Tetramethylpyrazine combined with bone marrow mesenchymal stem cell transplantation inhibits neuronal apoptosis in rats with cerebral ischemia

ATM: To investigate the effects of tetramethylpyrazine (TMP) combined with bone marrow mesenchymal stem cells (BMSCs) on neuronal apoptosis, and Bcl-2 and Bax expression in rats with cerebral ischemia. METHODS: The BMSCs were isolated by the whole bone marrow adherent method and cultured, and those in the 3rd passage were used for tail-vein transplantation. The rats were subjected to right middle cerebral artery occlusion (MCAO) using suture method, and the rats except sham group were randomly divided into model group, BMSCs (1×10⁹ cells/L) group, TMP (40 mg/kg) group and combination (TMP+BMSCs) group with 12 rats in each group. Neurological function was evaluated by modified neurological severity scoring (mNSS) on 1 d, 7 d and 14 d after cerebral ischemia. Toluidine blue staining was performed to detect cerebral infarct volume, HE staining was used to observe brain histopathological change, neuronal apoptosis was observed by TUNEL staining, and the mRNA and protein expression of Bcl-2 and Bax was detected by real-time fluorescence quantitative PCR and Western blot at 14 d after cerebral ischemia. RESULTS: Compared with BMSCs group and TMP group, TMP combined with BMSCs significantly reduced the score of mNSS (P<0.01) and the infarct volume (P<0.01), alleviated the pathological damage in the peripheral area of cerebral ischemia, decreased the number of TUNEL positive cells (P<0.01), increased the expression of Bcl-2 and decreased the expression of Bax at mRNA and protein levels (P<0.01).CONCLUSION: Tetramethylpyrazine combined with transplantation of BMSCs improves the functional recovery, reduces the infarct volume, relieves the ischemic injury of the brain tissue, and attenuates neuronal apoptosis in the rats with cerebral ischemia. The mechanism may be related to regulating the expression of Bcl-2 and Bax.     

Migration of enhanced green fluorescent protein labeled bone marrow after transplantation into rat cerebral infarct

AIM:To investigate the role of SDF-1α in migrating of bone marrow stromal cells to the injured areas.METHODS:Ischemic brain lesion model was created in rats by permanent middle cerebral artery occlusion (MCAO).48 SD rats were divided randomly into 2 groups.Group 1:phosphate buffered saline (PBS 1 mL) for control (n=25); Group 2:BMSCs (2×106) were injected intravenously at 24 h after MCAO (n=24).After propagated in BMSCs, Ad5/F35 GFP (green fluorescent protein) was infected to BMSCs.The expression of SDF-1α (stromal cell-derived factor-1α) mRNA in the penrumbral tissue was assayed by real-time quantitative PCR.The expression of CXCR4 on MSCs was detected by flow cytometry.Confocal microscopy was used to detect the GFP-labeled MSCs migration.RESULTS:Ad5/F35 GFP signals was observed in almost infected BMSCs.The expressions of SDF-1α mRNA in the thalamus and hippocampus of the ischemic brains were peaked at 3rd day after stroke, followed by a decrease at 14th day post-ischemia.The expression of SDF-1α mRNA in the cortex of the ischemic brains was peaked at 7th day post-ischemia, still at high level at 14th day post-ischemia.The median percentage of surface CXCR4 expression in BMSCs was 14%.GFP labeled BMSCs were detected in the origination of the middle cerebral artery (olfactory area) at 6 h, after 3 days in the prenumbra tissue such as thalamus, and in the cortex more labeled cells were found after 14 d post-ischemia.CONCLUSION:BMSCs can pass through the blood brain barrier of ischemic rats.Its mechanism might be associated with the expression of SDF-1α in the ischemic brain.     

