Dose-dependent effects of procyanidin on nerve growth factor expression following cerebral ischemia/ reperfusion injury in rats★

BACKGROUND： Recently, grape seed procyanidin （GSP） has been shown to be exhibit antioxidant effects, effectively reducing ischemia/reperfusion injury and inhibiting brain cell apoptosis. OBJECTIVE： To study the effects of GSP on nerve growth factor （NGF） expression and neurological function following cerebral ischemia/reperfusion injury in rats. DESIGN： Randomized controlled study based on SD rats. SETTING： Weifang Municipal People＇s Hospital. MATERIALS： Forty-eight healthy adult SD rats weighing 280-330 g and irrespective of gender were provided by the Experimental Animal Center of Shandong University. GSP derived from grape seed was a new high-effective antioxidant provided by Tianjin Jianfeng Natural Product Researching Company （batch number： 20060107）. Rabbit-anti-rat NGF monoclonal antibody was provided by Beijing Zhongshan Biotechnology Co., Ltd., and SABC immunohistochemical staining kit by Wuhan Boster Bioengineering Co., Ltd. METHODS： The present study was performed in the Functional Laboratory of Weifang Medical College from April 2006 to January 2007. Forty-eight SD rats were randomly divided into the sham operation group, ischemia/reperfusion group, high-dose GSP （40 mg/kg） group, or low-dose GSP （10 mg/kg） group （n = 12 per group）. Ischemia/reperfusion injury was established using the threading embolism method of the middle cerebral artery. Rats in the ischemia/reperfusion model group were given saline injection （2 mL/kg i.p.） once daily for seven days pre-ischemia/reperfusion, and once more at 15 minutes before reperfusion. Rats in the high-dose and low-dose GSP groups were injected with GSP （20 or 5 mg/mL i.p., respectively, 2 mL/kg） with the same regime as the ischemia/reperfusion model group. The surgical procedures in the sham operation group were as the same as those in the ischemia/reperfusion model group, but the thread was approximately 10 mm long, thus, the middle cerebral artery was not blocked. MAIN OUTCOME MEASURES： NGF expression in the     

Effects of kallikrein gene transfer on penumbral microvascular proliferation and on regional cerebral blood flow following cerebral ischemia/reperfusion injury

BACKGROUND： Recent findings have demonstrated that the kallikrein-kinin system （KKS） participates in the pathological process of cerebral ischemia/reperfusion injury. Kallikrein gene transfer exhibits neural protective effects following cerebral infarction. OBJECTIVE： To observe the effects of kallikrein gene transfer on vascular proliferation in the peripheral infarct focus and on regional cerebral blood flow （rCBF） following cerebral ischemia/reperfusion injury. DESIGN, TIME AND SETTING： The completely randomized, controlled experiment was performed at the Lin Baixin Laboratory Center, the Second Affiliated Hospital of Sun Yat-sun University between September 2007 and April 2008. MATERIALS： pUCI9-HTK plasmid was constructed and maintained in the Laboratory for Neurology, the Second Affiliated Hospital of Sun Yat-sen University, China. Mouse anti-human kallikrein 1 monoclonal antibody was purchased from R＆D Systems, USA. METHODS Ninety healthy, male, Sprague Dawley rats were used. Middle cerebral artery occlusion （MCAO） was established in all rats to induce cerebral ischemia/reperfusion injury. Following MCAO establishment, all rats were randomly divided into three groups （n = 30）： blank control, saline, and pAdCMV-HTK. The saline and pAdCMV-HTK groups were stereotactically micro-injected with 5μL of physiological saline or with pAdCMV-HTK [multiplicity of infection （MOI） = 20], respectively, into the ischemic penumbra. In the blank control group, only sham injection was performed. MAIN OUTCOME MEASURES： At 12, 24, and 72 hours after treatment, cerebral infarction volume was measured by 2, 3, 5-triphenyltetrazolium chloride （TTC） staining. Exogenous HTK expression, as well as regional vascular endothelial growth factor （VEGF） expression, was detected by immunohistochemistry. rCBF was examined by 14C-iodoantipyrine micro tracing. In addition, neurological severity score （NSS） was performed. Higher scores indicated more severe neurological deficits. RESULTS： NSS res     

