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.

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.

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.

Total flavonoid of Litsea coreana leve exerts anti-oxidative effects and alleviates focal cerebral ischemia/reperfusion injury

In this study, we hypothesized that total flavonoid of Litsea coreana leve （TFLC） protects against focal cerebral ischemia/reperfusion injury. TFLC （25, 50, 100 mg/kg） was administered orally to a rat model of focal ischemia/reperfusion injury, while the free radical scavenging agent, edaravone, was used as a positive control drug. Results of neurological deficit scoring, 2,3,5-triphenyl tetrazolium chloride staining, hematoxylin-eosin staining and biochemical tests showed that TFLC at different doses significantly alleviated cerebral ischemia-induced neurological deficits and histopathological changes, and reduced infarct volume. Moreover, it suppressed the increase in the levels of nitrates plus nitrites, malondialdehyde and lactate dehydrogenase, and it diminished the reduction in glu- tathione, superoxide dismutase and catalase activities induced by cerebral ischemia/reperfusion injury. Compared with edaravone, the protective effects of TFLC at low and medium doses （25, 50 mg/kg） against cerebral ischemia/reperfusion injury were weaker, while the protective effects at high dose （100 mg/kg） were similar. Our experimental findings suggest that TFLC exerts neuroprotective effects against focal cerebral ischemia/reperfusion injury in rats, and that the effects may be asso- ciated with its antioxidant activities.

Expression profiles of microRNAs after focal cerebral ischemia/reperfusion injury in rats

Rat models of focal cerebral ischemia/reperfusion injury were established by occlusion of the middle cerebral artery. Microarray analysis showed that 24 hours after cerebral ischemia, there were nine up-regulated and 27 down-regulated microRNA genes in cortical tissue. Bioinformatic analysis showed that bcl-2 was the target gene of microRNA-384-5p and microRNA-494, and caspase-3 was the target gene of microRNA-129, microRNA-320 and microRNA-326. Real-time PCR and western blot analyses showed that 24 hours after cerebral ischemia, bcl-2 mRNA and protein levels in brain tissue were significantly decreased, while caspase-3 mRNA and protein levels were significantly increased. This suggests that following cerebral ischemia, differentially expressed microRNA-384-5p, microRNA-494, microRNA-320, microRNA-129 and microRNA-326 can regulate bcl-2 and caspase-3 expression in brain tissue.

Comparison of the anti-apoptotic effects of 15-and 35-minute suspended moxibustion after focal cerebral ischemia/reperfusion injury

Heat-sensitive suspended moxibustion has a neuroprotective effect against focal cerebral ischemia/reperfusion injury, but the underly- ing mechanisms remain unclear. The duration of heat-sensitive suspended moxibustion （usually from 30 minutes to 1 hour） is longer than traditional suspended moxibustion （usually 15 minutes）. However, the effects of 15- and 35-minute suspended moxibustion in rats with cerebra/ischemia/reperfusion injury are poorly understood. In this study, we performed 15- or 35-minute suspended moxibustion at acupoint Dazhui （GV14） in an adult rat model of focal cerebral ischemia/reperfusion injury. Infarct volume was evaluated with the 2,3,5-triphenyltetrazolium chloride assay. Histopathological changes and neuronal apoptosis at the injury site were assessed by hematoxy- lin-eosin staining and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Caspase-9 and caspase-3 expression at the in- jury site was detected using immunofluorescent staining. Bax and Bcl-2 expression at the injury site was assessed using western blot assay. In the 35-minute moxibustion group, infarct volume was decreased, neuronal apoptosis was reduced, caspase-9, caspase-3 and Bax expres- sion was lower, and Bcl-2 expression was increased, compared with the 15-minute moxibustion group. Our findings show that 35-minute moxibustion has a greater anti-apoptotic effect than 15-minute moxibustion after focal cerebral ischemia/reperfusion injury.

Hydrogen sulfide intervention in focal cerebral ischemia/reperfusion injury in rats

The present study aimed to explore the mechanism underlying the protective effects of hydrogen sulfide against neuronal damage caused by cerebral ischemia/reperfusion. We established the middle cerebral artery occlusion model in rats via the suture method. Ten minutes after middle cerebral artery occlusion, the animals were intraperitoneally injected with hydrogen sulfide donor compound sodium hydrosulfide. Immunofluorescence revealed that the immunoreactivity of P2X7 in the cerebral cortex and hippocampal CA1 region in rats with cerebral ischemia/reperfusion injury decreased with hydrogen sulfide treatment. Furthermore, treatment of these rats with hydrogen sulfide significantly lowered mortality, the Longa neurological deficit scores, and infarct volume. These results indicate that hydrogen sulfide may be protective in rats with local cerebral ischemia/reperfusion injury by down-regulating the expression of P2X7 receptors.

