Neuroprotective effect of L-serine on rats with permanent middle cerebral artery occlusion

AIM: To investigate the neuroprotective effect, therapeutic dosage and time window of L-serine against permanent cerebral injury in rats. METHODS: Permanent middle cerebral artery occlusion (pMCAO) was induced in the rats to determine the efficacy of L-serine (ip) by neurological evaluation, TTC staining and Nissl staining.L-serine was used at different doses (56 mg/kg, 168 mg/kg and 504 mg/kg) and for different time periods (1 h, 3 h, 6 h, 12 h and 24 h after pMCAO). Aminooxyacetic acid (AOAA), an inhibitor of serine racemase, was used to alter the efficacy of L-serine. Laser Doppler perfusion monitor was used to observe the regional cerebral blood flow (rCBF) in the ischemic cerebral cortex under the condition with or without L-serine treatment. RESULTS: Treatment with L-serine at doses of 168 mg/kg and 504 mg/kg at time point of 3 h after pMCAO greatly decreased the neurological deficit score and infarct volume,and attenuated the loss of hippocampal CA1 neuronal cells. In the observation of therapeutic time window, L-serine displayed a significant neuroprotective effect if used within 6 h after pMCAO, but did not exert any notable effect if used over 12 h after pMCAO. AOAA hardly changed the effect of L-serine. L-serine treatment notably raised rCBF in the area of ischemic cerebral cortex when it was injected 30 min after pMCAO. However, strychnine, an antagonist of strychnine-sensitive glycine receptor, did not alter this effect of L-serine. CONCLUSION: L-serine has neuroprotective effect on permanent ischemic brain injury in rats if administered early and sufficiently by augmentation of rCBF in the ischemic cerebral cortex.     

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.     

Experimental study on how brain-dead state affects the heart structure and function of Ba-Ma mini pigs and the mechanism

AIM:To investigate how brain-dead state affects the heart structure and function and the effect of PKC-α in BA-Ma mini pigs.METHODS:Ten Ba-Ma mini pigs were randomized into 2 groups: brain-dead group (n=5),and control group (n=5). The brain-dead model was made by increasing intracranial pressure,while the control group was maintained anesthesia for 24 h. The concentrations of cTnT,TNF-α,IL-1β and IL-6 in serum were determined at 6,12 and 24 h after brain death. At 24 h,heart tissues were observed by HE staining and electron microscope. The expression of PKC-α was detected by immunohistochemistry and RT-PCR.RESULTS:(1) Histological changes of myocardium: flaky bleeding under endocardium and dissolution of myocardium were found in optical microscope. In electron microscope dropsical mitochondria and confluent muscle fiber were found. (2) Changes of serum cTnT: serum cTnT for brain-dead group began to increase gradually since 6 h,and were significantly higher at each time point than those in control group (P<0.05). (3) Changes of inflammatory factors: IL-1β,IL-6,and TNF-α in brain-dead group began to increase gradually since 6 h,and were significantly higher at each time point than those in control group (P<0.05). (4) Changes of PKC-α expression: PKC-α mRNA and protein expressions in brain-dead group increased significantly at 24 h (P<0.05).CONCLUSION:Brain death may evoke heart structure and functional injury,and increase the levels of inflammatory factors and PKC-α. The activation of PKC-α may participate in the process of heart injury.     

An experimental exploration of focal inflammatory response in cerebral ischemic foci in the rat

AIM: To study the pathological relationship of vascular cell adhesion molecule-1 (VCAM-1) expression and monocyte/macrophage infiltration with focal brain ischemia. METHODS: Immunohistochemical technique and focal brain ischemia/reperfusion model were used in the study in order to explore profiles and time-course of VCAM-1 expression and monocyte macrophage (ED2 positive cell) infiltration in ischemic rat brain. RESULTS: VCAM-1 was up-regulated in microvascular endothelial cells in ischemic cortex at 1h postischemia, and continuously expressed during the time of reperfusion. ED2 positive cells infiltrated into ischemic cortex at 1h iscehmia/ 2h reperfusion and then ED2 positive cells increased gradually with the time of reperfusion, ED2 positive cell infiltration showed apparently relationship with VCAM-1 expression, and both of them exhibited the some changes of time-dependence. CONCLUSION: Cerebral ischemia induced VCAM-1 expression and ED2 positive cell infiltration and VCAM-1 may regulate the recruitment of ED2 positive cells in the ischemic brain region. The results suggested that VCAM-1 and ED2 positive cells may be participated in the pathogenesis of cerebral ischemic injury.     

Effect of Fu-Sheng powder on NPY and its mRNA in brain of rats with hyperlipidemia after cerebral ischemia and reperfusion

AIM:To observe the changes in neuropeptide Y(NPY) and the effect of Fu-Sheng powder(FSP) on NPY in the rat brain in a steady cerebral ischemia and reperfusion(I/R) model. METHODS: The models of rat brain injury were established by repeated cerebral I/R in rats with hyperlipidemia. Radioimmunoassay was performed to determine the level of NPY, while NPY mRNA expression was observed by in situ hybridization. RESULTS: After 1 day of I/R, compared with control group, the content of NPY in the model animals were significantly increased by 51.86% (P＜0.01) and lasting 7 days after I/R, and the expression of NPY mRNA was greatly increased. FSP treatment decreased the contents of NPY (P＜0.05,P＜0.01) and its mRNA expression. CONCLUTION: There were obvious imbalances of NPY in the rat brain after cerebral I/R and the FSP might antagonize ischemia injury of brain through modulating NPY, which may be one of the mechanisms underlying FSP treatment for cerebral vascular diseases.     