Neuroprotective effect of Cerebralcare Granule after cerebral ischemia/reperfusion injury

Cerebralcare Granule(CG) improves cerebral microcirculation and relieves vasospasm,but studies investigating its therapeutic effect on cerebral ischemia/reperfusion injury are lacking.In the present study,we administered CG(0.3,0.1 and 0.03 g/m L intragastrically) to rats for 7 consecutive days.We then performed transient occlusion of the middle cerebral artery,followed by reperfusion,and administered CG daily for a further 3 or 7 days.Compared with no treatment,high-dose CG markedly improved neurological function assessed using the Bederson and Garcia scales.At 3 days,animals in the high-dose CG group had smaller infarct volumes,greater interleukin-10 expression,and fewer interleukin-1β-immunoreactive cells than those in the untreated model group.Furthermore,at 7 days,high-dose CG-treated rats had more vascular endothelial growth factor-immunoreactive cells,elevated angiopoietin-1 and vascular endothelial growth factor expression,and improved blood coagulation and flow indices compared with untreated model animals.These results suggest that CG exerts specific neuroprotective effects against cerebral ischemia/reperfusion injury.     

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

Proanthocyanidins have been shown to effectively protect ischemic neurons, but its mechanism remains poorly understood. Ginkgo proan-thocyanidins (20, 40, 80 mg/kg) were intraperitoneally administered 1, 24, 48 and 72 hours before reperfusion. Results showed that ginkgo proanthocyanidins could effectively mitigate neurological disorders, shorten infarct volume, increase superoxide dismutase activity, and de-crease malondialdehyde and nitric oxide contents. Simultaneously, the study on grape seed proanthocyanidins (40 mg/kg) conifrmed that different sources of proanthocyanidins have a similar effect. The neurological outcomes of ginkgo proanthocyanidins were similar to that of nimodipine in the treatment of cerebral ischemia/reperfusion injury. Our results suggest that ginkgo proanthocyanidins can effectively lessen cerebral ischemia/reperfusion injury and protect ischemic brain tissue and these effects are associated with antioxidant properties.     

水通道蛋白4在大鼠脑缺血再灌注损伤中的作用

目的研究水通道蛋白4(AQP4)在缺血再灌注损伤大鼠脑内表达及作用。方法以大脑中动脉线栓法建立大鼠缺血再灌注模型,采用干湿法测定模型的脑组织含水量及伊文氏蓝含量;免疫蛋白印记(WesternBlot)技术分析在缺血再灌注不同时程脑内AQP4的表达情况,以及AQP4与脑含水量和伊文氏蓝水平的相关性,并与对照组比较。结果与对照组相比,实验组大鼠脑组织含水量及伊文氏蓝水平在缺血再灌注后不同时间点明显高于对照组(P<0.05～0.01);AQP4蛋白表达明显增高(均P<0.05),并随着缺血再灌时间的延长,其表达量亦逐渐增加,在再灌后24～48h达到高峰。缺血再灌注后AQP4脑内的表达与脑组织含水量及伊文氏蓝水平呈正相关(r=0.38、r=0.45,均P<0.05)。结论AQP4的高表达参与了脑缺血再灌注后继发的血脑屏障的开放和脑水肿的发生,是脑水肿产生的重要分子基础。     