Astrocyte proliferation in the rat hippocampus after middle cerebral artery occlusion

AIM: To study rat astrocyte proliferation in ipsilateral hippocampus following focal cerebral ischemia. METHODS: Ischemia was induced by temporary middle cerebral artery occlusion (MCAO). In hippocampus of rats at 3, 7 and 30 days after MCAO, the numbers and anatomic distribution of glial fibrillary acidic protein (GFAP) were detected by immunohistochemistry. The protein expression of GFAP and proliferating cell nuclear antigen (PCNA) in the ipsilateral hippocampus were analyzed by Western blot analysis. RESULTS: Astrocytes appeared hypertrophic, with increased process thickness and numbers at 7 days after MCAO, and the highest density of astrocytes were seen at 30 days in the CA1, CA2 regions of the ipsilateral hippocampus. Western blot analysis revealed that GFAP levels were normal at 3 days, but increased by 7 days and remained elevation at 30 days. Western blot analysis of PCNA protein also revealed identified upregulation PCNA at 3 days after MCAO and the expression peaked at 7 days. CONCLUSION: This study demonstrates that focal cerebral ischemia in the rat results in a rapid response, a process often referred to as reactive astrogliosis or glial scarring, from resident astrocytes of the ipsilateral hippocampus to the side of ischemia.     

Effect of naoluo xintong on expression of Fas,Fas L in hippocampus CA1 area and Fas mRNA in the cortex of frontal or parietal lobe after local cerebral ischemia/reperfusion in MCAO rats

AIM: To investigate the effets of naoluo xintong on the expression of Fas, FasL protein in hippocampus CA1 area and Fas mRNA in the cortex of frontal or parietal lobe after local cerebral ischemia/reperfusion in MCAO rats. METHODS: The local cerebral ischemia /reperfusion model was established by intraluminal thread occlusion of the middle cerebral arteries (MCAO), the middle cerebral arteries of rats were occluded for 2 hours and reperfused for 1, 3 and 7 days. The animals were divided into pseudo surgery group(sham group), model group, Yiqi group, Huoxue group and naoluo xintong group. Using the techniques of immuno-histochemical staining and in situ hybridization, the expression of Fas and FasL was observed in hippocampus CA1 area, the expression of Fas mRNA was also observed in the cortex of frontal and parietal lobe. RESULTS: A value of Fas and FasL protein expression or A value and positive unit of Fas mRNA expression in control group were higher than those in sham in hippocampus CA1 area, the cortex of frontal or parietal lobe after local cerebral ischemia/reperfusion in MCAO rats (P<0.01). A value and/or positive unit of their expression in naoluo xintong group were lower than those in control group (P<0.05 or P<0.01). A value and/or positive unit of their expression in Yiqi and Huoxue groups were higher than those in naoluo xintong group for 3 and/or 7 days (P<0.05 or P<0.01). CONCLUSION: naoluo xintong could resist neuron apoptosis, alleviate pathologic injury after local cerebral ischemia/reperfusion in MCAO rats by inhibiting the expression of Fas, FasL protein and Fas mRNA.     

Effect of ginsenoside Rg1 on activity and protein expression of NOS in rats with cerebral ischemic reperfusion injury

AIM: To observe the neuroprotective effects of ginsenoside Rg1 on focal cerebral ischemia reperfusion (I/R) injury in rats. METHODS: SD rats were applied to right middle cerebral artery occlusion (MCAO) for 2 h followed by 24 h of reperfusion. The rats were randomly divided into sham-operation group, I/R group and ginsenoside Rg1 pretreatment groups. The rats in ginsenoside Rg1 pretreatment groups were pretreated with ginsenoside Rg1 at doses of 10, 20 or 40 mg/kg once a day for 7 days and then subject to MCAO. The neurological deficit score was measured by Longa's method. The neurons were observed with Nissel staining. The nitric oxide (NO) content, the activity of nitric oxide synthase (NOS) and inducible NOS (iNOS) in the brain tissues were determined. The expression of neuronal NOS(nNOS) and iNOS was detected by Western blotting. RESULTS: Compared with sham-operation group, ginsenoside Rg1 significantly reduced the neurological deficit score and increased the neuron number in the hippocampus. The activity of NOS and iNOS, and NO content were decreased. Ginsenoside Rg1 also down-regulated the expression of nNOS and iNOS. CONCLUSION: Ginsenoside Rg1 has protective effect on the brain during cerebral I/R injury in rats. The mechanism may be related to reducing the content of NO and the activiy of NOS dose-dependently.     