Therapeutic window of Qingkailing injection for focal cerebral ischemia/reperfusion injury

The time window in which a drug is effective varies between drugs. The present study investigated the therapeutic window of Qingkailing injection for focal cerebral ischemia/reperfusion in mice. Animals underwent middle cerebral artery occlusion and were injected with Qingkailing （1.5, 3, 6 mL/kg）. Infarct volume and neurological function were assessed after 24 hours of ischemia. In addition, to establish the therapeutic time window, mice were injected with 3 mL/kg Qingkailing at 0, 1, 3, 4, 6, 9 and 12 hours after occlusion. Results revealed that Qingkailing injection significantly reduced infarct volume and improved neurological function in model mice after cerebral infarction for up to 9 hours, demonstrating that the therapeutic window of Qingkailing injection can extend to 9 hours for cerebral ischemia/reperfusion in mice.

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.

Neuroprotective role of edaravone and the effects of endoplasmic reticulum stress in an adult rat model of focal cerebral ischemia/reperfusion

BACKGROUND： Endoplasmic reticulum （ER） stress impairs ER functions and leads to the accumulation of unfolded or misfolded proteins in the ER lumen. ER stress-induced cell death plays an important role in cerebral ischemia. Edaravon （3-methyl-1-phenyl-2-pyrazolin-5-one） is a potent and novel scavenger of free radicals that inhibit delayed neuronal death, as demonstrated by in vitro and animal studies. However, its effect on ER stress and induced neuronal apoptosis in a rat model of brief middle cerebral artery occlusion remains unclear. OBJECTIVE： To explore the effects of edaravone on the expression of ER stress-related factors and neuronal apoptosis, based on the hypothesis that edaravone influences ER stress in a rat model of cerebral ischemia/reperfusion. DESIGN, TIME AND SETTING： A randomized, controlled, animal study was performed at the Laboratory of Department of Neurology, Xiangya Hospital and the Department of Laboratory Animals, Xiangya Medical College, Central South University in China from June 2005 to May 2006. MATERIALS： Edaravone was purchased from Simcere Pharmaceutical Group, China. METHODS： A total of 216 adult, male, Sprague Dawley rats were randomly assigned to sham-surgery, model and edaravone groups, with 72 rats in each group, Brief middle cerebral artery occlusion was established in the model and edaravone groups. In addition, the edaravone group rats were injected with 3 mg/kg edaravone through the tail vein. MAIN OUTCOME MEASURES： RNA-dependent protein kinase-like endoplasmic reticulum eukaryotic translation initiation factor 2a kinase （PERK） and C/EBP homology protein （CHOP） mRNA expression in the ischemic parietal cortex was determined by reverse transcriptionpolymerase chain reaction; phosphorylated PERK and CHOP protein expression was detected by immunohistochemistry; neuronal apoptosis was detected by TdT-mediated-dUTP nick end labeling. RESULTS： Neurological deficit scores were significantly reduced in the edaravone group compared to the model group at 12, 24, and 72 hours following reperfusion （P〈 0.05）. In addition, PERK and CHOP mRNA as well as phosphorylated PERK and CHOP protein expression were significantly reduced in the edaravone group compared to the model group at 1,3, and 6 hours following reperfusion （P 〈 0.05, P 〈 0.01）. CHOP mRNA expression was decreased in the edaravone group compared to the model group at 3, 6, 12, and 24 hours following reperfusion （P〈 0.01）, while CHOP protein expression was less than the model group at 6, 12, and 24 hours following reperfusion （P 〈 0.05）. CONCLUSION： Edaravone treatment resulted in decreased PERK and CHOP expression following ischemia/reperfusion, as well as reduced neuronal apoptosis. Edaravone exhibited a neuroprotective role by inhibiting endoplasmic reticulum stress.

Changes in corticocerebral morphology in a rat model of focal cerebral ischemia/reperfusion injury following “Xingnao Kaiqiao” acupuncture

