Acute Cerebral Ischemia Reperfusion Injury Research Articles

Protective effect and underlying mechanism of muscone on acute cerebral ischemia-reperfusion injury in rats.

Musk is a widely used traditional Chinese medicine, which has resuscitation, activating blood, and disperse swelling effects. Musk is commonly used in the prevention of myocardial infarction and ischemic stroke, and muscone is its main active component. The effect and mechanism of muscone to improve the condition of ischemic stroke is not clear, accordingly, we verified its efficacy in ischemia-reperfused rats, and investigated its mechanism by PC12 and THP-1cells. A transient middle cerebral artery occlusion (tMCAO) rat model was established for in vivo experiments. 2,3,5-Triphenyl Tetrazolium Chloride (TTC) staining was used to calculate infarct rate. Neuroprotection and angiogenesis were assessed by Hematoxylin-eosin (HE) staining, nissl staining, immunofluorescence staining, and quantitative real-time PCR (qRT-PCR). Oxygen glucose deprivation-reperfusion (OGD/R) model of PC12cells was established for neuroprotection analysis, where CCK-8 assay was used to measure cell viability, flow cytometry and Hoechst 33258 staining were used to demonstrate apoptosis, and protein levels were detected by Western blot. For angiogenesis analysis, enzyme-linked immunosorbent assay (ELISA) and qRT-PCR were used to detect angiogenic factors expressed by THP-1. Cell viability assay, scratch wound assay, and tube formation assay were used to evaluate angiogenic effect of HUVECs treated with medium of THP-1. And the angiogenic pathway in HUVECs was detected by Western blot. According to the results, in cerebral ischemia-reperfusion rats, the infarct rate and tissue damage were significantly reduced by muscone, and the expression of neurotrophic factors and angiogenesis-related factors were all elevated. In OGD/R-PC12cell models, muscone could increase cell viability and inhibit apoptosis via Bax/Bcl-2/Caspase-3 pathway. In THP-1-mediated angiogenesis of HUVECs, muscone promoted the secretion of angiogenesis-related factors in THP-1 and thus indirectly promoted the proliferation, migration and tube formation of HUVECs, and then regulated phosphorylation of VEGFR2 and Akt in HUVECs. Our study indicated that muscone may be a potential neuroprotective and proangiogenic agent in cerebral ischemia.

Pre exposure to enriched environment alleviates brain injury after ischemia-reperfusion by inhibiting p38MAPK/STAT1 pathway

BackgroundStroke is one of the major diseases that endangers human health. It is widely reported that enriched environment (EE) can improve the neurological function in different brain injury models. Recently, relevant researches have indicated that MAPK pathway is closely related to the inflammatory response in nervous system related diseases. However, whether pre exposure to EE (EE pretreatment) has a preventive effect, and its mechanism are not clear. Therefore, this study aimed to determine the possible benefits and related mechanisms of EE in preventing brain injury after acute ischemia-reperfusion.MethodsAdult Sprague Dawley rats were kept in enriched or standardized environments for 21 days. Then the middle cerebral artery of rats was occluded for one hour and 30 min, and then reperfusion was performed. Then their neurological deficit score was evaluated. Cerebral edema, along with ELISA and protein quantities of p38MAPK, JNK, ERK, IL-1β, TNF-α, and co-localization of Iba1 were assessed. Changes in neuroinflammation and apoptosis were also detected in the penumbra cortex.ResultsOur research showed that EE pretreatment significantly alleviated acute cerebral ischemia-reperfusion injury in rats. Including the reduction of brain edema and apoptosis, and the improvement of neurological scores. In addition, the protein level of p38MAPK was significantly down regulated in EE pretreatment group, and the downstream protein STAT1 had the same trend. In addition, immunofluorescence results showed that Iba1 in EE pretreatment group decreased, the ELISA results showed that the classical proinflammatory cytokines increased significantly, while anti-inflammatory cytokines in EE pretreatment group increased, and the same results were obtained by Western blot analysis.ConclusionOn the whole, our research demonstrated that EE pretreatment can have a protective effect on the organism by inhibiting the p38 MAPK/STAT1 pathway. Thus, EE can be one of the most promising means of disease prevention. Secondly, p38MAPK/STAT1 pathway may be a latent target for the prevention of acute ischemic stroke.

