Fluorocitrate inhibits regulatory effect of ischemic postconditioning on HIF-1α/iNOS signaling pathway after cerebral ischemia in tree shrews with brain injury

AIM: To investigate the regulatory effect of HIF-1α/iNOS signaling pathway on the neuroprotection of ischemic postconditioning (PC) in tree shrews, and to explore the mechanisms of deteriorated cerebral injury after inhibiting astrocyte (AS) metabolism. METHODS: Thrombotic cerebral ischemia was induced by photochemical reaction in tree shrews. Fluorocitrate (FC) was used to inhibit AS metabolism and the ischemic PC was established at 4 h after ischemia followed by clipped ipsilateral common carotid artery on the ischemia side for 3 times, 5 min/time. A total of 67 male tree shrews were randomly divided into 7 groups:control (n=9), ischemia (4 h and 24 h, n=9 for each group), ischemia with PC (4 h and 24 h, n=9 for each group), and FC pretreatment (4 h and 24 h, n=11 for each group). The cerebral infarction size was detected by TTC staining, and the histological changes of hippocampal neurons were observed under light microscope. The regional cerebral blood flow (rCBF) in ischemic cortex was monitored by laser Doppler brain flowmetry. The protein expression of iNOS in hippocampus was detected both by immunohistochemistry and Western blot. The production of NO detected by spectrophotometer. The level of HIF-1α in hippocampus analyzed by ELISA. RESULTS: The cerebral infarct volume was increased with prolonged duration of ischemia, and the changes of ischemia at 24 h were significant (P<0.05). The cortical rCBF was progressively decreased, and it was decreased at 4 h and 24 h after ischemia (P<0.05). The expression of HIF-1α and iNOS in hippocampus was enhanced, and the production of NO was increased significantly (P<0.05). Ischemic PC restored the cortical rCBF (P<0.05), reduced cerebral infarction volume (P<0.05), down-regulated iNOS expression and reduced NO production in the hippocampus (P<0.05). However, the cortical rCBF in FC pretreatment group was significantly lower than that in ischemic group (P<0.05), the neuronal damage was aggravated, and the infarction volume was increased after pretreatment with FC (P<0.05). CONCLUSION: Ischemic PC may reduce cerebral injury by regulating the expression of HIF-1α and iNOS. Inhibition of AS function may attenuate the protective effect mediated by ischemic PC and aggravate brain injury.     

Effects of ischemic postconditioning on neuronal Akt signaling pathways in hippocampus CA1 area after cerebral ischemia in tree shrews

AIM: To investigate the phosphatidylinositol 3-kinase/Akt (PI-3K/Akt) Ser-473/Thr-308/ phosphorylation (Akt /Akt ) and the intensity of the neurons in happocampus CA1 area under the conditions of thrombotic cerebral ischemia and postconditioning in tree shrews. METHODS: The thrombotic focal cerebral ischemia was induced by photochemical reaction in tree shrews. Two hundred and ten minutes after cerebral ischemia, ischemic postconditioning was established by repeated cliping of ipsilateral carotid. The distribution of Akt and Akt , and neuronal ultrastructure in hippocampus CA1 area were observed by the methods of electronic microscopy and immunohistochemistry. The phosphorylation intensity was measured by determining the optical gray value. RESULTS: The photochemical reaction induced cerebral ischemia and resulted in obvious lesions in hippocampus CA1 neurons. The damages of ultrastructure in the hippocampus were diminished by postconditioning. Correspondingly, in ischemia group, although the Akt showed positive during 72 h, the positive Akt was only observed at the time point of 4 h, and went negative at the time points of 24 h and 72 h. In postconditioning group, Akt at the time points of 4 h, 24 h and 72 h were positive,and Akt at the time points of 24 h and 72 h was also positive. CONCLUSION: Cerebral ischemia leads to neuron lesions in tree shrew hippocampus and the postconditioning decreases the damage. The Akt and Akt may play important roles in the protective mechanism.     