Combination of taurine and diazepam has neuroprotective effect on focal cerebral ischemia-reperfusion in rats

AIM: To observe the neuroprotective effect of combined treatment with taurine and diazepam against focal cerebral ischemia-reperfusion in rats. METHODS: Sixty male Sprague-Dawley rats were randomly divided into five groups: sham-operation group, vehicle group, taurine group (200 mg/kg, ip), diazepam group (10 mg/kg, ip) and combined treatment group (taurine 100 mg/kg+diazepam 5 mg/kg). Focal cerebral ischemia was induced by the method of middle cerebral artery occlusion (MCAO) in rats, and reperfusion was emerged by removing the thread 2 h later. The drugs were administered respectively at the time of reperfusion, and subsequently repeated once 12 h later. The animals in vehicle group were intraperitoneally injected with isodose normal saline. The neurological deficit score, the brain water content and cerebral infarction were measured 48 h after MCAO. Other 5 group animals of focal cerebral ischemia-reperfusion (n=16 in each group) were set up as mentioned above and accepted treatments 10 h after reperfusion, likewise repeated once 12 h later. Twelve animals in each group were adopted the same management as the previous 5 groups at 48 h after MCAO. The remained 4 animals in each group were sacrificed until two weeks after MCAO to observe the histopathological changes by nissl staining. RESULTS: Compared to vehicle group, the animals in combined treatment group at 2 h or 12 h after MCAO both decreased the neurological deficit score, reduced the brain water content and infarct volume (P<0.01 or P<0.05). The combined treatment significantly alleviated the neurological necrosis as well. The neuroprotective effect of the combined treatment was superior to that of using taurine or diazepam alone. CONCLUSION: These results suggest that combination of taurine and diazepam treatment has a coordinate neuroprotective effect on both the acute and chronic brain damage of focal cerebral ischemia-reperfusion.     

Relationship between morphologic changes in neuron or neuroglial cells and expression of TNF-α and c-Myc in cortex after focal cerebral ischemia/reperfusion in MCAO rats

AIM: To investigate the relationship between morphologic changes in neuron or neuroglial cells and expression of tumor necrosis factor α (TNF-α) and c-Myc in cortex after focal cerebral ischemia/reperfusion in MCAO rats. METHODS: The focal cerebral ischemia/reperfusion model was established by intraluminal thread occlusion of the middle cerebral artery (MCAO). The middle cerebral arteries of rats were occluded for 2 hours and reperfused for 1, 3 and 7 days. Using the techniques of immunohistochemical staining and optical microscopy, the morphologic changes in neuron or neuroglial cells were observed in the cortex of frontal or parietal lobe; the cell types which dynamicaly expressed TNF-α, c-Myc in the different period were also observed. RESULTS: The degeneration or necrosis of neuron or neuroglial cells were observed at the center of infarction, it was very serious at 3 d after reperfussion. Astrocyte and microglial cell proliferation were observed at the broder of infarction. TNF-α and c-Myc positive cells, most of which were astrocytes and microglial cells, increased significantly at 3 d after reperfusion. CONCLUSION: TNF-αand c-Myc may play an important role in the regulation of neuron or neuroglial cells after focal cerebral ischemia with reperfusion.     

Effects of Astragalus polysaccharide on expression of HSP70, PKB and P53 in rat cerebral cortex with cerebral ischemia and reperfusion