TJ-M2010-5, a novel CNS drug candidate, attenuates acute cerebral ischemia-reperfusion injury through the MyD88/NF-κB and ERK pathway.

Background: Cerebral ischemia-reperfusion injury (CIRI) inevitably occurs after vascular recanalization treatment for ischemic stroke. The accompanying inflammatory cascades have a major impact on outcome and regeneration after ischemic stroke. Evidences have demonstrated that TLR/MyD88/NF-κB signaling contributes to CIRI. This study aimed to investigate the druggability of MyD88 in the central nervous system (CNS) and the neuroprotective and anti-neuroinflammatory effects of the MyD88 inhibitor TJ-M2010-5 on CIRI. Methods: A middle cerebral artery occlusion (MCAO) model was used to simulate CIRI in mice. BV-2 cells were stimulated with oxygen glucose deprivation/reoxygenation (OGD/R) or lipopolysaccharide, and SH-SY5Y cells were induced by OGD/R in vitro. Neurological deficit scores and cerebral infarction volumes were evaluated. Immunofluorescence staining was performed to measure neuronal damage and apoptosis in the brain. The anti-neuroinflammatory effect of TJ-M2010-5 was evaluated by analyzing the expression of inflammatory cytokines, activation of microglia, and infiltration of peripheral myeloid cells. The expression of proteins of the MyD88/NF-κB and ERK pathway was detected by Simple Western. The concentrations of TJ-M2010-5 in the blood and brain were analyzed by liquid chromatography-mass spectrometry. Results: The cerebral infarction volume decreased in mice treated with TJ-M2010-5, with the most prominent decrease being approximately 80% of the original infarction volume. Neuronal loss and apoptosis were reduced following TJ-M2010-5 treatment. TJ-M2010-5 inhibited the infiltration of peripheral myeloid cells and the activation of microglia. TJ-M2010-5 also downregulated the expression of inflammatory cytokines and inhibited the MyD88/NF-κB and ERK pathway. Furthermore, TJ-M2010-5 showed good blood-brain barrier permeability and no neurotoxicity. Conclusion: TJ-M2010-5 has an excellent therapeutic effect on CIRI as a novel CNS drug candidate by inhibiting excessive neuroinflammatory responses.

Eye acupuncture improves cerebral ischemia reperfusion injury by improving autophagy via ATF6 pathway in cerebral ischemia reperfusion injury rats

To explore the effect of eye acupuncture on autophagy and expressions of autophagy-related proteins Beclin1, LC3B, ATF6 and XBP1 in the infarction area of brain tissue in rats with acute cerebral ischemia-reperfusion injury (CIRI), so as to explore its mechanisms underlying improvement of CIRI. Male SD rats were randomly divided into sham operation, model and eye acupuncture groups (n=16 in each group). The CIRI model was prepared by occlusion of the middle cerebral artery. Eye acupuncture stimulation was applied to bilateral "Gan"(Liver), "Shangjiao"(Upper-energizer), "Xiajiao"(Lower-energizer) and "Shen"(Kidney) regions at 0, 12 and 24 h after CIRI, 30 min each time. The neurological deficit score was given by referring to Longa's method, and TTC staining used to determine the success of model replication. After the treatment, the pathological changes of the cerebral infarction area were observed under light microscope, and the autophagosomes were observed by electron microscope. The protein expression levels of LC3B, Beclin1, ATF6 and XBP1 in the infarction area of brain tissue were detected by Western blot. The immunoactivity of Beclin1 and the immunofluorescence density of ATF6 and XBP1 in the infarction area of brain tissue were determined by immunohistochemistry. The Longa's score, and the protein expression levels of LC3B, Beclin1, ATF6 and XBP1 and immunoactivity or immunofluorescence density of Beclin1, ATF6 and XBP1 were significantly higher in the model group than those in the sham operation group (P<0.01), and considerably lower in the eye acupuncture group than those in the model group (P<0.01). Under light microscope, the model group had typical ethmoidal reticular cerebral infarction, while the eye acupuncture group had significantly smaller areas and clearer edges. Under electron microscope, there were more autophagosomes in the cytoplasm of neurons in the model group, and fewer autophagosomes in the eye acupuncture group (in contrast to the model group). Eye acupuncture can improve the neurological function and mitigate cerebral injury in CIRI rats which may be associated with its function in inhibiting autophagy in the brain tissue by regulating ATF6 pathway.