BACKGROUND： Cerebral ischemia/reperfusion injury has been shown to induce inflammatory reactions, including white blood cell activation and adhesion molecule expression. These reactions often lead to aggravated neuronal injury. OBJECTIVE： To observe corticocerebral pathology, as well as ultrastructural changes, in a rat model of focal cerebral ischemia/reperfusion injury through optical and electron microscopy, and to investigate interventional effects of ＂Xingnao Kaiqiao＂ acupuncture （a brain-activating and orifice-opening acupuncture method）. DESIGN, TIME AND SETTING： A randomized, controlled, neuropathology, animal experiment was performed at the Laboratory of Molecular Biology, First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine between April and June 2004. MATERIALS： A total of 50 healthy, male, Wistar rats were randomized into 5 groups, with 10 rats per group： control, sham-operated, model, non-acupoint, and ＂Xingnao Kaiqiao ＂. Transmission electron microscope （TEM 400ST） was provided by Philips, Netherlands. Electro-acupuncture treatment apparatus （KWD-8082） was provided by Changzhou Wujin Great Wall Medical Instrument, China. METHODS： Focal cerebral ischemia/reperfusion injury was induced by occlusion of the middle cerebral artery in the model, non-acupoint, and ＂Xingnao Kaiqiao＂ groups. Rats from the control group did not undergo any treatment. The sham-operated group received identical experimental procedures as the model group, except that the nylon suture was not inserted into the right internal carotid artery. At 1, 3, 6, and 12 hours following focal cerebral ischemia/reperfusion injury induction, rats from the Xingnao Kaiqiao group underwent 1-minute acupuncture at the bilateral ＂Neiguan＂ （PC 6） acupoint, using a reducing method of lifting-thrusting and twirling-rotating. Subsequently, the rats were subjected to acupuncture at the ＂Renzhong＂ （DU26） acupoint 10 times by a heavy bird-pecking method. The non-acupoint group received acupuncture administration at the bilateral costal region. MAIN OUTCOME MEASURES： After ischemia for 1 hour and reperfusion for 24 hours, corticocerebral morphology and ultrastructural changes were observed on the injured side through the use of optical and electron microscopy. RESULTS： Cerebral ischemia/reperfusion resulted in damage to neurons, glial cells, and capillary vessels in the rat brain. ＂Xingnao Kaiqiao＂ acupuncture produced superior curative effects when it was performed 3 hours after cerebral ischemia/reperfusion induction, resulting in slightly recovered neuronal structures, alleviated cellular interstitial edema, and more capillary vessels. At each corresponding time point, the ＂Xingnao Kaiqiao＂ group exhibited improved neuronal structure and cellular interstitial edema, compared with the non-acupoint group. CONCLUSION： ＂Xingnao Kaiqiao＂ acupuncture results in protective effects on corticocerebral neuronal morphology and ultrastructure in rats following focal cerebral ischemia/reperfusion.

The effect of hyperglycemia on blood brain barrier of rats with focal cerebral ischemia/reperfusion injury

Objective： To determine whether hyperglycemia could aggravate the microvascular damage in ischemic stroke. Methods： Hyperglycemia model was made by injection of streptozocin through subcutaneous injection in wistar rats. Using the suture model, the rats were subjected to 3h of focal ischemia and different times of reperfusion, including 6,12,24,48,96h and 7d. TIC dyeing was used to Show the infarction area of rats. The infarctive volume of rats were calculated by computer imaging analysis system;Matrix metalloproteinase （MMP-2） and （MMP-9）were detected by immunohistochemistly and in situ hybridization histochemistly in Wistar rats. Results： The infarctive volume was siginificantly larger in hyperglycemic rats than that of nonhyperglycemic rats. The level of MMP-2, MMP-9 expression in the group of hyperglycemic rats was higher than that of nonhyperglycemic rats. Conclusion： Hyperglycemia aggravated the injury of focal ischmia-repeffusion in wistar rats and the higher expression of MMP-2,MMP-9 might be one of the mechanism in aggravation of focal ischemia/repeffusion injury.

EFFECTS OF PHYCOCYANIN ON EXPRESSION OF CytC mRNA AND CASPASE-3 mRNA AFTER FOCAL CEREBRAL ISCHEMIA/REPERFUSION IN RATS

Objective To study the effects of phycocyanin on the expression of Cytochrome C (CytC)genes and Caspase-3 genes after focal cerebral ischemia/reperfusion in rats. Methods A rat middle cerebral ar-tery occlusion (MCAO)/reperfusion model was produced using the intraluminal filament method. The rats were di-vided into three groups: sham operation group, model control group and phycocyanin group. After MCAO, the neu-robehavioral testing of all rats was made. The infarction area was evaluated with the method of 2,3,7-triphenyltet-razolium chloride (TTC) staining. The expression of CytC mRNA and Caspase-3 mRNA were determined by in situhybridization. Results In the sham operation group and the model control group, there was only a few CytC-positive cells were seen in the normal cerebral tissue. In the model control group, the upregulation of CytC mRNAbegan 6h after ischemia, reached a maximum at 12h (cortex) -24h (striatum) , then subsided gradually, but stillin high level. In the phycocyanin group, CytC-positive cells were also mainly in cortex and striatum, but the numberof the cells was significantly lower than the number of the model control group. The time-phase pattern of CytCmRNA in the phycocyanin group was similar to the pattern of the model control group. In the sham operation groupand the model control group, there was only a few Caspase-3-positive cells were seen in the normal cerebral tissue.In the model control group, the upregulation of Caspase-3 mRNA began 6h after ischemia, reached a maximum at24h and subsided at 48h, but still in high level. In the phycocyanin group, Caspase-3-positive cells were also mainlyin the penumbral area, but the number of the cells were significantly lower than the number of the model controlgroup. The time-phase pattern of Caspase-3 mRNA in the phycocyanin group was similar to the pattern of the modelcontrol group. Conclusion The over-expression of CytC mRNA and Caspase-3 mRNA might play a key role inischemic cerebral injury after MCAO. Phycocyanin could inhibit the over-expression of CytC mRNA and Caspase-3mRNA in the cerebral cortex, and might play an important role in the protection of ischemic neurons.

