Microvessel changes in the gerbil hippocampus after cerebral ischemia and reperfusion by Buyang Huanwu decoction pretreatment

Buyang Huanwu decoction （BYHWD） is a classic recipe for the prevention and treatment of ischemic cerebrovascular disease. Gerbils were pretreated with BYHWD, and then subjected to cerebral ischemia and reperfusion. Microvascular changes were determined with laser Doppler monitoring, tannic acid-ferric chloride mordant, and electron microscopy. Results showed that BYHWD pretreatment could enhance the function of hippocampal microvessels, prevent injury, and increase microvasular density and microvasular area density. Thus, these results suggest that BYHWD pretreatment could prevent microvascular occlusion, enhance the capacity of microvascular reperfusion, increase cerebral blood flow, and inhibit neuronal damage, and may be an effective therapy against brain ischemic injury.

Expression of neurocan mRNA and ultrastructure of brain tissue after cerebral ischemia and reperfusion in stroke-prone renovascular hypertensive rats treated by electroacupuncture

We established a stroke-prone renovascular hypertensive rat model by bilateral constriction of the renal artery with sliver loop clips. After ten weeks, middle cerebral artery occlusion was induced for 2 hours. The rats then received electro-acupuncture at Baihui （DU 20） and Dazhui （DU 14） after onset of ischemia for 30 days. In situ hybridization study showed that electroacupuncture significantly reduced the number of neurocan mRNA-positive cells in the ischemic penumbra and hippocampal tissues of rats. Electron microscopy results demonstrated that the structure of neurons and blood vessels in the ischemic tissues were restored with electroacupuncture. Overall, these data suggest that electroacupuncture may protect neurons against ischemic reperfusion injury in stroke-prone renovascular hypertensive rats, which may be regulated by downregulation of expression of nerve inhibitory factor neurocan mRNA.

Effects of L-Tetrahydropalmatine on the Expressions of bcl-2 and bax in Rat after Acute Global Cerebral Ischemia and Reperfusion

To investigate the effects of L-Tetrahydropalmatine (L-THP) on the expressions of bcl-2, bax and neuronal apoptosis after cerebral ischemia and reperfusion, 60 Wistars rats were randomly divided into 3 groups: sham-operation group (group S, n = 20), ischemic-reperfusion group treated with saline (group I, n=20) and ischemia-reperfusion group treated with L-THP (group T, n=20) .The rat model of global cerebral ischemia and reperfusion was induced by Pulsinelli's four-vessel occlusion method. The expression of bcl-2 and bax mRNA was detected by in situ hybridization and reverse transcriptional polymerase chain reaction (RT-PCR). The number of apoptotic neurons was examined by terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) method. Compared with group S, the expression of bcl-2 and bax mRNA in group I was increased significantly (P<0.01), and the number of apoptotic neurons increased either (P< 0.01). After L-THP treatment, the expression of bcl-2 mRNA was up-regulated (P<0.01) and that of bax mRNA was down-regulated (P<0.01); the number of apoptotic neurons was decreased (P<0.01). Our results indicated that bcl-2 may suppress apoptosis and bax promote apoptosis after cerebral ischemia and reperfusion. L-THP could ameliorate cerebral ischemia and reperfusion damage by reducing the apoptosis through regulating bcl-2 and bax.

Expression of cyclin-dependent protein kinase 5 in the hippocampus of vascular dementia mice after cerebral ischemia and reperfusion