Postconditioning modulates TLR4 expression in hippocampus of tree shrews with thrombotic cerebral ischemia

AIM: To observe the effects of thrombotic cerebral ischemia and postconditioning on the expression of toll-like receptor 4 (TLR4) in hippocampus of tree shrews.METHODS: The model of thrombotic focal cerebral ischemia was established by photochemical reaction.Four hours after the onset of photochemical reaction, ischemic postconditioning was induced by 3 repeated cycles of carotid artery occlusion for 5 min and reperfusion for 5 min. The histological changes of hippocampus (by HE staining), TLR4 protein level (by Western blotting) and TLR4 mRNA expression (by semiquantitative RT-PCR) were observed.RESULTS: The extensive neuronal degeneration in hippocampus was observed from 4 h to 72 h and peaked at 24 h after cerebral ischemia, but was significantly attenuated after postconditioning. Cerebral ischemia caused a progressive increase in the expression of TLR4 protein at 4 h and 24 h (P<0.05), and decreased at 72 h (P<0.05). In contrast to ischemia groups, postconditioning decreased the expression of TLR4 protein at 4 h and 24 h (P<0.05), but an increase in the expression of TLR4 at 72 h (P<0.05) was observed. Simultaneously, the level of TLR4 mRNA in hippocampus showed the tendency of approximate variation in accordance with the protein expression.CONCLUSION: The expression of TLR4 increases by cerebral ischemia. The protection mechanisms of postconditioning may be associated with modulating TLR4 expression.     

Ischemia postconditioning induces tight junction protein expression and inhibits brain edema after thrombotic cerebral ischemia in tree shrews

AIM: To assess whether the expression of tight junction(TJ) proteins, occludin/zonula occludins(ZO)-1, and regional cerebral blood flow(rCBF) link to brain edema in tree shrews during thrombotic cerebral ischemia and ischemic postconditioning(PC), and to explore how TJ affects brain edema and cerebral infarction. METHODS: Tree shrews were randomly grouped into control, ischemia and cerebral ischemia+PC(n=23), and the remaining 3 animals were used for magnetic resonance imaging(MRI). The local cerebral thrombosis were induced by photochemical reaction in the tree shrews, and ischemic PC was established at 4 h after induction of cerebral ischemia followed by clipped ipsilateral common carotid artery(5 min×3). The changes of the neural ultrastructure were observed under electron microscope. The neuronal apoptosis was analyzed by the method of TUNEL. Laser Doppler brain flowmetry was used to monitor the rCBF. The protein levels of occludin/ZO-1 were determined by immunochemistry and Western blot. The cerebral infarction volume was detected by MRI. The brain water content was measured by dry-wet weight method. RESULTS: Induction of cerebral ischemia led to a significant reduction of the normal neuron numbers in the hippocampal CA1 area, and conversely, the number of neurons with abnormal ultrastructure was increased. The TUNEL positive cells were increased significantly(P<0.01) in ischemia group. Moreover, the rCBF decreased significantly(P<0.01), and occludin/ZO-1 protein expression decreased(P<0.01). The brain water content and cerebral infarction volume were significantly increased(P<0.01). Ischemic PC increased the rCBF and the occludin/ZO-1 expression, but reduced the brain water content, the TUNEL positive cells, and the infarction volume(P<0.01). CONCLUSION: Ischemic PC increases the rCBF but not the local water content, suggesting that reduced cerebral infarction volume after ischemia PC is associated with the attenuation of cerebral edema by the enhancement of occludin/ZO-1 protein expression.     

Effects of cerebral ischemia and postconditioning on signaling molecules PERK and GRP78 in hippocampus tissue of tree shrew during endoplasmic reticulum stress

AIM: To investigate the effects of cerebral ischemia and postconditioning on protein kinase R-like endoplasmic reticulum kinase (PERK) and glucose-regulated protein 78 (GRP78) in the hippocampus tissue of tree shrew during endoplasmic reticulum stress and the mechanism of post-conditioning protecting the brain from damage. METHODS: The focal cerebral ischemic model was duplicated by photochemical reaction in tree shrew and the postconditioning was induced by alternatively occluding and opening the carotid artery of ischemic side for 3 cycles (5 min each cycle) at 3.5 h after ischemia. The damage and ultrastructural changes of the hippocampal neurons were observed by HE staining. The expression of PERK and GRP78 at mRNA and protein levels in the hippocampal tissue at different time points after cerebral ischemia and postconditioning was determined by RT-PCR, immunohistochemistry and Western blot. RESULTS: The injuries of hippocampal neurons were aggravated with prolonged cerebral ischemia, which was most severe at 24 h after ischemia while the postconditioning alleviated these damages correspondingly. The expression of PERK at mRNA and protein levels was upregulated at 4 h, 24 h and 72 h after ischemia (P<0.05), while postconditioning downregulated the expressions of PERK at ischemia and postconditioning 4 h (IP4 h) gruop and IP24 h group (P<0.05). The expression of GRP78 at mRNA and protein levels was not changed at 4 h, 24 h and 72 h after ischemia, while postconditioning upregulated the expressions of GRP78 at IP24 h group (P<0.05). CONCLUSION: The focal thrombotic cerebral ischemia activates the endoplasmic reticulum stress in ischemic hippocampus of tree shrews, leading to the changes in mRNA and protein expression of PERK in the PERK/eIF2α signal transduction pathway. The postconditioning treatment alleviates endoplasmic reticulum stress and neuronal damages by downregulating PERK and upregulating GRP78, thereby protecting the brain from injury.     