Effect of SAPK/JNK signal pathway on neuronal apoptosis in cerebral infarction rats

AIM: To investigate the molecular mechanism of neuronal apoptosis by observing the changes of key proteins in SAPK/JNK and Bcl-2/Bax signal pathways after brain infarction. METHODS: The cortical infarction was induced by photochemistry, namely photothrombotic cortical injury (PCI). Thirty-six Sprague-Dawley rats were randomly divided into 2 groups: PCI group and sham-operated group. The ipsilesional cortex was harvested for histomorphometry and transmission electron microscopy 7 days after PCI. Some key proteins including p-JNK1, p-JNK2, p-c-Jun, p-ATF-2, total JNK1, total JNK2, Bcl-2 and Bax were detected by Western blotting analysis.RESULTS: The cortical infarction in rats was successfully induced by photochemistry. The apoptosis of neurons in cortex was more obvious in PCI group than that in sham-operated group 7 days after PCI. The levels of p-JNK1, p-JNK2, p-c-Jun and p-ATF-2 in PCI group were significantly higher than those in sham-operated group, whereas the ratio of Bcl-2/Bax was significantly lower(P<0.05). CONCLUSION: Apoptosis is a major contributor to neuronal loss induced by cerebral hypoxia-ischemia for a long period after cortical infarction. The process is related to some apoptotic proteins such as Bcl-2/Bax and the SAPK/JNK signal pathways activated by ischemic injury.     

Protective effect of BNDF on vascular endothelial cells with H2O2-induced oxidative injury

AIM: To study the protective effect of brain-derived neurotrophic factor (BDNF) on vascular endothelial cells with H₂O₂-induced oxidative injury. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured in vitro, and the oxidation injury model of HUVECs was established by treatment with H₂O₂. The oxidatively injured HUVECs were cultured with different concentrations (1, 10 and 100 μg/L) of BDNF. At the same time, the control group (no injury), PBS treatment after H₂O₂ injury group and TrkB inhibitor group (with 100 μg/L BDNF and 1: 1 000 TrkB inhibitor) were also set up. The viability of the HUVECs was detected by MTT assay. The levels of LDH, MDA, SOD and GSH were measured. The releases of NO, ET-1 and ICAM-1 were analyzed by ELISA. The changes of ROS production and cell apoptosis were evaluated by flow cytometry. The protein levels of TrkB, p-TrkB, cleaved caspase-3, Bcl-2 and Bax were determined by Western blot. RESULTS: Compared with uninjured control group, in H₂O₂ oxidative injury plus PBS treatment group, the viability of the cells was decreased significantly, the LDH and MDA levels were increased significantly and the activities of SOD and GSH were decreased significantly. The NO secretion was decreased, and the ET-1 and ICAM-1 concentrations were increased significantly. The ROS content and apoptotic rate were increased significantly. The protein levels of cleaved caspase-3 and Bax were increased but Bcl-2 protein expression was decreased significantly. Compared with PBS treatment group, in H₂O₂-injured HUVECs treated with different concentrations of BDNF, the cell viability was gradually increased, the LDH and MDA levels were decreased and the activities of SOD and GSH were increased gradually. The secretion of NO was increased but ET-1 and ICAM-1 were decreased gradually. The ROS content and apoptotic rate were decreased significantly. The TrkB and p-TrkB levels were significantly increased significantly, the protein expression of cleaved-caspase 3 and Bax was decreased gradually and the Bcl-2 protein expression increased gradually. The role of BDNF was inhibited by TrkB inhibitor. CONCLUSION: BDNF protects HUVECs from oxidative injury by binding with TrkB to activate the BDNF-TrkB signaling pathways.     

Effects of ischemic postconditioning on hippocampus CA1 area rCBF and astrocyte activation after cerebral ischemia in tree shrews

AIM: To study the effects of ischemic postconditioning (PC) on regional cerebral blood flow (rCBF) and astrocyte (AS) activation in hippocampus CA1 area and to explore the possible mechanism of ischemic PC affecting glial fibrillary acidic protein (GFAP) expression during focal cerebral thrombosis. METHODS: The thrombotic focal cerebral ischemia was induced by photochemical reaction in tree shrews, and ischemic postconditioning was established by cliped ipsilateral carotid of the animal at 4 h after cerebral ischemia. The rCBF and GFAP expressions in hippocampus CA1 area were detected, respectively, by laser-Doppler (LD) fowmeter and immunohistochemistry. RESULTS: The numbers of GFAP positive cells were increased markedly and GFAP expression enhanced (P<0.01). AS oncosis was apparent 24 h after cerebral ischemia. Postconditioning increased hippocampus rCBF from (2.55±0.28) PU to (10.42±3.75) PU (P<0.05) at 24 h and from (9.84±1.22) PU to (18.74±1.60) PU (P<0.05) at 72 h after the cerebral ischemia, and AS oncosis was inhibited markedly. CONCLUSION: Multiple, short, regional carotid occlusions may prolong “time window” of therapeutic cerebral ischemia. The protection mechanism of the ischemic postconditioning may be associated with the increase in rCBF and improvement of hippocampus microenvironment by regulating AS activation.     

Cell proliferation and nestin expression in cortex and SEZ of MCAO rats

AIM:To explore the effect of brain ischemia injury on cell proliferation and nestin expression in cortex and subependymal zone (SEZ).METHODS:Using a local brain ischemia model(MCAO), BrdU positive cells of cortex and subependymal zone (SEZ), also nestin positive cells, were observed by immunohistochemistry.RESULTS:BrdU and nestin positive cells in SEZ of MCAO rats were obviously increased. In cortex, only nestin positive cells were observed.CONCLUSION:Neural stem cells in SEZ and cortex were activated after brain ischemia, it may be related with neural recovery after brain ischemia injury.     

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.