麻黄碱干预脑缺血再灌注损伤的不良反应：不同剂量比较

Ephedrine has protective effect to rats suffered from cerebral ischemia, the aim of the present study was to evaluate the side effects of ephedrine on heart, liver, kidney and cerebrum of rats after ischemic-reperfusion. Sprague-Dawley (SD) rats, suffered from ischemic-reperfusion were randomly divided into 4 groups: A group for ephedrine, B group for ephedrine, C group for ephedrine, and control group （D group）.At each week, hematoxylin eosin staining was used to observe the tissue of all the organs, then, the blood pressure, aspartate aminotransferase (AST), alkaline phosphatase (AKP) and creatinine were tested, also immunohistochemical method was used to test the expression of brain-derived neurotrophic factor (BDNF) on hippocampal CA3 area. The blood pressure values were lower in ephedrine groups than those in control group all the time (P<0.05). The biochemistry results showed that AST、AKP and serum creatinine in ephedrine groups were higher than those in control groups (P<0.05). The expression levels of BDNF on hippocampal CA3 area in ephedrine groups were higher than that in control group at the last three weeks (P<0.05). The pathological section showed that in all the ephedrine groups, we can see congestion, degeneration and edema of tissues. These findings indicate that ephedrine may have side effects on heart, liver, kidney and cerebrum in ischemic/reperfusion rats. There may be correlation between the side effects and dose, the side effects are enhanced with an increased dose of ephedrine. The injury of the above organs also may be transient, which can be recovered after discontinuation of treatment.     

Transcription factor changes following long term cerebral ischemia/reperfusion injury

The present study established a rat model of cerebral ischemia/reperfusion injury using four-vessel occlusion and found that hippocampal CA1 neuronal morphology was damaged, and that there were reductions in hippocampal neuron number and DNA-binding activity of cAMP response element binding protein and CCAAT/enhancer binding protein, accompanied by decreased learning and memory ability. These findings indicate that decline of hippocampal cAMP response element binding protein and CCAAT/enhancer binding protein DNA-binding activities may contribute to neuronal injury and learning and memory ability reduction induced by cerebral ischemia/reperfusion injury.     

Triptolide for cerebral ischemia/reperfusion injury

BACKGROUND: Studies have demonstrated that triptolide has good anti-inflammatory and immunosuppressive effects. However, the effect of triptolide on cerebral ischemia/reperfusion injury is still unclear. OBJECTIVE: To observe the effects of triptolide on neurologic function, infarct volume, water content of brain tissue, neutrophil number in microvascular wall and interleukin-1β(IL-1β) expression in rat models of local ischemia/reperfusion, and analyze the mechanism of triptolide for protecting brain. DESIGN: Randomized controlled experiment. SETTING: Department of Pathology, Medical School of Ningbo University; Department of Forensic Medicine, Tongji Medical College of Huazhong University of Science and Technology. MATERIALS: Sixty Wistar rats of either gender, aged 4 months old, weighing from 200 to 250 g, were provided by the Experimental Animal Center, Tongji Medical College, Huazhong University of Science and Technology. Triptolide was purchased from Fujian Institute for Medical Science (purity 99.98%; Batch No. 2000215). It was dissolved in 20 g/L propanediol, and filtered with 200-mesh filter for later use. METHODS: This experiment was carried out in the laboratory of Forensic Medicine, Tongji Medical College of Huazhong University of Science and Technology, Department of Pathology, Medical School of Ningbo University between January 2001 and September 2004. ① Sixty Wistar rats were randomized into 4 groups: sham-operation group, model group, low-dose triptolide group and high-dose triptolide group. Rats in each group, except for sham-operation group, were developed into rat models of cerebral ischemia/reperfusion according to the method of Longa et al. In the first 3 days of modeling, rats in the low- and high-dose triptolide groups were intraperitoneally injected with 0.2 and 0.4 mg/kg triptolide respectively, once a day, 3 days in total. ② At ischemia 1 hour and reperfusion 24 hours, infarct volume, neurologic deficit (five-point scale, higher scores indicated poor neurologic function), water content of brain tissue, neutrophil number in microvascular wall in the middle cerebral artery occlusive side of rats were detected, meanwhile, brain tissue injury degree and IL-1βimmunohistochemical staining changes in brain-derived nerve cells were observed under the optical microscope. MAIN OUTCOME MEASURES: Neurologic deficit, infarct volume percentage, water content of brain tissue, neutrophil number in microvascular wall and positive rate of IL-1β immunoreaction. RESULTS: Sixty rats were all involved in the final analysis. ① Neurologic deficit scores of rats in the low- and high-dose triptolide groups were （1.96±0.14） and （1.75±0.16）points respectively , which were both significantly lower than those in model group [（2.58±0.11）points，P < 0.05，0.01]. ② Infarct volume percentages of rats in low- and high-dose triptolide groups were significantly lower than that in model group separately (P < 0.05, 0.01). ③Water content of brain tissue of rats in model group was significantly higher than that in the sham-operation group [（82.35±1.26）% vs. （76.65±1.17）%，P < 0.01]; Water content of brain tissue of rats in the low- and high-dose triptolide groups was respectively（80.15±1.43）%，（78.23± 1.15）%, which was significantly lower than that in the model group (P < 0.05, 0.01). ④Pathological changes of brain tissue of rats: Under the optical microscope, infarct focus was not found in the brain tissue of rats in the sham-operation group, while clear infarct focus could be found in the brain tissue of rats in the model group; Although infarct focus was found in the brain tissue of rats in the low- and high-dose triptolide groups, the whole infarct area was contracted as compared as that in the model group. ⑤Neutrophil number in microvascular wall of brain tissue of rats in the low- and high dose triptolide groups was 10.60±2.12，8.11±1.21 respectively, which was significantly less than that in model group（16.25±1.96，P < 0.05，0.01）. ⑥Positive rate of IL-1βimmunoreaction in the brain tissue of rats in model group was significantly higher than that in the sham-operation group (P < 0.01); and positive rate of IL-1β immunoreaction in the brain tissue of rats in low- and high-dose triptolide groups was significantly lower than that in the model group (P < 0.05, 0.01), but higher than that in the sham-operation group, without significant difference (P > 0.05). CONCLUSION: Triptolide protects against cerebral ischemia/reperfusion injury of rats that may be related with anti-inflammations. Triptolide inhibits IL-1β expression in brain tissue and reduces the attachment and aggregation of neutrophils in blood capillary, and further inhibits the infiltration of blood white cells, thus, it will lessen cerebral injury, contract cerebral infarct and improve cerebral function.     

