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.

Effect of ephrin-B2 on the expressions of angiopoietin-1 and-2 after focal cerebral ischemia/reperfusion

Ephrin-B2 has been shown to participate in angiogenesis, but the underlying mechanisms involved remain unclear. In this study, a rat model of local cerebral ischemia was prepared by focal middle cerebral artery occlusion, followed by 24-hour reperfusion. Then, ephrin-B2 protein was administered intracerebroventricularly for 3 consecutive days via a micro-osmotic pump. Western blot assay and quantitative real-time reverse transcription PCR demonstrated the expression levels of angiopoietin-1 （Ang-1） mRNA and protein in the penumbra cortex of the ephrin-B2 treated group were decreased at day 4 after reperfusion, and increased at day 28, while the expression levels of angiopoietin-2 （Ang-2） were highly up-regulated at all time points tested. Double immunofluorescent staining indicated that Ang-1 and Ang-2 were both expressed in vascular endothelial cells positive for CD31. These findings indicate that ephrin-B2 influences the expressions of Ang-1 and Ang-2 during angiogenesis following transient focal cerebral ischemia.

An Early Continuous Experimental Study on Magnetic Resonance Diffusion-weighted Image of Focal Cerebral Ischemia and Reperfusion in Rats

Summary： The chronological and spatial rules of changes during focal cerebral ischemia and reperfusion in different brain regions with magnetic resonance diffusion-weighted imaging （DWI） in a model of occlusion of middle cerebral artery （MCAO） and the development of cytotoxic edema in acute phase were explored. Fifteen healthy S-D rats with MCA occluded by thread-emboli were randomly divided into three groups. 15 min after the operation, the serial imaging was scanned on DWI for the three groups. The relative mean signal intensity （RMSI） of the frontal lobe, parietal lobe, lateral cauda-putamen, medial cauda-putamen and the volume of regions of hyperintense signal on DWI were calculated. After the last DWI scanning, T2WI was performed for the three groups. After 15 rain ischemia, the rats was presented hyperintense signals on DWI. The regions of hyperintense signal were enlarged with prolonging ischemia time. The regions of hyperintense signal were back to normal after 60 min reperfusion with a small part remaining to show hyperintense signal. The RMSIs of parietal lobe and lateral cauda-putamen were higher than that of the frontal lobe and medial cauda-putamen both in ischemia phase and recanalization phase. The three groups were normal on T2WI imaging. DWI had good sensitivity to acute cerebral ischemia, which was used to study the chronological and spatial rules of development of early cell edema in ischemia regions.

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.

Effects of fastigial nucleus electrical stimulation on lateral ventricle nestin expression after focal cerebral ischemia/reperfusion in adult rats