AIM:To explore the molecular effects of Astragalus polysaccharide(AP) on improving nervous functions and preventing neuronal apoptosis in rat cerebral cortex with cerebral ischemia and reperfusion. METHODS:One hundred and twenty male Wister rats were randomly divided into sham operation group(SOG), model groups(MG-1 d, 3 d and 7 d), low-dose AP treatment groups(L-APTG-1 d, 3 d and 7 d), and high-dose AP treatment groups(H-APTG-1 d, 3 d and 7 d). The right middle cerebral artery of the rats in MG and AGTG was intercepted by operation to induce ischemic brain injury. The rats in L-APTG and H-APTG were treated with AP at the doses of 5 mg/kg and 15 mg/kg by intraperitoneal injection, respectively. On the 1st day, 3rd day and 7th day after operation, those animals were sacrificed to collect the brain specimens for the study after cerebral blood flow reperfusion and determination of neurological deficit scores. The structural changes of the neurons were observed under electron microscope. Apoptosis was analyzed by flow cytometry. The protein levels of heat-shock protein 70(HSP70), protein kinase B(PKB) and P53 in cerebral corical neurons were determined by immunohistochemical staining and Western blotting. RESULTS:The neurological deficit scores and the apoptotic rate of cerebral cortical neurons in H-APTG were significantly lower than those in MG and L-APTG(P<0.05). The structures of the neurons in H-APTG, such as ribosome endoplasmic reticulum, nucleolus, Golgi complex, mitochondria, etc, were better than those in MG and L-APTG. On the 1st day, 3rd day and 7th day, the protein levels of HSP70 and PKB in cerebral cortical neurons in H-APTG were significantly higher than those in L-APTG, which were significantly higher than those in MG(P<0.05). However, the P53 protein level in H-APTG was significantly lower than that in L-APTG, which was significantly lower than that in MG(P<0.05). CONCLUSION:AP improves nervous functions and inhibits neuronal apoptosis during ischemia and reperfusion. The molecular mechanisms are associated with variations of protein expression in cerebral cortical neurons, such as promotion of HSP70 and PKB and inhibition of P53.     

Effect of aminoguanidine on TNF-α,IL-1β and neuronal apoptosis after focal cerebral ischemic injury in rats

AIM: To evaluate the effect of aminoguanidine (AG) on inflammatory factors and neuronal apoptosis after focal cerebral ischemic injury in rats and the possible mechanism of protective effect of AG against cerebral ischemic injury.METHODS: Thirty male SD rats (weighing 250 g-280 g) were randomly divided into three groups: (1) sham operated group (SH group,n=10),(2) ischemic groups (IS group,n=10),(3) AG group (n=10).In AG group,AG at dose of 100 mg·kg-1 was given intraperitoneally twice a day for 3 consecutive days.In IS group,normal saline was given instead of AG.Focal cerebral ischemia was produced by middle cerebral artery occlusion (MCAO) for 12 h.A nylon thread with rounded tip was inserted into left internal carotid artery cranially until resistance was felt.The distance from bifurcation of common carotid artery to the tip of the thread was about 18-19 mm.Focal cerebral ischemia was confirmed by left Horners syndrome and right side hemiplegia.In SH group,the carotid artery was exposed but no thread was inserted.The expression of tumor necrosis factor-α(TNF-α) was determined by immunochemistry and the content of interleulin-1β(IL-1β) was measured by radioimmunoassay.The expressions of Bcl-2 and Bax protein were detected by flow cytometry.RESULTS: The expression of TNF-α and the content of IL-1β were markedly increased after MCAO.Significantly increased DNA fragmentation,the indication of apoptosis,was detected after MCAO.The expression of TNF-α and the content of IL-1β were significantly lower in AG group than those in IS group.The percentage of apoptosis cells and expression of Bax protein were markedly lower in AG group than those in IS group but still significantly higher than those in SH group.The expression of Bcl-2 protein was markedly higher in AG group than that in IS group.No significant difference in the expression of Bcl-2 protein between IS and SH group was observed.CONCLUSION: AG inhibits the increase in the expression of TNF-α and the content of IL-1β,and protects neurons from apoptosis induced by focal cerebral ischemia through increasing the Bcl-2 protein expression and inhibiting the Bax protein expression.     

Neuronal damage and cell death following ischemia/reperfusion in diabetic rats

AIM:To study forms of cell death following cerebral ischemia/reperfusion in diabetic rats. METHODS:Based on the modles of diabetes and middle cerebral artery occlusion(MCAO), characteristics of cell death after ischemia/reperfusion were evaluated synthetically by the pathological, flow cytometry(FCM), TUNEL and the DNA agarose electrophoresis.RESULTS:The occurrence of cerebral injury after ischemia/reperfusion were accompanied by cell necrosis and cell apoptosis. And cell apoptosis was mainly located in ischeamic penumbra(IP) zone around the densely ischemic focus. Ischemic centre(IC)was characterized by cell necrosis. At the same time, the results showed that the process of ischemic cerebral injury worsen by diabetes mellitus was related to inducing cell apoptosis in IP and Mid zone.CONCLUSION:Neuronal damage following focal cerebral ischemia/reperfusion included cell necrosis and apoptosis, IC zone was mainly characterized by the former, however IP zone by the latter, and there had close internal relationship between them. Brain damage following cerebral ischemia/reperfusion was worsen instinctly under diabetic condition.     

