立大鼠脑缺血再灌注损伤(CIRI)模型,将造模成功大鼠随机分为模型组和丙泊酚低(1 mg/ml)、中(2.5 mg/ml)、高剂量(5 mg/ml)组,各12只,另设含有12只大鼠的假手术组。分组后即开始给药,1次/d,共4周,末次给药12 h后,采用改良神经功能评分(mNSS)法进行神经缺损评分;采用TTC染色法检测脑梗死面积;HE、Nissl染色进行神经元细胞及尼氏小体形态学观察;Tunel法进行神经元细胞凋亡检测;Elisa法检测脑组织cAMP、脑源性神经营养因子(BDNF)、神经生长因子(NGF)含量;免疫荧光法检测脑组织环磷酸腺苷(cAMP)、p-PKA、p-CREB阳性细胞数及其蛋白共表达阳性细胞数;Western blot法检测脑组织PKA、p-PKA、CREB、p-CREB蛋白表达量。结果:模型组大鼠比较假手术组大鼠的mNSS评分、脑梗死面积百分比显著增加(均P<0.05),HE染色和Nissl染色可见明显的神经元细胞损伤和尼氏小体破坏,脑组织cAMP、BDNF、NGF含量和PKA、p-PKA、CREB、p-CREB蛋白表达量显著下降,模型组大鼠比较假手术组大鼠的cAMP、p-PKA、p-CREB阳性细胞数和蛋白共表达阳性细胞数也明显下降(均P<0.05)。与模型组比较,丙泊酚给药组大鼠mNSS评分、脑梗死面积百分比显著降低(均P<0.05),HE染色和Nissl染色可见神经元细胞损伤和尼氏小体破坏有不同程度改善,脑组织cAMP、BDNF、NGF含量和PKA、p-PKA、CREB、p-CREB蛋白表达量显著升高(均P<0.05),cAMP、p-PKA、p-CREB阳性细胞数及其蛋白共表达阳性细胞数均显著升高(均P<0.05)。结论:丙泊酚可能通过cAMP/PKA/CREB通路改善CIRI大鼠神经功能。

二甲双胍对局灶性脑缺血再灌注损伤大鼠神经保护作用机制研究

目的探讨二甲双胍对局灶性脑缺血再灌注损伤大鼠神经保护作用的机制。方法选择30只8周龄健康雄性SPF级SD大鼠为研究对象,按随机数字表法分为假手术组、大脑中动脉闭塞(MCAO)组及二甲双胍组(MCAO+Met组),每组各10只。在构建大鼠MCAO模型前3周,MCAO+Met组行10 mg/kg二甲双胍腹腔注射,假手术组及MCAO组不作任何药物处理,随后参考Longa线栓法制备大鼠MCAO模型,假手术组除不插入线栓,余下操作同MCAO组、MCAO+Met组。依据造模成功标准,在造模成功24 h后采用改良神经功能评分(mNSS)评定神经功能缺损程度;采用氯化三苯基四氮唑(TTC)染色测定大鼠脑梗死体积;采用蛋白质印迹法测定磷脂酰肌醇3激酶(PI3K)、蛋白激酶B(AKT)及血管内皮生长因子(VEGF)蛋白的表达水平;采用酶联免疫吸附试验测定白细胞介素(IL)-6、IL-1β、肿瘤坏死因子-α(TNF-α)水平。比较3组大鼠mNSS评分、脑梗死体积百分数、PI3K、AKT、VEGF蛋白表达、IL-6、IL-1β及TNF-α水平。结果与假手术组比较,MCAO组及MCAO+Met组mNSS评分均更高,差异均有统计学意义(P<0.05);与MCAO组比较,MCAO+Met组mNSS评分更低,差异有统计学意义(P<0.05)。与假手术组比较,MCAO组及MCAO+Met组脑梗死体积百分数均更高,差异均有统计学意义(P<0.05);与MCAO组比较,MCAO+Met组脑梗死体积百分数更低,差异有统计学意义(P<0.05)。与假手术组比较,MCAO组及MCAO+Met组PI3K、AKT及VEGF蛋白表达水平均更低,差异均有统计学意义(P<0.05);与MCAO组比较,MCAO+Met组脑梗死体积PI3K、AKT及VEGF蛋白表达水平均更高,差异均有统计学意义(P<0.05)。与假手术组比较,MCAO组及MCAO+Met组IL-6、IL-1β及TNF-α水平均更高,差异均有统计学意义(P<0.05);与MCAO组比较,MCAO+Met组IL-6、IL-1β及TNF-α水平均更低,差异均有统计学意义(P<0.05)。结论局灶性脑缺血再灌注损伤应用二甲双胍有利于神经保护,其作用机制可能与激活PI3K/AKT/VEGF通路减轻炎症反应有关。