BACKGROUND： Previous studies have confirmed that fastigial nucleus electrical stimulation can induce endogenous neuroprotective mechanisms and produce wide and long-lasting neuroprotective effects. Nevertheless, the precise mechanisms remain poorly understood. OBJECTIVE： This study was designed to observe the effects of fastigial nucleus electrical stimulation on nestin-positive cell expression in adult rat lateral ventricle after focal cerebral ischemia/reperfusion, as well as neurological functional changes as a function of time. DESIGN： A randomized controlled animal experiment. SETTING： Department of Neurology, First Affiliated Hospital of Chongqing Medical University; Chongqing Key Laboratory of Neurology. MATERIALS： This study was performed in the Department of Neurology, First Affiliated Hospital of Chongqing Medical University and Chongqing Key Laboratory of Neurology from September 2004 to February 2006. A total of 180 healthy, adult, male Wistar rats, aged 8 weeks old, were provided by the Laboratory Animal Center of Chongqing Medical University. The main reagents and equipments were as follows： rabbit anti-rat nestin monoclonal antibody （Wuhan Boster Company, China）. METHODS： The included male Wistar rats were randomly divided into 5 groups： normal control, sham-operated, model, fastigial nucleus sham-stimulation （sham-stimulation for short）, and fastigial nucleus electrical stimulation （stimulation for short） groups. Six time points （1 hour of ischemia and 1, 3, 7, 14, 21, and 28 days of reperfusion, 6 rats per time point） were allotted to each group. Cerebral ischemia/reperfusion was performed by occlusion to the right middle cerebral artery with suture, followed by suture removal. In the stimulation group, subsequent to reperfusion, the rat left cerebellar fastigial nucleus was immediately subjected to 1 hour of stimulation. After anesthesia, the rat left cerebellar fastigial nucleus was stimulated for 1 hour using a square-wave electronic stimulator with a current intensity of 50 μ A, frequency of 50-100 Hz, and duration of 0.5 ms. In the sham-stimulation group, the procedure was identical to the stimulation group, except the needle was retained for 1 hour and current stimulation was withdrawn. In the model group, rats were subjected to cerebral ischemia/reperfusion, but electrical stimulation was omitted. In the sham-operated group, the internal carotid artery, rather than the middle cerebral artery, was inserted with suture, and simultaneously, electrical stimulation was omitted. In the normal control group, the rats received no treatments. MAIN OUTCOME MEASURES： Nestin-positive cells were detected by immunohistochemical staining in the rat ischemic lateral cerebral ventricle at 1, 3, 7, 14, 21, and 28 days post-reperfusion. RESULTS： Morphological changes of nestin-positive cells in the ischemic lateral ventricle： in the normal control group, very few nestin-positive cells were detected in the choroid plexus, ependyma, and subependymal region of the lateral ventricle. In the model group, the number of nestin-positive cells exhibited a tendency towards a single peak, i.e., cells increased at day 1, reached peak levels by day 7, and then decreased sharply. Fastigial nucleus electrical stimulation was administered following focal cerebral ischemia/reperfusion, and results revealed that nestin-positive cell morphology was similar to horizontal cell morphology on day 7. The number of nestin-positive cells decreased after 14 days; however, the proportion of horizontal cell-like cells increased. In the sham-stimulation group, there was no change in nestin-positive cells, nestin-positive cell expression in the ischemic lateral ventricle： nestin-positive cell expression increased in the ischemic lateral cerebral ventricle, exhibiting a tendency towards unimodality; the number of cells peaked on day 7 （P 〈 0.01） and gradually decreased after 14 days （P 〈 0.01）. Following fastigial nucleus electrical stimulation, the number of nestin-positive cells increased significantly （P 〈 0.05-0.01）, reached peak levels by day 7 （P 〈 0.01）, and remained at very high levels after 14 days （P 〈 0.01）. Neurofunctional changes： neurofunctional deficits were gradually alleviated with prolonged focal cerebral ischemia/reperfusion time. At 1 hour of ischemia, and 6 hours to 7 days of reperfusion, rat neurological scores were significantly lower in the stimulation group than in the model and sham-operation groups （P 〈 0.05-0.01）. CONCLUSION： Fastigial nucleus electrical stimulation increased nestin-positive cells in the rat lateral ventricle after focal cerebral ischemia/reperfusion in a time-dependent manner and simultaneously improved neurological deficits, as well as promoted differentiation of nestin-positive cells towards a cell type with neuronal and neuroglial cellular morphology.

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 calcitonin gene-related peptide and nerve growth factor on spatial learning and memory abilities of rats following focal cerebral ischemia/reperfusion