Effects of ginsenoside RH2 on neovascularization of rats with middle cerebral artery occlusion

AIM: To investigate the effects of ginsenoside RH2 (GS-RH2) on neovascularization of rats with middle cerebral artery occlusion (MCAO) and its potential mechanisms. METHODS: SPF Sprague-Dawley rats were randomly divided into sham operation (sham) group, MCAO model (MCAO) group and GS-RH2 group, with 18 rats in each group. After surgery, the general condition and neurological function score of the rats were assessed. At the 1st day, 3rd day and 7th day after intervention, the microvessel density (MVD), the content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were examined. The protein expression of kelch-like ECH-associated protein 1 (Keap1), nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) was determined by Western blot. RESULTS: Compared with sham group, the rats in MCAO group showed significant neurobehavioral obstacles and ischemic brain infarction with higher neurological function score, while treatment with GS-RH2 significantly improved behavioral impairment and reduced the infarction volume with lower neurological function score. The MVD score in GS-RH2 group was increased as the animal survival time prolonged, while the MVD score in MCAO group was decreased. After intervention for 7 d, the MVD score in GS-RH2 group was significantly higher than that in MCAO group (P<0.05). Compared with sham group, the content of MDA was increased and the activities of SOD and GSH-Px were decreased in MCAO group at each time point. After intervention for 7 d, the MDA content was decreased and the SOD and GSH-Px activities were increased in GS-RH2 group compared with MCAO group. After intervention for 7 d, the protein expression of Nrf2 and HO-1 was increased, while the protein expression of Keap1 was decreased in GS-RH2 group compared with MCAO group(P<0.05). CONCLUSION: Ginsenoside RH2 promotes neovascularization of MCAO model rats. The mechanism may be related to the activation of Keap1/Nrf2 signaling pathway, promotion of the antioxidant enzyme activity and inhibition of oxidative stress.     

Expression of nerve growth inhibiting factor Nogo-A mRNA and protein in the brain ischemic infarction rats

AIM: To investigate the changes of expression of Nogo-A at different time points in brain ischemic infarct rats. METHODS: The model of 80 cases of middle cerebral artery occlusion (MCAO) in rats was established. The expression of Nogo-A mRNA and protein were determined by Western blotting and hybridization, and the relationship between functional scoring and Nogo-A was also evaluated. RESULTS: In the brain of MCAO rats, Nogo-A mRNA expression was decreased on day 3 and increased significantly on day 7. The highest level was observed at the 21th d, keeping the same level at the 28th d. Nogo-A protein expression showed the same results. These results were correlated with the brain function scoring. CONCLUSION: Expression of Nogo-A does not increase in the early stage, but increases significantly in the late stage of MCAO, suggesting that Nogo-A expression may play an important role in the nerve regeneration of brain ischemic injury.     

Effects of Le Er Mai on apoptosis of hippocampus neuronal cells in anaphase of cerebral ischemic reperfusion injury in rats