西格列汀对局灶性脑缺血再灌注小鼠血-脑脊液屏障通透性的影响及抗凋亡作用

目的研究西格列汀(SIT)在非糖尿病的小鼠脑缺血再灌注(I/R)时的神经保护作用。方法采用大脑中动脉栓塞(MCAO)模型,脑缺血60 min后实施再灌注。SIT按低剂量(40 mg·kg^(-1))、高剂量(80 mg·kg^(-1))在手术前口服给药3d,术后再次给药。术后24 h检测小鼠神经功能、梗死体积和脑水肿,通过Western blot评估Bcl-2和Akt的蛋白表达,RT-qPCR评估Bax和Bcl-2的转录水平。SIT对血-脑脊液屏障(BCFB)通透性的影响通过伊文思蓝渗出和Claudin-5的蛋白表达来测量。结果本研究结果表明,SIT可减轻CD-1小鼠大脑中动脉栓塞再灌注24h后的肢体功能障碍,减少脑梗死体积,减轻脑水肿。SIT显著增加Bcl-2和磷酸化Akt的蛋白表达水平,下调Bax、上调Bcl-2的基因表达。减少小鼠脑组织伊文思蓝渗出并增加Claudin-5的蛋白表达水平(^(均)P<0.05)。结论SIT在非糖尿病小鼠的脑I/R急性期,降低神经功能缺损评分、减少脑梗死体积、保护BCFB,具有脑保护作用,机制可能与抗凋亡有关。这些发现为急性缺血性脑血管病的治疗提供新的策略。

TREM2通过激活Th17细胞调节神经免疫炎症并保护局灶性脑缺血再灌注损伤

目的:探讨髓细胞触发受体2(TREM2)调节局灶性脑缺血再灌注损伤和神经免疫炎症的作用和机制。方法:对小鼠的大脑行中动脉闭塞术(MCAO)以建立局灶性缺血再灌注损伤模型,分为假手术组(sham组)和MCAO组(n=6)。小干扰RNA(siRNA)构建TREM2的沉默质粒(TREM2 siRNA组),构建TREM2过表达质粒(pcDNA-TREM2组),以及各自的阴性对照(CON siRNA)组和pcDNA组分别感染MCAO小鼠。采用IL-17抑制剂苏金单抗(SEC)联合pcDNA-TREM2处理MCAO组(MCAO+pcDNA-TREM2+SEC组)。qPCR和Western blot检测TNF-α、IL-1β、IL-17和TREM2表达;免疫荧光法检测脑部TREM2定位;TTC染色评价脑损伤程度;流式细胞术检测Th17细胞比例。结果:TREM2表达于小胶质细胞,且MCAO组TREM2表达均增加(P<0.05)。沉默TREM2诱导神经元细胞凋亡(F=206.971,P=0.001)、梗死体积和神经功能障碍评分升高(均P<0.05),TNF-α和IL-1β的mRNA水平升高,IL-10降低(均P<0.05)。过表达TREM2导致TNF-α、IL-1β表达降低,而IL-10和IL-17表达及Th17阳性细胞比例升高(均P<0.05)。与MCAO+pcDNA-TREM2组比较,MCAO+pcDNA-TREM2+SEC组的Th17细胞比例降低、神经元百分比降低,脑损伤程度增加,TNF-α、IL-1β表达升高,IL-10和IL-17表达降低(均P<0.05)。结论:过表达TREM2可通过激活Th17细胞并抑制神经炎症从而保护局灶性脑缺血再灌注损伤。