BACKGROUND： The p25-activated cyclin-dependent protein kinase 5 （Cdk5） may induce neuronal cell death and cause the development of dementia following cerebral ischemia and reperfusion. OBJECTIVE： To observe changes in the expression of Cdk5 and p25 in hippocampal tissue of vascular dementia mice at different time points following cerebral ischemia and reperfusion. DESIGN, TIME AND SETTING： A randomized, controlled animal experiment was performed in the clinical trial center of Hebei Provincial People＇s Hospital between September 2007 and October 2008. MATERIALS： Cdk5 rabbit anti-mouse polyclonal antibody, p35 rabbit anti-mouse polyclonal antibody, and β-actin mouse monoclonal antibody were purchased from Santa Cruz Biotechnology, Inc., USA; horseradish peroxidase-labeled goat anti-rabbit IgG and horseradish peroxidase-labeled goat anti-mice IgG were offered by Beijing Zhongshan Geldenbridye Biotechnology Co.,Ltd., China; the protein quantitative kit was produced by Applygen Gene Technology Corp., Beijing, China; cDNA reverse transcription and PCR amplification reagents were products of TianGen＆ Biotech （Beijing） Co.,Ltd., China. METHODS： One hundred and sixty male Kunming mice were randomly divided into two groups： a sham-operated group （n = 65） and a model group （n = 95）. Vascular dementia was induced with three periods of transient ischemia and reperfusion of the bilateral common carotid arteries. In the sham-operated group, the bilateral common carotid arteries were not blocked. MAIN OUTCOME MEASURES： Behavioral tests were done at four and six weeks post surgery. Pathological changes in the hippocampal CA1 region were observed with hematoxylin-eosin staining Cdk5 mRNA expression was examined by RT-PCR, and Western blots were used to evaluate Cdk5 and p25 expression. Learning and memory performance were assayed using the Morris water maze. RESULTS： Vascular dementia reduced learning and memory performance at 4 and 6 weeks post surgery. Vascular dementia also caused severe, time-dependent neuronal damage and death in the hippocampal CA1 region. Dementia induction also increased mRNA and protein expression of Cdk5 and p25 at both 4 and 6 weeks after surgery. CONCLUSION： Cdk5/p25 is involved in the development of vascular dementia in mice following cerebral ischemia and reperfusion.

Correlation between expression of two transforming growth factor-beta 1 receptors and microvascular density in a rat model of cerebral ischemia and reperfusion injury

The effects of transforming growth factor-β1 （TGF-β1） are currently controversial. Whether TGF-β1 promotes or inhibits revascularization under different conditions remains poorly understood. Based on previous studies, the current experiment established rat models of cerebral ischemia and reperfusion injury （IRI）, and demonstrated that pathological and functional damage was also increased after IRI. The most serious damage was observed at 3 days after reperfusion, at which time microvascular density fell to its lowest level. Soon afterwards, microvascular density increased, new collateral circulation was gradually established at 4 to 7 days after reperfusion, and pathological damage and neurological deficits were improved. TGF-β1, activin receptor-like kinase 5 （ALK5） mRNA and protein expression levels increased gradually over time. In contrast, ALK1 mRNA and protein expression decreased over the same period. A significant negative correlation was detected between microvascular density and expression of the ALK5 gene transcript. There was no correlation between microvascular density and ALK1 gene transcriptional expression following cerebral IRI in a rat model. These findings suggest that ALK5, rather than ALK1, is the critical receptor in the TGF-β1 signal pathways after cerebral IRI.

Protective effects of combination of Xuesaitong and aspirin on cerebral ischemia and reperfusion injury in rats