AIM: To investigate the effects and mechanism of Le Er Mai (LEM) on the apoptosis of hippocampus neuronal cells in the anaphase of cerebral ischemic reperfusion injury in rats.METHODS: A rat model of middle cerebral artery occlusion reperfusion (MCAO) was produced with the intraluminal filament. During reperfusion for 30 d after 2 h of ischemia, the TUNEL staining methods were used to detect apoptosis of hippocampus neuronal cells, and immunohistochemical technique were employed to examine the protein expression of Fas, Bax, caspase-3 and caspase-9 in the hippocampial. The gene expressions of fas, bax, caspase-3 and caspase-9 in hippocampial were examined by RT-PCR. RESULTS: After 2 h ischemia and 30 d reperfusion, compared with sham-operated group, TUNEL-positive staining cells and expression levels of Fas, Bax as well as caspase-3 and caspase-9 obviously increased, and the mRNA expressions of fas, bax, caspase-3 and caspase-9 in hippocampial markedly up-regulated in model group. Compared with model group, LEM at dose of 2.00 g/kg or 0.87 g/kg, and flunarizinum significantly reduced apoptosis and decreased the protein expressions of Fas, Bax, caspase-3 and caspase-9 in hippocampial, and down-regulated the mRNA expressions of fas, bax, caspase-3 and caspase-9 (P<0.05), those action of LEM in 0.87 g/kg dosage group was lower than those in 2.00 g/kg dosage group.CONCLUSION: LEM obviously lower the injury of hippocampial in the anaphase of cerebral ischemia reperfusion through inhibiting the apoptosis of hippocampus neuronal cells. The mechanism of LEM may be related to regulate the expression of signal transduction pathway correlated gene of apoptosis in neuronal cells.     

Expression of nerve growth inhibiting factor Nogo-A in ischemic infarct brain in rats

AIM: To investigate the dynamic changes of Nogo-A expression in ischemic infarct brain in rats. METHODS: The model of middle cerebral artery occlusion (MCAO) in 80 rats was established and expression of Nogo-A mRNA was measured by immunohistochemistry and hybridization. RESULTS: In the brain of MCAO rats, Nogo-A mRNA expression was decreased at the third day and increased significantly at the 7th day, and reached high level at the 21th day, then remained the high level to the 28th day. Nogo-A protein expression showed the same results. CONCLUSION: Expression of Nogo-A did not change in the early stage of MCAO, but increased significantly in the late stage of MCAO, suggesting that Nogo-A expression may play an important role in the nerve regeneration of brain ischemic injury.     

Effects of astragaloside IV on autophagy in rats with cerebral ischemia/reperfusion injury

AIM: To investigate the effects of astragaloside IV (AS-IV) on autophagy in rats with cerebral ischemia/reperfusion (I/R) injury. METHODS: The focal cerebral ischemia/reperfusion of rat left middle cerebral artery occlusion (MCAO) was induced by suture method. Male SD rats (n=70) were randomly divided into sham operation group, I/R group, solvent control group, AS-IV group, AS-IV+autophagy inhibitor (3-methyladenine, 3-MA) group, 3-MA group and autophagy activator (rapamycin, Rapa) group. Except for sham operation group, the rats in other groups were subjected to ischemia for 2 h and reperfusion for 24 h. The rats with successful modeling were selected according to Zea Longa scoring criteria. The volume of cerebral infarction was measured by TTC staining. The morphological changes of nerve cells in the rats were observed with Nissl staining. The phenomenon of autophagy was observed under transmission electron microscope. The protein expression of beclin-1 and LC3-Ⅱ was determined by Western blot. RESULTS: No neurological deficit in sham operation group was observed, and the cerebral infarction was not found. Compared with sham operation group, obvious cerebral infarction was observed, the Nissl bodies were small in size and number and stained light, typical autophagosomes were observed, and the protein expression of beclin-1 and LC3-Ⅱ was increased in I/R group (P<0.05). Compared with I/R group, the volume of cerebral infarction was decreased obviously, neurological deficit restored significantly, and the number of autophagosomes and the protein expression of beclin-1 and LC3-Ⅱ were increased in AS-IV group and Rapa group (P<0.05). However, no significant difference between solvent control group and I/R group was observed (P>0.05). Compared with AS-IV group, the neurological deficit was serious, the volume of cerebral infarction and the number of autophagosomes were increased, while the expression of beclin-1 and LC3-Ⅱ was decreased in AS-IV+3-MA group and 3-MA group (P<0.05). CONCLUSION: Astragaloside IV may play an important role in atte-nuating cerebral ischemia/reperfusion injury by activating autophagy.     