脑泰通组方对局灶性脑缺血再灌注大鼠模型的影响及保护机制

目的:观察脑泰通组方对局灶性脑缺血再灌注大鼠模型的影响及保护机制。方法:构建局灶性脑缺血再灌注大鼠模型,成模后随机分为模型对照组、阳性药物组、中药组、联合用药组,另选取健康斯泼累格·多雷(SD)大鼠作为假手术组。观察并评估各组大鼠的美国国立卫生研究院卒中量表(NIHSS)评分、脑组织含水量、脑梗死体积百分比,免疫组织化学法检测脑组织Bcl-2相关X蛋白(Bax)、B细胞淋巴瘤-2(Bcl-2)的平均光密度值,蛋白质免疫印迹法、实时定量PCR(QT-PCR)检测脑组织叉头框转录因子3a(FoxO3a)基因、低氧诱导因子-1α(HIF-1α)、核因子κB(NF-κB)、脑源性神经营养因子(BDNF)蛋白及mRNA的表达水平。结果:与假手术组比较,模型对照组NIHSS评分,脑组织含水量,脑组织缺血体积百分比,脑组织FoxO3a、HIF-1α、NF-κB、BDNF蛋白和mRNA的表达水平,Bax、Bcl-2平均光密度值以及Bax/Bcl-2值均显著升高(均P<0.05);与模型对照组比较,药物干预各组的NIHSS评分、脑组织含水量、脑梗死体积百分比、Bax平均光密度值、Bax/Bcl-2值、脑组织NF-κB蛋白和mRNA的表达水平显著降低(均P<0.05),其中联合用药组的降低程度最大(均P<0.05),Bcl-2平均光密度值,脑组织FoxO3a、HIF-1α、BDNF蛋白和mRNA的表达水平显著升高(均P<0.05),其中联合用药组的升高程度最大(均P<0.05)。结论:脑泰通组方能有效保护局灶性脑缺血再灌注大鼠模型脑组织损伤,改善局部脑缺血,其作用机制可能与脑泰通组方调控FoxO3a/HIF-1α/NF-κB信号通路相关。

栀子提取物对局灶性脑缺血损伤模型大鼠凝血功能及脑血管内皮功能的影响

目的:观察栀子提取物(GE)对局灶性脑缺血损伤模型大鼠凝血功能及脑血管内皮功能的影响。方法:采集新鲜干燥栀子,制备GE。将48只SD大鼠随机分为假手术组(sham组)、模型组(model组)、GE高剂量组(GE-H组)、GE低剂量组(GE-L组),每组12只。构建大鼠局灶性脑缺血损伤模型,术后采用Longa神经学评分对符合局灶性脑缺血损伤标准大鼠进行实验。采用Longa神经学评分标准比较用药前后神经功能改善情况;采用断尾法及玻片法检测大鼠凝血时间(CT)和出血时间(BT);凝血酶活性荧光检测凝血酶时间(TT)及凝血酶含量,观察大鼠凝血因子Ⅹa含量,判断大鼠凝血功能变化情况;检测大鼠大脑动脉环(Willis)一氧化氮(NO)、内皮型一氧化氮合酶(eNOS)、血管壁血栓素A_(2)(TXA_(2))、血浆前列环素I_(2)(PGI_(2))、内皮组织纤维溶酶原激活物(t-PAT)及其抑制剂(PAI)活性,判断大鼠脑血管内皮功能变化情况;苏木精-伊红(HE)染色法检测各组大鼠脑部损伤情况。结果:sham组大鼠脑组织正常,与sham组比较,model组CT、BT、TT缩短,t-PAT、PGI_(2)、NO、eNOS降低,凝血酶生成及内源性、外源性凝血因子Ⅹa生成、PAI、TXA_(2)升高(P<0.05),且大鼠脑组织出现局灶性脑缺血损伤。与model组比较,GE-H组和GE-L组CT、BT、TT延长,t-PAT、PGI_(2)、NO、eNOS升高,凝血酶生成及内源性、外源性凝血因子Ⅹa生成、PAI、TXA_(2)降低(P<0.05),脑组织损伤情况较model组明显好转。与sham组比较,model组治疗后Longa神经学评分升高;与model组比较,GE-H组和GE-L组Longa神经学评分降低(P<0.05)。结论:GE作用可延长CT、BT、TT,降低凝血酶和凝血因子含量,增强NO、eNOS活性,提高t-PAT、PGI_(2)表达同时抑制PAI、TXA_(2)表达,可改善局灶性脑缺血损伤凝血功能,恢复脑血管内皮功能。