The effect of mesenchymal stromal cells of various origins on mortality and neurologic deficit in acute ischemia-reperfusion in rats

Stroke is a global epidemic issue and the second leading cause of death in the world and in Ukraine. According to official statistics, every year 100-110 thousand Ukrainians suffer acute cerebrovascular disorders. One third of such patients are of working age, up to 50 % will have a disability, and only one in ten will fully return to full life. So far, promising experimental data on the treatment of neurological dysfunction using mesenchymal stromal cells (MSCs) have been obtained. The aim of study is to compare the effect of MSCs of different origins on mortality and neurologic deficit in rats with acute cerebral ischemia-reperfusion injury (CIRI). Materials and methods. Transient bilateral 20-minute occlusion of internal carotid arteries was modeled in male Wistar rats aged 4 months and animals were injected intravenously with MSCs derived from human umbilical cord Wharton's-jelly (hWJ-MSC), human and rat adipose tissue. Other groups of experimental animals were injected intravenously with rat fetal fibroblasts and cell lysate from hWJ-MSC. The last group of rats received Citicoline at a dose of 250 mg/kg as a reference drug. Control animals were injected intravenously with normal saline. The cerebroprotective effect of therapy was assessed by mortality and neurologic deficit in rats on the McGraw's stroke index score. Results. After 12 hours of observation in the crucial period in the development of experimental acute cerebrovascular disorders with the administration of hWJ-MSC, mortality was only 10 % against 45 % of animals in the control group. The use of rat fetal fibroblasts reduced the mortality of animals compare to the control group by an average of 25 %. CIRI in rats caused severe neurologic deficits: paralysis, paresis, ptosis, circling behavior. On the 7th day of observation in the control group of animals, the mean score on the McGrow's stroke index indicated severe neurological disorders. On the 14th day of observation in this group of animals there was no complete recovery of lost central nervous system functions. Compared with the control group of animals, all the treatment agents for acute CIRI (MSCs of various origins, MSC's lysate and Citicoline) contributed to a significant regression of neurologic deficit. Conclusions. Thus, transplantation of human Wharton's jelly-derived MSCs and rat fetal fibroblasts reduced mortality and alleviated neurological symptoms in rats with experimental ischemic stroke. hWJ-MSC, rat fetal fibroblasts, and rat adipose-derived MSCs reduced the incidence of neurological disorders better than Citicoline, which was accompanied by a regression of neurologic deficit dynamics on the 14th day of follow-up. The ability of stem cells of different origins to reduce neurologic deficit indicates the feasibility of their use in experimental acute cerebral ischemia.

Abstract TP286: The Study of the Role and Mechanism of TRPM7 in Acute Cerebral Ischemia-Reperfusion Injury

Although the NMDA receptor was identified to mediate excitotoxicity for ischemia-reperfusion (IR) injury, its antagonists have not shown clinical efficacy. Unrelated to NMDA, the transient receptor potential cation channel, subfamily M, member 7 (TRPM7) has been reported to cause intracellular Ca 2+ overloading, oxidative stress and neuronal apoptosis. We postulated that TRPM7 could be a target for treatment of IR injury. To investigate the potential neuroprotective role of TRPM7 in IR injury, 1) qRT-PCR, Western blot and immuno-staining were used to identify the expression and localization of TRPM7 in normal rat forebrain cortex and cultured primary cortical neurons; 2) the oxygen glucose deprivation and reperfusion (OGD/R) model of primary cortical neurons was used to detect changes in TRPM7 expression and study the effect of two TRPM7 ion channel inhibitors, carvacrol and 2-Aminoethoxydiphenyl borate (2-APB), on neurons in OGD/R; 3) the rat middle cerebral artery (MCAO/R) model was used to study TRPM7 involvement and the effect of its inhibitor during the course of IR. Our results showed that: 1) TRPM7 was expressed in normal neurons and astrocytes mainly as a cell membrane protein in the rat cortex; 2) TRPM7 expression was enhanced in OGD/R treated neurons; treatment with carvacrol or 2-APB reversed the overexpression of TRPM7 in OGD/R neurons and reduced the level of Ca 2+ overload in neuronal IR injury and neuronal apoptosis; 3) TRPM7 expression was also augmented in rat cortical tissues during MCAO/R and peaked between 24-48 hours during the reperfusion stage; 4) treatment with TRPM7 inhibitors reversed TRPM7 overexpression, alleviated neuronal apoptosis, and improved neurobehavioral scores in MCAO/R rats. Our conclusions are: 1) TRPM7 is expressed in normal rat forebrain cortex and functions mainly as a cell membrane protein in neurons and astrocytes; 2) TRPM7 is overexpressed during cerebral ischemia-reperfusion in rats; 3) TRPM7 ion channel inhibitors carvacrol and 2-APB can alleviate OGD/R injury in neurons in vitro as well as cortical damage by MCAO/R in rat forebrain; 4) the mechanism of TRPM7 on neuronal injury in OGD/R and MCAO/R can contribute to its role in neuronal apoptosis and Ca 2+ influx toxicity.