Effects of Salvia miltiorrhiza Bunge.f.alba. on mitochondrial damage and apoptosis induced by cerebral ischemia and reperfusion

AIM: To observe the effects of Salvia miltiorrhiza Bunge.f.alba. (Sal) on the mitochondrial ultra-structure, oxidative stress and apoptosis induced by ischemia injury in a rat model of focal cerebral ischemia and reperfusion.METHODS: The middle cerebral artery occlusion/reperfusion (MCAO/R) rat model was established by a modified Longa occlusion method. Adult male SD rats were randomly divided into control group, simple ischemia reperfusion group, Sal with ischemia reperfusion group and butylphthalide with ischemia reperfusion group. To study the protective effects of Sal and its mechanism, the intervention of Sal was given and the ultra-structure of mitochondria, functions of mitochondria under oxidative stress and the incidence of apoptosis of brain cells were determined.RESULTS: Many electron dense toxic granulation and vacuolus in mitochondria were observed in the rat brain of focal cerebral ischemia and reperfusion. Under the condition of ischemia and reperfusion, the mitochondria membrane was disaggregative, and the tubular cristae of mitochondrion disappeared. MDA content was obviously increased and the activity of glutathione peroxidase decreased significantly. The apoptosis of brain cells were observed in a great quantity. The changes of ultra-structure of mitochondria and the activity of GSH-Pxase were significantly improved by the treatment of Sal. Furthermore, treatment with Sal delayed the decrease of GSH-Pxase activity, and inhibited the increase in MDA content in brain tissue after ischemia and reperfusion. The incidence of apoptosis of brain cells was also decreased.CONCLUSION: Sal protects the brain tissue from ischemia injury.     

Changes of MARCKS mRNA expression in rat hippocampus with acute multi-cerebral infarction

AIM: To observe the dynamic alteration of myristoylated alanine-rich C kinase substrate （MARCKS） mRNA expression in rat hippocampus with acute multi-cerebral infarction, and discuss the relationship between the alteration of hippocampus MARCKS gene and ischemia damage. METHODS: The acute multi-cerebral infarction model was established by method of Kaneko. Neurological function deficits were evaluated in the behavior test. The consequences of cerebral ischemic damage were examined by histopathological analyses. The MARCKS mRNA expression was measured by semi-quantitative PCR. RESULTS: The rats in acute multi-cerebral infarction group showed different level changes of neurological function deficits. The hippocampus damage of histopathology became significant 24h after ischemia. At the same time, the MARCKS mRNA expression was upregulated at the area of rats hippocampus during ischemia, and its overexpression started 1h after ischemia, and reached maximum7d after ischemia. CONCLUSION: MARCKS mRNA of rat hippocampus overexpresses during acute cerebral ischemia. This MARCKS mRNA overexpression is related with hippocampus ischemia damage.     

Expression and histogenesis of matrix metalloproteinase-9 and transforming growth factor-beta 1 in acute cerebral ischemia model of rats

AIM:To observe the expression and tissue localization of matrix metalloproteinase 9 (MMP-9) and transforming growth factor beta 1 (TGF-β1) in the rat acute cerebral ischemia model. METHODS:Male Wistar rats were used to establish acute cerebral ischemia model by a suturing method. The rats were divided into normal control group, sham group and ischemia 6 h, 12 h, 1 d, 2 d, 6 d and 14 d groups. The rat cerebral cortex and hippocampus of the brain were collected at different time points.The mRNA and protein levels of MMP-9 and TGF-β1 in the brain tissues were detected by real-time PCR and in situhistochemistry staining, respectively. The levels of MMP-9 and TGF-β1 in the plasma were also measured by ELISA. RESULTS:The results of real-time PCR showed that the mRNA levels of MMP-9 began to increase 6 h after acute ischemia and reached to a peak 2 d after acute ischemia. Similarly, the mRNA level of TGF-β1began to rise 12 h after acute ischemia and reached to the highest level 6 d after acute ischemia. Compared with the sham rats, the mRNA levels of MMP-9 and TGF-β1 in the rat brains that collected at ischemic time of 12 h, 1 d, 2 d, 6 d and 14 d were significantly increased. Moreover, results of in situhistochemical staining showed that the expression of MMP-9 was detected at cerebral cortex and hippocampus 1 d after acute cerebral ischemia.Further studies showed that MMP-9 dyeing of the rat cerebral cortex was most obvious 2 d after the acute cerebral ischemia. Similarly, the rat cortex and hippocampus began to express TGF-β1 2 d after acute ischemia and TGF-β1 staining at rat cerebral cortex was most obvious 6 d after the acute cerebral ischemia. In addition, ELISA showed that the increase in MMP-9 and TGF-β1 was detected in the plasma 12 h after ischemia. Compared with the sham rats, the level of these 2 factors significantly upregulated since 1 d after ischemia. CONCLUSION: The brain tissue itself contributes to the upregulation of MMP-9 and TGF-β1 post acute cerebral ischemia, which shed light on the related research in the field.     