BACKGROUND: Calcitonin gene-related peptide (CGRP) and nerve growth actor (NGF) cam improve spatial learning and memory abilities of rats with cerebral ischemia/reperfusion; however, the effect of combination of them on relieving learning and memory injury following cerebral ischemia/reperfusion should be further studied. OBJECTIVE: To study the effects of exogenous CGRP and NGF on learning and memory abilities of rats with focal cerebral ischemia/reperfusion. DESIGN: Randomized controlled animal study. SETTING: Department of Neurosurgery, the Second Hospital of Xiamen; Department of Neurosurgery, the Second Affiliated Hospital of China Medical University; Department of Neurobiology, Basic Medical College of China Medical University. MATERIALS: A total of 30 healthy male SD rats, aged 8 weeks, of clean grade, weighing 250-300 g, were provided by Experimental Animal Department of China Medical University. All rats were randomly divided into sham-operation group, ischemia/reperfusion group and treatment group with 10 in each group. The main reagents were detailed as the follows: 100 g/L chloral hydrate, 0.5 mL CGRP (2 mg/L, Sigma Company, USA), and NGF (1× 106 U/L, 0.5 mL, Siweite Company, Dalian). METHODS: The experiment was carried out in the Department of Neurobiology, Basic Medical College of China Medical University from February to July 2005. Rat models of middle cerebral artery occlusion were established by method of occlusion, 2 hours after that rats were anesthetized and the thread was slightly drawn out for 10 mm under direct staring to perform reperfusion. Rats in the ischemia/reperfusion group received intraperitoneal injection of 1 mL saline via the abdomen at two hours later, while rats in the treatment group at 2 hours later received intraperitoneal injection of 2 mg/L CGRP (0.5 mL) and 1×106 U/L NGF (0.5 mL) once a day for 10 successive days. First administration was accomplished within 15 minutes after ischemia/reperfusion. Rats in the sham-operation group were separated of the vessels without occlusion or administration. The neural function was evaluated with Zea Longa 5-grade scale. Animals with the score of one, two and three points received Morris water-maze test to measure searching time on platform (omitting platform-escaping latency). Meanwhile, leaning and memory abilities of animals were reflected through testing times of passing through platform per minute. MAIN OUTCOME MEASURES: Experimental results of omitting platform-escaping latency and spatial probe. RESULTS: Three and two rats in the ischemia/reperfusion group and treatment group respectively were not in accordance with the criteria in the process, and the rest were involved in the final analysis. ① Comparisons of platform-escaping latency during Morris water-maze test in all the three groups: Ten days after modeling, the platform-escaping latency in the ischemia/reperfusion group was obviously longer than that in sham-operation group (P < 0.01), and was significantly shorter than that in the treatment group (P < 0.01). ② Comparisons of times of passing through platform in all the three groups: Times of passing through platform were remarkably less in the ischemia/reperfusion group than those in the sham-operation group [(1.79±0.39), (4.30±0.73) times/minute, P < 0.01], and those were markedly more in the treatment group than the ischemia/reperfusion group [(3.16±1.03), (1.79±0.39) times/minute, P < 0.01]. CONCLUSION: CGRP and NGF are capable of ameliorating the abilities of spatial learning and memory in MCAO rats, which indicates that CGRP and NGF can protect ischemic neurons.

Time-dependent changes of glial fibriliary acidic protein and cytosolic phospholipase A2 in hippocampal area of focal cerebral ischemia/reperfusion in rats

BACKGROUND： Interaction between astrocyte and neuron may two-dimensionally influence on ischemic injury; however, glial fibriliary acidic protein （GFAP） and cytosolic phospholipase A2 （cPLA2） are both important markers to reflect changes of astrocyte and neuron after cerebral ischemia, respectively. OBJECTIVE： To observe the changes of GFAP and positive cPLA2 cells in hippocampal area of model rats with focal cerebral ischemia in various phases of cerebral ischemia/reperfusion. DESIGN ： Randomized contrast observation SETTING： Department of Basic Medical Science of Human Anatomy and Histology ＆ Embryology, Medical College of Wuhan Polytechnic University; Faculty Medical College of Wuhan University. MATERIALS： The experiment was carried out in the Department of Basic Medical Science, Medical College of Wuhan Industry College from May to June 2004. A total of 28 healthy SD rats of either gender and weighing 200-250 g were provided by Animal Department of Medical College of Jianghan University. METHODS： All 28 rats were randomly divided into 7 groups, including sham operation group, 2-, 6-, 12-, 24- and 48-reperfusion groups, and triphenyltetrazolium chloride （TTC） group, with 4 in each group. Two hours after ischemia, ischemia/reperfusion models were established in left middle cerebral artery （MCA）; common carotid artery was ligated and line cork was inserted into it with the depth of （1.8±0.5） cm. Rats in sham operation group were inserted with the depth of 1.0 cm, and other operations were as the same as those in 2-hour ischemia/reperfusion groups. Models in TTC group were established as the same as those in 2-hour ischemia/24-hour reperfusion group, and they were used to evaluate the therapeutic effect. Changes of GFAP and cPLA2 in hippocampal area in various phases were detected with immunohisto- chemical method. MAIN OUTCOME MEASURES ： Changes of GFAP and positive cPLA2 cells in hippocampal area of rats with focal cerebral ischemia in various phases of ischemia/reperfusion. RESULTS： All 28 rats were involved in the final analysis without any loss. （1） Animal models successfully showed the effect of focal cerebral ischemia. （2） Changes of GFAP and cPLA2 in hippocampal area in various phases： Two hours after ischemia/reperfusion, changes of GFAP and cPLA2 were increased gradually, reached at peak at 24 hours, and decreased gradually. CONCLUSION ： Courses of GFAP and cPLA2 are changed at the onset of focal cerebral ischemia, and this suggests that both of them participate in injury or protection of brain tissue of focal cerebral ischemia.