Angiopoietin-1 mRNA and Bcl-2 expression following estradiol treatment in ovariectomized rats with focal cerebral ischemia/reperfusion injury

BACKGROUND： Estrogen has been clinically demonstrated to attenuate ischemic brain injury. However, the precise mechanisms remain controversial. OBJECTIVE： To investigate the effects of estradiol on angiopoietin-1 mRNA and Bcl-2 expression, as well as apoptosis and cerebral blood flow, in ovadectomized rats with focal cerebral ischemia following reperfusion. DESIGN, TIME AND SETTING： Randomized, controlled, animal experiment. The study was performed at the Central Laboratory, Chongqing Medical University from September to December 2005. MATERIALS： Estradiol benzoate was purchased from Shanghai Ninth Pharmaceutical Factory; corn oil was purchased from Walmart Supercenter; TUNEL kit, rabbit anti-rat Bcl-2 polyclonal antibody, and biotin-labeled goat anti-rabbit antibody were purchased from Wuhan Boster, China. METHODS： Healthy, female, 6-month-old Wistar rats-wild-type and estrogen alpha receptor gene knockout （ERKO）-were randomly divided into estradiol and control groups with 25 animals in each group. The rats were intramuscularly injected with estradiol benzoate （100 μg/kg per day） at 30 days following bilateral ovariectomy or corn oil （1 mL/kg per day） for seven consecutive days. Following administration, cerebral ischemia/reperfusion models were established using the right middle cerebral artery occlusion （MCAO） method. After 30 minutes of MCAO, estradiol and control groups were separately injected with estradiol benzoate and corn oil with the above-mentioned doses. MAIN OUTCOME MEASURES： Cell apoptosis was determined by TUNEL; angiopoietin-1 mRNA and Bcl-2 gene expression was determined, respectively, by immunohistochemical staining and RT-PCR. In addition, changes in cerebral blood flow were measured by laser Doppler flowmetry. RESULTS： Changes in angiopoietin-1 mRNA and cerebral blood flow in estradiol-treated, wild-type, MCAO rats following ischemia/reperfusion were greater than in control rats （P 〈 0.01 or 0.05）. However, no significant difference was observed between estradiol-treated ERKO MCAO rats and control rats. In addition, estradiol-treated wild-type and ERKO MCAO rats exhibited significantly increased Bcl-2 expression （P 〈 0.05） and decreased number of apoptotic cells in brain tissues compared with control groups （P 〈 0.05）. CONCLUSION： Estradiol upregulated angiopoietin-1 mRNA and Bcl-2 expression, suggesting that estradiol might be involved in protective mechanisms of cerebral ischemia/reperfusion injury.

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.

Lactiplantibacillus plantarum AR113 alleviates microbiota dysbiosis of tongue coating and cerebral ischemia/reperfusion injury in rat

Stroke is one of the leading causes of death and disability worldwide.However,information on stroke-related tongue coating microbiome(TCM)is limited,and whether TCM modulation could benefit for stroke prevention and rehabilitation is unknown.Here,TCM from stroke patients(SP)was characterized using molecular techniques.The occurrence of stroke resulted in TCM dysbiosis with significantly reduced species richness and diversity.The abundance of Prevotella,Leptotrichia,Actinomyces,Alloprevotella,Haemophilus,and TM7_[G-1]were greatly reduced,but common infection Streptococcus and Pseudomonas were remarkably increased.Furthermore,an antioxidative probiotic Lactiplantibacillus plantarum AR113 was used for TCM intervention in stroke rats with cerebral ischemia/reperfusion(I/R).AR113 partly restored I/R induced change of TCM and gut microbiota with significantly improved neurological deficit,relieved histopathologic change,increased activities of antioxidant enzymes,and decreased contents of oxidative stress biomarkers.Moreover,the gene expression of antioxidant-related proteins and apoptosis-related factors heme oxygenase-1(HO-1),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),nuclear factor erythroid 2-related factor 2(Nrf2),NAD(P)H:quinone oxidoreductase-1(NQO-1),and Bcl-2 was significantly increased,but cytochrome C,cleaved caspase-3,and Bax were markedly decreased in the brain by AR113 treatment.The results suggested that AR113 could ameliorate cerebral I/R injury through antioxidation and anti-apoptosis pathways,and AR113 intervention of TCM may have the application potential for stroke prevention and control.

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.