Effects of ischemic postconditioning on cerebral ischemia/reperfusion injury in rats

AIM: To study the effects of ischemic postconditioning on cerebral ischemia following middle cerebral artery occlusion in rats. METHODS: 21 rats were randomly divided into three groups: middle cerebral artery occlusion (MCAO), MCAO+transient unilateral common carotid artery occlusion (u-CCA-O), MCAO+transient bilateral common carotid artery occlusion (b-CCA-O)(n=7, respectively). u-CCA-O/b- CCA-O was generated by transient middle cerebral artery occlusion plus transient unilateral/bilateral common carotid artery (CCA) occlusion. After the suture was removed, ischemic postconditioning was performed by occluding CCA for 10s, reperfusion 10s, and then allowing for another 4 cycles of 10s of reperfusion and 10s of CCA occlusion. Rats were sacrificed 2 d later and infarct size was measured. Cerebral blood flow (CBF) was measured in different 15 time points: 0 min, 10 min, 1 h after MCA occlusion, 0 min after MCA reperfusion, 10s of CCA occlusion and 10s of CCA reperfusion in all five cycles, 30 min after MCA reperfusion. Functional neurological outcome was determined 1 h and 48 h after reperfusion. Infarct volume was measured 48 h after reperfusion. RESULTS: The infarct volumes in u-CCA-O group and b-CCA-O group diminished compared to the control group. The results of CBF demonstrated that b-CCA-O group diminished 9% compared with control and u-CCA-O group when 30 min after intervention. The rats in u-CCA-O and b-CCA-O group had better neurological performance at 1 h after reperfusion. CONCLUSION: Ischemic postconditioning reduces infarct size, improves functional neurological outcome, most plausibly by diminishing cerebral blood flow.     

Effects of Astragalus injection on neuronal apoptosis and expression of JNK3 in rat hippocampus after cerebral ischemia reperfusion

AIM:To investigate the effects of Astragalus injection on neuronal apoptosis and expression of c-Jun N-terminal kinase 3(JNK3) in the rat hippocampus after cerebral ischemia reperfusion. METHODS:The rat model of cerebral ischemia reperfusion was set up by a four-vessel occlusion method. The SD rats were randomly divided into 4 groups:sham operation group, cerebral ischemia reperfusion group(model group), cerebral ischemia reperfusion+Astragalus injection group(Astragalus injection group) and cerebral ischemia reperfusion+vehicle group(vehicle group). The rats in model group, Astragalus injection group and vehicle group after transient global cerebral ischemia(30 min) were then divided into 7 subgroups according to the reperfusion time of 0 h, 0.5 h, 2 h, 6 h, 24 h, 72 h and 120 h. The apoptosis of the neuron in the hippocampus was measured by the method of TUNEL staining. The expression of JNK3 at mRNA and protein levels was determined by real-time PCR and Western blotting,respectively. RESULTS:Compared with sham operation group, the number of apoptotic neurons increased in model group(P<0.05). Compared with model group, the number of apoptotic neurons decreased obviously in Astragalus injection group(P<0.05). Compared with sham operation group, the expression of JNK3 at mRNA and protein levels in the hippocampus increased obviously in model group at all time points except 120 h(P<0.05). Compared with model group, the expression of JNK3 at mRNA and protein levels in the hippocampus decreased obviously in Astragalus injection group at all time points except 120 h(P<0.05). CONCLUSION:Astragalus injection decreases neuronal apoptosis in rat hippocampus after cerebral ischemia reperfusion by inhibiting the expression of JNK3 at mRNA and protein levels.