Neuroprotective effects of atorvastatin against cerebral ischemia/reperfusion injury through the inhibition of endoplasmic reticulum stress

Cerebral ischemia triggers secondary ischemia/reperfusion injury and endoplasmic reticulum stress initiates cell apoptosis. However, the regulatory mechanism of the signaling pathway remains unclear. We hypothesize that the regulatory mechanisms are mediated by the protein kinase-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α in the endoplasmic reticulum stress signaling pathway. To verify this hypothesis, we occluded the middle cerebral artery in rats to establish focal cerebral ischemia/reperfusion model. Results showed that the expression levels of protein kinase-like endoplasmic reticulum kinase and caspase-3, as well as the phosphorylation of eukaryotic initiation factor 2α, were increased after ischemia/reperfusion. Administration of atorvastatin decreased the expression of protein kinase-like endoplasmic reticulum kinase, caspase-3 and phosphorylated eukaryotic initiation factor 2α, reduced the infarct volume and improved ultrastructure in the rat brain. After salubrinal, the specific inhibitor of phosphorylated eukaryotic initiation factor 2α was given into the rats intragastrically, the expression levels of caspase-3 and phosphorylated eukaryotic initiation factor 2α in the were decreased, a reduction of the infarct volume and less ultrastructural damage were observed than the untreated, ischemic brain. However, salubrinal had no impact on the expression of protein kinase-like endoplasmic reticulum kinase. Experimental findings indicate that atorvastatin inhibits endoplasmic reticulum stress and exerts neuroprotective effects. The underlying mechanisms of attenuating ischemia/reperfusion injury are associated with the protein kinase-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/caspase-3 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.

Baicalin and jasminoidin effects on gene expression and compatibility in the hippocampus following focal cerebral ischemia

The compound traditional Chinese medicine Qingkailing, which is an ingredient used to treat cerebral ischemia, has been limited to studies concerning single genes or single pathways. Interactions and pharmacological mechanisms of the compound ingredients （baicalin and jasminoidin） remain poorly understood. In the present study, baicalin and jasminoidin, as well as the combination, were used to separately treat mouse models of cerebral ischemia, cDNA microarray analyses of 374 cerebral ischemia-related genes were utilized to determine changes in gene-expression profiles. Arraytrack 3.40 and Ingenuity Pathway Analysis （IPA） databases were utilized to analyze changes in gene molecular functions and network path functions. Baicalin or jasminoidin alone effectively reduced infarct area, and the combination resulted in significantly better outcomes. IPA showed inhibited cell apoptosis in the baicalin group and Ca^2＋ channel regulation in the jasminoidin group. The combination of baicalin and jasminoidin activated HTR3A and F5 expression, regulated Ca^2＋ channels, activated kappa light polypeptide gene enhancer inhibitor IKBKG in B cells to control IkB kinase/nuclear factor-kB cascade, suppressed activation of inflammatory cytokine interleukin-6 receptors and activated transduction of guanine-nucleotide- binding protein （G protein） signal. Results suggested that the combination of baicalin and jasminoidin resulted in similar molecular mechanisms to baicalin and jasminoidin alone. However, novel pharmacological actions of compatibility were detected, demonstrating significant protection against cerebral ischemia.

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.

Spatiotemporal expression of Nogo-66 receptor after focal cerebral ischemia

Ng R, the receptor for the neurite outgrowth inhibitor Nogo-66, plays a critical role in the plasticity and regeneration of the nervous system after injury such as ischemic stroke. In the present study, we used immunohistochemistry to investigate the regional expression of Ng R in rat brain following middle cerebral artery occlusion（MCAO）. Ng R protein expression was not observed in the center of the lesion, but was elevated in the marginal zone compared with control and sham-operated rats. The cerebral cortex and hippocampus（CA1, CA2, and CA3） showed the greatest expression of Ng R. Furthermore, Ng R expression was higher in the ipsilesional hemisphere than on the control side in the same coronal section. Although time-dependent changes in Ng R expression across brain regions had their own characteristics, the overall trend complied with the following rules： Ng R expression changes with time showed two peaks and one trough; the first peak in expression appeared between 1 and 3 days after MCAO; expression declined at 5 days; and the second peak occurred at 28 days.

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.