Protective effects of scopolamine and penehyclidine hydrochloride on acute cerebral ischemia-reperfusion injury after cardiopulmonary resuscitation and effects on cytokines

The objective of this study was to investigate the protective effects of scopolamine and penehyclidine hydrochloride on acute cerebral ischemia-reperfusion injury after cardiopulmonary resuscitation, and the effect on cytokine levels. Eighty patients with cardiac arrest admitted to our hospital from June 2011 to December 2015 were recruited and randomly divided into two groups (n=40 each). Following cardiopulmonary resuscitation, scopolamine was administered in the control group, whereas penehyclidine hydrochloride was administered in the observation group. After intervention, the following medical indicators were compared between the groups: Intracranial pressure, cerebral oxygen partial pressure, cerebral perfusion pressure, assessment of the balance of cerebral oxygen supply and demand, levels of neuron-specific enolase (NSE) and blood lactic acid, levels of oxidative stress markers, and levels of inflammatory-related factors. Additionally, the areas of brain tissue edema and National Institutes of Health Stroke Scale (NIHSS) scores before and after intervention were compared. Rescue success rates of the groups were recorded. After intervention, the following indicators were lower in the observation group than in the control group: Intracranial pressure (p<0.05), levels of NSE (p<0.05), levels of blood lactic acid (p<0.05), levels of malondialdehyde (p<0.05), and levels of interleukin 6 (IL-6), tumor necrosis factor-α, IL-1, and hs-CRP (p<0.05). However, the following indicators were higher in the observation group than in the control group: Cerebral oxygen partial pressure, cerebral perfusion pressure (p<0.05), levels of CaO2, CjvO2, and CERO2 (p>0.05), and levels of superoxide dismutase and glutathione peroxidase (p<0.05). Additionally, the areas of brain tissue edema after intervention were smaller in the observation group than those before intervention and those after intervention in the control group (p<0.05). Similarly, the NIHSS scores after intervention in the observation group were lower than those before intervention and those after intervention in the control group (p<0.05). Rescue success rate was significantly higher in observation group than in control group (p<0.05). In conclusion, administration of penehyclidine following cardiopulmonary resuscitation can effectively improve cerebral perfusion pressure, lower intracranial pressure, reduce brain tissue edema and inflammation, and improve neurological function.

Pre-treatment with spermine for acute cerebral ischemia/reperfusion injuries.

Acute cerebral ischemia/reperfusion injury (ACIR) rat model was used to investigate the effect of spermine on oxidative stress and apoptosis. Sixty healthy and clean male Sprague-Dawley rats were randomly divided into 3 groups: The sham-operated group (n=12), the model group (n=12) and the experiment group (n=36). The experiment group was further divided into 3 subgroups: The SPE-10 group (n=12), the SPE-25 group (n=12) and the SPE-50 group (n=12). Rats in the experimental sub-groups SPE-10, SPE-25 and SPE-50 were injected with 10, 25 and 50 mg/kg of spermine, respectively, one week before the establishment of rat models. Rats in the sham-operated and model groups were injected with 0.9% NaCl solution. We evaluated the effect of spermine on malondialdehyde (MDA) level and superoxide dismutase (SOD) activity using ELISA kits. Bax and Bcl-2 levels were measured using western blot analysis. Our results showed that after spermine injection, MDA levels markedly decreased, while SOD activity increased significantly. The variations in MDA levels and SOD activity were dose (spermine)-dependent. Bax protein levels increased significantly, while Bcl-2 levels decreased significantly after the onset of ACIR injuries. After spermine injection, there was a significant decrease in Bax levels. Bcl-2 levels in these rats markedly increased. The observed decline in Bax levels and the increase in Bcl-2 levels in the experimental groups were dose-dependent. We concluded that spermine protected nerve tissues in rats with ACIR by decreasing the MDA level, increasing SOD activity and modifying the balance between Bax and Bcl-2 proteins.