Atorvastatin protects against cerebral ischemia/ reperfusion injury through anti-inflammatory and antioxidant effects

In addition to its lipid-lowering effect, atorvastatin exerts anti-inflammatory and antioxidant effects as well. In this study, we hypothesized that atorvastatin could protect against cerebral isch-emia/reperfusion injury. The middle cerebral artery ischemia/reperfusion model was established, and atorvastatin, 6.5 mg/kg, was administered by gavage. We found that, after cerebral ischemia/ reperfusion injury, levels of the inflammation-related factors E-selectin and myeloperoxidase were upregulated, the oxidative stress-related marker malondialdehyde was increased, and super- oxide dismutase activity was decreased in the ischemic cerebral cortex. Atorvastatin pretreatment significantly inhibited these changes. Our findings indicate that atorvastatin protects against ce-rebral ischemia/reperfusion injury through anti-inflammatory and antioxidant effects.     

Puerarin protects brain tissue against cerebral ischemia/reperfusion injury by inhibiting the inflammatory response

Puerarin, a traditional Chinese medicine, exerts a powerful neuroprotective effect in cerebral ischemia/reperfusion injury, but its mechanism is unknown. Here, we established rat models of middle cerebral artery ischemia/reperfusion injury using the suture method. Puerarin(100 mg/kg) was administered intraperitoneally 30 minutes before middle cerebral artery occlusion and 8 hours after reperfusion. Twenty-four hours after reperfusion, we found that puerarin significantly improved neurological deficit, reduced infarct size and brain water content, and notably diminished the expression of Toll-like receptor-4, myeloid differentiation factor 88, nuclear factor kappa B and tumor necrosis factor-α in the ischemic region. These data indicate that puerarin exerts an anti-inflammatory protective effect on brain tissue with ischemia/reperfusion damage by downregulating the expression of multiple inflammatory factors.     