Synthesis, characterization and in vivo evaluation of honokiol bisphosphate prodrugs protects against rats’ brain ischemia-reperfusion injury

Honokiol(HK)usage is greatly restricted by its poor aqueous solubility and limited oral bioavailability.We synthesized and characterized a novel phosphate prodrug of honokiol(HKP)for in vitro and in vivo use.HKP greatly enhanced the aqueous solubility of HK(127.54±15.53 mg/ml)and the stability in buffer solution was sufficient for intravenous administration.The enzymatic hydrolysis of HKP to HK was extremely rapid in vitro(T 1/2=8.9±2.11 s).Pharmacokinetics studies demonstrated that after intravenous administration of HKP(32 mg/kg),HKP was converted rapidly to HK with a time to reach the maximum plasma concentration of^5 min.The prodrug HKP achieved an improved T 1/2(7.97±1.30 h)and terminal volume of distribution(26.02±6.04 ml/kg)compared with direct injection of the equimolar parent drug(0.66±0.01 h)and(2.90±0.342 ml/kg),respectively.Furthermore,oral administration of HKP showed rapid and improved absorption compared with the parent drug.HKP was confirmed to maintain the bioactivity of the parent drug for ameliorating ischemia-reperfusion injury by decreasing brain infarction and improving neurologic function.Taken together,HKP is a potentially useful aqueous-soluble prodrug with improved pharmacokinetic properties which may merit further development as a potential drug candidate.

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.

Iptakalim,an ATP-sensitive potassium channel opener,confers neuroprotection against cerebral ischemia/reperfusion injury in rats by protecting neurovascular unit cells

Objective:To investigate the role of iptakalim,an ATP-sensitive potassium channel opener,in transient cerebral ischemia/reperfusion (I/R) injury and its involved mechanisms.Methods:Intraluminal occlusion of middle cerebral artery (MCAO) in a rat model was used to investigate the effect of iptakalim at different time points.Infarct volume was measured by staining with 2,3,5-triphenyltetrazolium chloride,and immunohistochemistry was used to evaluate the expressions of Bcl-2 and Bax.In vitro,neurovascular unit (NVU) cells,including rat primary cortical neurons,astrocytes,and cerebral microvascular endothelial cells,were cultured and underwent oxygen-glucose deprivation (OGD).The protective effect of iptakalim on NVU cells was investigated by cell viability and injury assessments,which were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and release of lactate dehydrogenase.Caspase-3,Bcl-2 and Bax mRNA expressions were evaluated by real-time polymerase chain reaction (PCR).Results:Administration of iptakalim 0 or 1 h after reperfusion significantly reduced infarct volumes,improved neurological scores,and attenuated brain edema after cerebral I/R injury.Iptakalim treatment (0 h after reperfusion) also reduced caspase-3 expression and increased the ratio of Bcl-2 to Bax by immunohistochemistry.Iptakalim inhibited OGD-induced cell death in cultured neurons and astrocytes,and lactate dehydrogenase release from cerebral microvascular endothelial cells.Iptakalim reduced mRNA expression of caspase-3 and increased the ratio of Bcl-2 to Bax in NVU cells.Conclusions:Iptakalim confers neuroprotection against cerebral I/R injury by protecting NVU cells via inhibiting of apoptosis.

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。

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。

Wistar大鼠插线法局灶性脑缺血/再灌注模型的实验研究

目的：局灶性脑缺血／再灌注模型是研究缺血性脑血管病的重要模型。本文介绍一种简便易行的插线法Ｗｉｓｔａｒ 大鼠局灶性脑缺血／再灌注模型的制备方法，并通过神经病学评分、ＴＴＣ染色、病理形态学变化的观察，对该模型的可靠性进行评价。方法：用头端处理过的尼龙钓丝，从颈外动脉向颈内动脉插入，可逆性阻断大鼠一侧大脑中动脉（ＭＣＡ）。结果：大鼠ＭＣＡ阻断后１．５ｈ，同时出现Ｈｏｒｎｅｒ＇ｓ征阳性、提尾悬空时梗塞对侧前肢屈曲、内收、自主运动时身体向偏瘫侧划圈的动物，ＴＴＣ染色能清楚地显示梗塞范围，且相对较稳定，光镜下可见脑缺血后的病理改变。阻断后３ｈ及再灌注３、６ｈ时的病理改变加重，神经病学评分同前。结论：该改良法制备的模型简便易行、稳定性好、结果可靠，是用于研究局灶性脑缺血／再灌注的较为理想的实验动物模型。