Hippo/MST1 signaling mediates microglial activation following acute cerebral ischemia–reperfusion injury

Cerebral ischemia–reperfusion injury is a major public health concern that causes high rates of disability and mortality in adults. Microglial activation plays a crucial role in ischemic stroke-induced alteration of the immune microenvironment. However, the mechanism underlying the triggering of microglial activation by ischemic stroke remains to be elucidated. Previously, we demonstrated that the protein kinase Hippo/MST1 plays an important role in oxidative stress-induced cell death in mammalian primary neurons and that the protein kinase c-Abl phosphorylates MST1 at Y433, which increases MST1 kinase activity. Microglial activation has been implicated as a secondary detrimental cellular response that contributes to neuronal cell death in ischemic stroke. Here, we are the first, to our knowledge, to demonstrate that MST1 mediates stroke-induced microglial activation by directly phosphorylating IκBα at residues S32 and S36. We further demonstrate that Src kinase functions upstream of MST1–IκB signaling during microglial activation. Specific deletion of MST1 in microglia mitigates stroke-induced brain injury. Therefore, we propose that Src–MST1–IκB signaling plays a critical role in stroke-induced microglial activation. Together with our previous work demonstrating that MST1 is important for oxidative stress-induced neuronal cell death, our results indicate that MST1 could represent a potent therapeutic target for ischemic stroke.

Orally administrated pterostilbene attenuates acute cerebral ischemia–reperfusion injury in a dose- and time-dependent manner in mice

Pterostilbene (3,5-dimethoxy-4-hydroxystilbene) is a component of blueberry. It has been reported that long-term treatment with blueberry has a neuroprotective effect. However, it has not been reported whether pterostilbene is effective in attenuating cerebral ischemia/reperfusion (I/R) injury. In the present study, focal cerebral ischemia was induced by middle cerebral artery occlusion for 90min followed by reperfusion. To observe the dose-dependent effect, pterostilbene (2.5–80mg/kg, ig) was administered for 3days before ischemia. To determine the time-dependent effect, pterostilbene (10mg/kg, ig) was administered as a single dose at 0, 1, or 3h after reperfusion. Twenty-four hours after I/R, pterostilbene dose-dependently improved neurological function, reduced brain infarct volume, and alleviated brain edema. The most effective dose was 10mg/kg; the therapeutic time window was within 1h after I/R and treatment immediately after reperfusion showed the best protective effect. The protective effect is further confirmed by the results that post-ischemic treatment with pterostilbene (10mg/kg) significantly improved motor function, alleviated blood brain barrier disruption, increased neurons survival and reduced cell apoptosis in cortical penumbra after cerebral I/R. We also found that pterostilbene (10mg/kg) significantly reversed the increased content of malondialdehyde and the decreased activity of superoxide dismutase in the ipsilateral hemisphere. Furthermore, pterostilbene decreased the oxidative stress markers 4-hydroxynonenal and 8-hydroxyguanosine positive cells in the cortical penumbra. All these findings indicate that pterostilbene dose- and time-dependently exerts a neuroprotective effect against acute cerebral I/R injury. This neuroprotective effect of pterostilbene may be associated with its inhibition of oxidative stress and subsequent neuronal apoptosis in the cortical penumbra.