Objective：To investigate the protective effects of the combination of Xuesaitong（XST）and aspirin on cerebral ischemia and reperfusion injury（CIRI）in rats,and further explore the underlying mechanisms.Methods：A total of 150 male Sprague-Dawley（SD）rats were randomly divided into five groups with 30rats in each group：sham group,middle cerebral artery occlusion/reperfusion（MCAO/R）model group,XST group,aspirin group,and XST＋aspirin group.Rats were pretreated with XST,aspirin,or XST＋aspirin for7 d.One hour after the last administration,a model of CIRI was induced by MCAO/R.Neurological deficits were assessed using Longa’s five-point scale.Cerebral edema was detected by the measurement of brain water content.The volume of cerebral infarction was determined by 2,3,5-triphenyltetrazolium chloride（TTC）staining.The activities of superoxide dismutase（SOD）,catalase（CAT）,and glutathione peroxidase（GSH-Px）,as well as levels of malonaldehyde（MDA）were detected by commercial kits.Enzyme-linked immunosorbent assay（ELISA）was used to determine the levels of interleukin-1（）,interleukin-4（IL-4）,interleukin-6（IL-6）,interleukin-10（IL-10）,tumor necrosis factor-alphamonocyte chemotactic protein 1（MCP-1）,and kynurenine in serum,cerebral cortex,and hippocampus of MCAO/R rats.The protein expression of nuclear factor erythroid 2-related factor（Nrf2）,heme oxygenase-1（HO-1）,I-kappa B alpha（IκBα）,and nuclear factor kappa B（B）/p65 in the cortex were analyzed by western blotting.Results：Treatment of XST,aspirin,and XST＋aspirin significantly alleviated the neurological deficits,cerebral edema,and cerebral infarct volume induced by MCAO/R.Treatment of XST,aspirin,and XST＋aspirin also reduced MDA,,MCP-1,and kynurenine levels,and increased SOD,CAT,GSH-Px,IL-4,and IL-10 levels in serum,cerebral cortex,and hippocampus of MCAO/R rats.Furthermore,treatment of XST,aspirin,and XST＋aspirin decreased the expression of nuclearB/p65 and increased the expression of IκBα,nuclear Nrf2,and HO-1.Importantly,the combination of XST and aspirin enhanced the protective effects of XST or aspirin treatment alone on CIRI in rats.Conclusion：The combination of XST and aspirin significantly inhibited oxidative stress and inflammation in serum,cerebral cortex,and hippocampus of MCAO/R rats.The combination of XST and aspirin exerted more protective effects than XST or aspirin treatment alone.The combination of XST and aspirin might provide the synergistic therapeutic effects on CIRI,and deserve further clinical investigation.

Expression of netrin-1 and its receptors, deleted in colorectal cancer and uncoordinated locomotion-5 homolog B, in rat brain following focal cerebral ischemia reperfusion injury

Netrin-1 is currently one of the most highly studied axon guidance factors. Netrin-1 is widely expressed in the embryonic central nervous system, and together with the deleted in colorectal cancer and uncoordinated locomotion-5 homolog B receptors, netrin-1 plays a guiding role in the construction of neural conduction pathways and the directional migration of neuronal cells. In this study, we established a rat middle cerebral artery ischemia reperfusion model using the intraluminal thread technique. Immunofluorescence microscopy showed that the expression of netrin-1 and deleted in colorectal cancer in the ischemic penumbra was upregulated at 1 day after reperfusion, reached a peak at 14 days, and decreased at 21 days. There was no obvious change in the expression of uncoordinated locomotion-5 homolog B during this time period. Double immunofluorescence labeling revealed that netrin-1 was expressed in neuronal cells and around small vessels, but not in astrocytes and microglia, while deleted in colorectal cancer was localized in the cell membranes and protrusions of neurons and astrocytes. Our experimental findings indicate that netrin-1 may be involved in post-ischemic repair and neuronal protection via deleted in colorectal cancer receptors.

MicroRNA-670 aggravates cerebral ischemia/reperfusion injury via the Yap pathway

Apoptosis is an important programmed cell death process involved in ischemia/reperfusion injury.MicroRNAs are considered to play an important role in the molecular mechanism underlying the regulation of cerebral ischemia and reperfusion injury.However,whether miR-670 can regulate cell growth and death in cerebral ischemia/reperfusion and the underlying mechanism are poorly understood.In this study,we established mouse models of transient middle artery occlusion and Neuro 2a cell models of oxygen-glucose deprivation and reoxygenation to investigate the potential molecular mechanism by which miR-670 exhibits its effects during cerebral ischemia/reperfusion injury both in vitro and in vivo.Our results showed that after ischemia/reperfusion injury,miR-670 expression was obviously increased.After miR-670 expression was inhibited with an miR-670 antagomir,cerebral ischemia/reperfusion injury-induced neuronal death was obviously reduced.When miR-670 overexpression was induced by an miR-670 agomir,neuronal apoptosis was increased.In addition,we also found that miR-670 could promote Yap degradation via phosphorylation and worsen neuronal apoptosis and neurological deficits.Inhibition of miR-670 reduced neurological impairments after cerebral ischemia/reperfusion injury.These results suggest that microRNA-670 aggravates cerebral ischemia/reperfusion injury through the Yap pathway,which may be a potential target for treatment of cerebral ischemia/reperfusion injury.The present study was approved by the Institutional Animal Care and Use Committee of China Medical University on February 27,2017(IRB No.2017PS035K).