Pretreatment with scutellaria baicalensis stem-leaf total flavonoid protects against cerebral ischemia/ reperfusion injury in hippocampal neurons

Previous experimental studies have shown that cerebral infarction can be effectively reduced following treatment with scutellaria baicalensis stem-leaf total flavonoid （SSTF）. However, the mechanism of action of SSTF as a preventive drug to treat cerebral infarction remains unclear. In this study, Sprague-Dawley rats were pretreated with 50, 100, 200 mg/kg SSTF via intragastric ad- ministration for 1 week prior to the establishment of focal cerebral ischemia/reperfusion injury. The results showed that pretreatment with SSTF effectively improved neurological function, reduced brain water content and the permeability of blood vessels, ameliorated ischemia-induced morphology changes in hippocampal microvessels, down-regulated Fas and FasL protein expression, elevated the activity of superoxide dismutase and glutathione peroxidase, and decreased malondialdehyde content. In contrast to low-dose SSTF pretreatment, the above changes were most obvious after pretreatment with moderateand high-doses of SSTF. Experimental findings indicate that SSTF pretreatment can exert protective effects on the brain against cerebral ischemia/reperfusion injury. The underlying mechanisms may involve reducing brain water content, increasing microvascular recanalization, inhibiting the apoptosis of hippocampal neurons, and attenuating free radical damage.     

经鼻给予VEGF治疗脑缺血/再灌注大鼠的量效关系

目的探讨经鼻给予血管内皮生长因子(VEGF)治疗脑缺血/再灌注损伤大鼠的量效关系。方法48只SD大鼠随机分为四组:低剂量组(100μg/mL)、中剂量组(200μg/mL)、高剂量组(500μg/mL)及盐水对照组(n=12)。通过阻塞大脑中动脉制作大鼠局灶性脑缺血90 m in再灌注损伤模型。缺血后1d、7d和14 d行神经功能评价,14 d动物被麻醉,行组织学检查,应用图像分析系统计算梗死体积、评价血管形成。结果与对照组相比,经鼻给予中剂量VEGF,可明显降低梗死体积,改善神经功能(P<0.01);而低和高剂量组对比于对照组,不能降低脑缺血后大鼠脑梗死体积和改善神经功能(P>0.05)。与对照组相比,经鼻给予中和高剂量VEGF,可增加缺血后脑表面血管形成(P<0.01);而低剂量组对比于对照组,不能促进脑缺血后血管生成(P>0.05)。结论经鼻给予中剂量VEGF可有效降低脑缺血/再灌注损伤大鼠梗死体积,改善神经功能,增加血管密度。因此经鼻给予中等剂量(200μg/mL)VEGF是治疗脑缺血/再灌注损伤的最佳剂量,其可用于进一步评价VEGF作用的有效实验剂量。     

Neuronal apoptosis in cerebral ischemia/reperfusion area following electrical stimulation of fastigial nucleus

Previous studies have indicated that electrical stimulation of the cerebellar fastigial nucleus in rats may reduce brain infarct size, increase the expression of Ku70 in cerebral ischemia/ reperfusion area, and decrease the number of apoptotic neurons. However, the anti-apoptotic mechanism of Ku70 remains unclear. In this study, fastigial nucleus stimulation was given to rats 24, 48, and 72 hours before cerebral ischemia/reperfusion injury. Results from the electrical stim- ulation group revealed that rats exhibited a reduction in brain infarct size, a significant increase in the expression of KuT0 in cerebral ischemia/reperfusion regions, and a decreased number of terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL）-positive cells. Double immunofluorescence staining revealed no co-localization of Ku70 with TUNEL-positive cells. However, Ku70 partly co-localized with Bax protein in the cytoplasm of rats with cerebral ischemia/reperfusion injury. These findings suggest an involvement of Ku70 with Bax in the cy- toplasm of rats exposed to electrical stimulation of the cerebellar fastigial nucleus, and may thus provide an understanding into the anti-apoptotic activity of KuT0 in cerebral ischemia/reperfu- sion injury.     

Recombinant human granulocyte-colony stimulating factor and differential expression of cerebral cortical proteins in the subacute stage of cerebral ischemia/reperfusion

Recombinant human granulocyte-colony stimulating factor (hG-CSF) has been shown to protect the nervous system after brain ischemia. However, the neuroprotective mechanism of hG-CSF remains unclear. The present study established a rat model of cerebral ischemia/reperfusion and subcutaneously injected recombinant hG-CSF after reperfusion for 2 hours. Cerebral cortical protein was extracted following 14 days of reperfusion and subjected to two-dimensional electrophoresis. In brain ischemic rats, 56 different p...