Effect of DADLE on expressions of Bax and Bcl-2 proteins in rats with acute cerebral ischemia repeffusion

Objective To observe the expressions of Bax and Bcl-2 proteins in the brain tissue of the rats with acute cerebral ischemia reperfusion, and to explore the effect of DADLE, a delta opioid receptor ago- nist, on brain apoptotic pathways of acute cerebral ischemia reperfusion and the role of DADLE in the protection of brain. Methods 50 healthy female SD rats（ 180-220 g） were randomly divided into 5 groups： sham operation group （Sham, n = 10）： only exposed blood vessels without clipping; ischemia reperfusion group （I/R, n = 10）： modified two-vessel occlusion with hypotension to establish a model of cerebral ischemia, and reperfused 15 min after ischemia; DADLE treatment group was subdivided into group of 2 mg/kg（A, n = 10）, group of 3mg/kg （B, n= 10）, and group of 5 mg/kg （C, n-- 10）： on the basis of I/R group with reperfusion via external jugular vein injection of DADLE. Expressions of Bax and Bcl-2 proteins were detected by immunohistochemistry assay after the operation. Results The number of cells with Bax or Bcl-2 expression （20.38 ＋ 2.84 and 9.62 ＋ 2.10, P 〈 0.05 ）was significantly smaller in Sham group than in the other groups; In I/R group, the number of cells with Bax or Bcl-2 expression was 52.73 ＋ 6.08 and 19.49 ＋ 2.35 ; In DADLE treatment groups, the number of cells with Bax experssion was 43.71 -＋ 3.85 for group A, 37.92 ＋ 4.08 for group B, and 36.20 -＋ 3.94 for group C; the number of cells with Bel-2 expression was 28.74 _＋ 2.41,33.58 ＋ 2.16,and 34.96 ＋ 1.98, respectively. The ratio of Bel-2/Bax was significantly higher in Sham group than in I/R group （49.3% ＋ 3.4% vs. 35.8% ＋ 3.2%, P〈 0.05 ） ; and it was 66.4% ~ 6.8% in group A, 85.7% ：t 7.3% in group B, and 92.6% ＋ 6.9% in group C.Conelusions In rats with acute cerebral ischemia reperfusion injury, expressions of Bax and Bcl-2 proteins are obviously upregulated. DADLE can play a role in protection of brain by affecting the expression degree of Bax and Bcl-2 and thus lowering apoptosis. Bcl-2Bax ratio shows that the role and the dose is in a S-shape curve of dose-effect. Key words: DADLE ; Whole brain ischemia reperfusion; Bax; Bcl-2

Effects of Danhong mixture on water and calcium content in brain tissue of rats with acute cerebral ischemia reperfusion injury

Objective To Study the effects of Danhong mixture on water and calcium contents in brain tissue of rats with acute cerebral ischemia reperfusion injury.Methods 60 healthy SD rats were randomly divided into 6 groups,respectively,a sham operation group,a model control group,and 4 drug treatment groups.Models of cerebral ischemia reperfusion injury were established by 4 blood-vessel method,and administrated with different doses of Danhong mixture injection and compound salvia miltiorrhiza injection.Effects of Danhong mixture on water and calcium content in brain tissue of rats with acute cerebral ischemia and reperfusion injury were observed.Results Water and calcium content in brain tissue of rats with acute cerebral ischemia reperfusion injury were significantly decreased in Danhong mixture of high dose group (P＜0.01),(water was 75.84％ and calcium was 114.56 μg/g).Water and calcium content were also significantly decreased in Danhong mixture of medium dose group (P＜0.01),(water was 80.13％ and calcium was 132.35 μg/g)Water and calcium content showed decreasing tendency in Danhong mixture of low dose group,but without statistical significance,its numerical value was respectively 73.94％ and 106.32 μg/g.Conclusion Danhong mixture can reduce water and calcium content in brain tissue of rats with acute cerebral ischemia reperfusion injury,which indicated its neuroprotective effects. Key words: Danhong mixture; Cerebral ischemia reperfusion; Moisture content; Calcium content

Impact of DADLE on ERK signal transduction pathway in brain tissue in rats with acute global cerebral ischemia-reperfusion injury