Protective mechanisms of picroside Ⅱ on aquaporin-4 expression in a rat model of cerebral ischemia/reperfusion injury

BACKGROUND： Aquaporin-4 （AQP-4） over-expression following cerebral ischemia results in cerebral edema. Picroside Ⅱ has been shown to exhibit a neuroprotective effect on neuronal apoptosis. However, few reports have addressed the neuroprotective mechanisms and therapeutic times following cerebral ischemic reperfusion injury. OBJECTIVE： To explore the neuroprotective effects and ideal treatment window for picroside Ⅱ treatment of middle cerebral artery occlusion and reperfusion injury in rats. DESIGN, TIME AND SETTING; A randomized, controlled, animal experiment was performed at Institute of Cerebrovascular Diseases, Qingdao University Medical College from September 2008 to May 2009. MATERIALS： Picroside II was purchased from Tianjin Kuiqing Medical Technology, China. METHODS： A total of 165 adult, healthy, male, Wistar rats were randomly assigned to sham-surgery （n = 15）, model （n = 75）, and treatment groups （n = 75）. Rats in the model and treatment groups underwent middle cerebral artery occlusion and reperfusion through the use of an intraluminal monofilament suture on the left external-internal carotid artery, The treatment group was injected with 1.0% picroside Ⅱ （10 mg/kg） into the tail vein, and the model and sham-surgery groups were injected with 0.1 mol/L phosphate buffered saline （250 μL）. MAIN OUTCOME MEASURES： Neurological functional scores were evaluated using the Longa＇s method; cerebral infarction volume was detected through the use of tetrazolium chlodde staining; cellular apoptosis was determined through the use of the in situ end-labeling method; aquaporin-4 expression was measured using fluorescence labeling analysis and reverse transcription polymerase chain reaction technique. RESULTS： At 0.5 hour following cerebral ischemic injury, neurological functional scores were low, and a small infarction focus was detected in the ischemic cortex of the model group. Along with prolonged ischemia and an increased number of apoptosis-positive cells, AQP-4 mRNA and protein expression was increased. At 1-2 hours after ischemia, neurological scores and infarction sizes were significantly increased in the model group. Apoptotic-positive cells were widespread in the ipsilateral cortex and stdatum. In addition, AQP-4 mRNA and protein expression levels were increased. Picroside II treatment significantly decreased neurological scores and infarction volume, and reduced AQP-4 mRNA and protein expression levels compared with the model group （P 〈 0.05 or P 〈 0.01）. At 1 hour after ischemia, the therapeutic effect of picroside Ⅱ was notable （P 〈 0.01）. CONCLUSION： Picroside Ⅱ played a protective role in cerebral ischemic reperfusion injury by inhibiting apoptosis and regulating AQP-4 expression. The best therapeutic time window was 1 hour after cerebral ischemic reperfusion.

Differential expression of 114 oxidative stress-related genes in peripheral blood mononuclear cells of acute cerebral infarction patients A gene microarray experiment

Previous studies have focused on the analysis of single or several function-related genes in oxidative stress; however, little information is available regarding altered expression of oxidative stress-related genes in the process of ischemia-reperfusion injury from microarray experiments. The aim of the present study was to investigate the changes in cell oxidative stress- and toxicity-related gene expression utilizing microarray screening in patients with acute cerebral infarction during cerebral ischemia-reperfusion injury. Of the included 114 genes, expression was significantly upregulated in eight genes, including three heat shock protein-related genes, one oxidative and metabolic stress-related gene, one cell growth arrest/senescence related gene, two apoptosis signal-related genes, and one DNA damage and repair related gene. Expression was significantly downregulated in four genes, including one cell proliferation/cancer related gene, two oxidative and metabolic stress-related genes and one DNA damage and repair related gene. The results demonstrated that cerebral ischemia-reperfusion injury in patients with acute cerebral infarction was affected by many genes including oxidative stress-, heat shock-, DNA damage and repair-, and apoptosis signal-related genes. Therefore, it could be suggested that cerebral ischemia-reperfusion injury may be subjected to complex genetic regulation mechanisms.