Objective To investigate the impact of DADLE on ERK signal transduction pathway in brain tissue in rats with acute global cerebral ischemia-reperfusion injury and the association of DADLE with cerebral protection.Methods Fifty female Sprague-Dawley rats (180-220 g) were randomly divided into 5 groups.Ischemia/reperfusion group (I/R,n =10) was induced by bilateral common artery occlusion combined with hypotension for 15-minute global ischemia followed by 120-minute reperfusion ; sham group (Sham,n =10) exposed bilateral common artery without occlusion; DADLE group was subdivided into DADLE of 2 mg/kg group (A,n =10),DADLE of 3 mg/kg group (B,n =10),and DADLE of 5 mg/kg group (C,n =10).DADLE was applied through external jugular vein before reperfusion.Neuropathological changes in hippocampus CAI area were detected by Hernatoxylin-Eosin (H-E) staining; and the expressions of ERK and phosphorylated ERK (p-ERK) in brain tissue were determined by Western blot.Pesults Expression levels of ERK and p-ERK were significantly lower in sham group than in I/R group and DADLE group (P＜0.05).And the expression levels of ERK and p-ERK were increased in DADLE group as compared with I/R group (P＜0.05).Among the DADLE subgroups,the expression levels of ERK and p-ERK were higher in groups B and C than in group A,but no statistical difference was found between group B and group C.Conclusions DADLE activates ERK signal transduction pathway in brain tissue in rats with acute global cerebral ischemia-reperfusion injury and reduce neuronal cell damage.Tthe neuroprotective action of DADLE is dose-related and has ‘ceiling effect'. Key words: DADLE; Global cerebral ischemia and reperfusion; ERK

Effect of Shenxiong-Huayu capsule preconditioning on cell apoptosis and the expression of C-fos and C-jun in the hippocampal CA1 area of rats with cerebral ischemia reperfusion injury

[Abs Objective To observe the effect of Shenxiong-Huayu Capsule preconditioning on cell apoptosis and the expression of c-fos, c-jun in the hippocampal CA1 area of rats with acute cerebral ischemia reperfusion injury. Methods SD rats were divided into 3 groups by completely randomized method： sham operation group（n = 6）, ischemia reperfusion group （model group）, and Shenxiong-Huayu Capsule preconditioning group （preconditioning group）. The last two groups were divided randomly into 6 h, 24 h, 48 h and 72 h reperfusion subgroups （each n=6）. Intragastric administration for 7 days, once a day. Middle cerebral artery occlusion and reperfushion model was made by an intralurninal filament method. Cell apoptosis was detected by TUNEL method, the expression of c-fos, c-jun was observed by immunohistochemistry. Results ①The number of apoptosic cells at 6, 24, 48, 72 h in the hippocampal CA1 area of rats was respectively （11.17± 3.71, 39.83±5.67, 48.33 ±5.32 and 22.17±3.71） single/High power field in the model group, and was respectively （7.83±2.04, 15.00±3.58, 29.50±6.89 and 10.17 ±2.32） single/High power field in Shenxiong-Huayu Capsule preconditioning group. Compared with the model group, the number of apoptosic cells at each time point in thehippocampal CA1 area of rats was decreased in Shenxiong-Huayu Capsule preconditioning group （P〈 0.05 or P〈0.01 ） .②The number of c-los at 6、24、48、72 h in the hippocampal CA1 area of rats was respectively （14.50 ± 3.45,33.67 ± 1.63,42.33 ± 3.32 and 32.00 ± 2.90） single/High power field in the model group, and was respectively （10.17 ± 2.93, 21.50 ± 2.43, 30.83 ± 3.76 and 25.17 ± 5.27） single/High power field in Shenxiong-Huayu Capsule preconditioning group.The number of c-jun at 6, 24, 48, 72 h in the hippocampal CA1 area of rats was respectively （15.50±4.19, 22.83±5.64, 33.10±4.19 and 14.67±3.08） single/High power field in the model group, and was respectively （9.67 ± 3.63, 15.67 ± 2.73, 21.26 ± 3.63 and 9.33 ± 3.61） single/High power field in Shenxiong-Huayu Capsule preconditioning group.Compared with the model group, the expression of c-los, c-jun at each time point in the hippocampal CAI area of rats was decreased in Shenxiong-Huayu Capsule preconditioning group （P〈0.05 or P〈0.01 ） . Conclusion Shenxiong-Huayu capsule can inhibit apoptosis by restraining the expression of c-fos, c-jun and relive cerebral ischemia reperfusion injury in rats. Key words: Cerebral ischemia reperfusion injury; Cell apoptosis; c-los; c-jun; Shenxiong-Huayu capsule

Effect of hyperbaric oxygen preconditioning on expression of Nogo mRNA,Nogo-A and NgR protein in the cerebral cortex after acute global cerebral ischemia-reperfusion injury in aged rats

Objective To investigate the effect of hyperbaric oxygen preconditioning(HOP)on expression of Nogo mRNA,Nogo-A and NgR protein in the cerebral cortex after acute global cerebral ischemia-reperfusion(I/R)injury in aged rats.Methods Forty-two aged male SD rats aged 14 months weighing 480-530 g were randomly divided into 4 groups: group Ⅰ control(group C,n = 6);group Ⅱ HOP(n = 12);group Ⅲ cerebral I/R(n = 12)and group Ⅳ HOP+I/R(n = 12).Global cerebral I/R was induced by 4-vesasl occlusion method described by Pulsinelli.Bilateral vertebral arteries were permanently occluded by cauterization,aad bilateral carotid arteries were occluded for 10 min.Successful induction of cerebral ischemia was confirmed by bilateral dilated pupils unresponsive to light and EEG changes.In group Ⅱ and Ⅳ the animals received 1 h hyperbaric oxyyen at 2 absolute atmospheric pressure per day for 5 consecutive days.In group Ⅳ(HOP+I/R)global cerebral I/R was induced at 24 h after last hyperbaric oxyyen treatment.Six animals in each group were killed at24 h after I/R in each group and their brains were removed for detection of Nogo mRNA expression(by Q-PCR)and expression of Nago-A and NgR protein(by Western blot)in cerebral cortex.Cognitive function was determined at 5 days after cerebral ischemia using Morris Water Maze in the other animals.Results The learning ability and memory were significantly decreased while the expression of Nogo mRNA and Nogo-A protein was significantly increased in group Ⅲ(I/R)and Ⅳ(HOP+I/R)as compared with control group.HOP significantly ameliorated the cerebral I/R-induced decrease in learning ability and memory and increase in Nogo mRNA and Nogo-A protein expression.Conclusion Hyperbaric oxyyen preconditioning can ameliorate acute global cerebral I/R injury-induced cognitive impairment by decreasing Nogo mRNA and Nogo-A protein expression in the brain. Key words: Hyperbaric oxygenation; Ischemic preconditioning; Reperfusion injury; Brain; Aged; Nogo; Nogo-A; NgR

Effect of scalp acupuncture on inflammatory response in rats with acute cerebral ischemia-reperfusion injury

To explore the mechanism of scalp acupuncture (SA) in treating cerebral ischemia. Sixty SD rats with middle cerebral artery occlusion (MCAO) were randomized into untreated group and SA-treated group, with another group of 10 SD rats without artery occlusion as sham-operated control. Neurological severity score (NSS), hemetoxylin and eosin (HE) staining and enzyme-linked immunosorbent assay were applied to observing the changes of neurofunctional defect, inflammatory infiltration in cerebral tissue and content of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and IL-10 at 24, 48 and 72 h after ischemia-reperfusion. There existed significant difference in NSS between SA-treated group and untreated group (P<0.01), especially at 72 h after ischemia-reperfusion. The HE staining results of ischemic cerebral tissue showed an apparent reduction of inflamed lesions in SA-treated group as compared with the untreated group (P<0.01), especially at 72 h after ischemia-reperfusion. The content of TNF-alpha and IL-1beta at each phase point after ischemia-reperfusion in SA-treated was decreased as compared with that in the untreated group, and there were differences between SA-treated group and untreated group at 72 h after ischemia-reperfusion (P<0.01). An apparent increase was observed in IL-10 between SA-treated group and untreated group at each phase point after ischemia-reperfusion, and there were significant differences between SA-treated group and untreated group at 48 and 72 h after ischemia-reperfusion (P<0.05, P<0.01). Scalp acupuncture can improve neurofunctional rehabilitation, suppress leukocyte infiltration, decrease the content of TNF-alpha and IL-1beta within a certain range and enhance IL-10 expression so as to suppress cytokines-mediated inflammatory reaction and attenuate cerebral ischemia-reperfusion injury.