Hypothermia postconditioning protects hippocampal CA1 neurons from focal cerebral ischemia in tree shrew

AIM: To observe the changes in vascular endothelial growth factor (VEGF) expression and the cell numbers of cellular necrosis in hippocampus CA1 area after cerebral ischemia and hypothermia postconditioning (HPC). METHODS: The focal thrombotic cerebral ischemia was induced by photochemical reaction in tree shrews. 6 h after ischemia, HPC was executed by a focal homoeothermic equipment, which reduced the brain temperature and maintained at 31-32 ℃ for 1 h. VEGF expression in hippocampus CA1 area was detected by immunohistochemistry. The numbers of death cells were counted and the ultrastructure was observed under the electron microscope. RESULTS: Compared to control group, VEGF expressions increased in neuron of hippocampus CA1 area at 24 h, and decreased at 72 h in HPC group (P<0.01). Meanwhile the numbers of necrotic cells reduced at 24 h, and increased at 72 h. In accordance with this, ultrastructure of mitochondrion and endoplasmic reticulum became deterioration at 72 h. CONCLUSION: During the early stage, VEGF expression maybe directly protects neurons from cerebral ischemia. HPC has a remarkable significance to neuroprotective function in this time, but it may aggravate neuron injury in the last stage of cerebral ischemia. The HPC may prolong the treatment time-windows in the acute phase of cerebral ischemia.     

Effect of cholecystokinin-octapeptide on focal cerebral ischemia/reperfusion injury in rats

AIM:To examine the effect of cholecystokinin-octapeptide (CCK-8) on focal cerebral ischemia/reperfusion injury and its underlying mechanisms.METHODS:By using the suture model of focal cerebral ischemia and reperfusion, the effects of intracerebroventricular (icv) injection of CCK-8 and proglumide, nonselective CCK receptors antagonist, on the infarct size, regional cerebral blood flow (rCBF), and the levels of nitric oxide (NO), malondialdehyde (MDA) were observed in different brain regions of rats subjected to 1 h focal cerebral ischemia followed by 24 h reperfusion.RESULTS:(1) pretreatment with different doses of CCK-8 (0.3 μg,1.0 μg,2.0 μg or 4.0 μg) could attenuate the infarct size, but the statistically significant effects of CCK-8 were obtained only at the doses of 1.0 μg and 2.0 μg(P＜0.05). The neuroprotective effects of CCK-8 were blocked by pretreatment with proglumide. Administration of proglumide alone could worsen the ischemia/reperfusion injury. (2) CCK-8 (1.0 μg) inhibited the increase in NO, MDA levels in the ischemic core, and also inhibited the increase in NO level in the ischemic penumbra. The rCBF in the CCK-8 group was significantly higher than the normal value at 24 h after reperfusion (P＜0.05).CONCLUSIONS:These results suggest that both endogenous and exogenous CCK-8 alleviate focal cerebral ischemia/reperfusion injury. Such an action may be associated with inhibition of free radical-induced injuries and the improvement in rCBF.     

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.     

Relation between rCBF and neuronal apoptosis in penumbra following focal cerebral infarction

AIM: To study regional cerebral blood flow (rCBF) and neuronal apoptosis in penumbra following focal cerebral infarction in rats. METHODS: To establish the model of focal cerebral infarction based on the principle of photochemical initiation of thrombosis. Laser Doppler flowmetry was used to measure rCBF in ischemic core and penumbra. The numbers and anatomic distribution of apoptosis cells were examined in situ histologically with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-fluorescein nick end-labeling (TUNEL) assay in adjacent sections. RESULTS: rCBF in penumbra decreased to the minimum at 6 h in which it was 30.88%±7.11% of normal. From 9 h to 48 h, rCBF in penumbra increased gradually. At 48 h, 91.90%±4.56% of normal cerebral blood flow was recovered which was significant differences than others. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-fluorescein nick end-labeling (TUNEL) assay demonstrated that TUNEL-positive cells occurred at 6 h and were expending in a radial fashion only in penumbra. The numbers of apoptosis cells were progressively increased with time and to the maximum in penumbra after 48 h following focal cerebral infarction. CONCLUSION: There were delayed neuronal apoptosis following focal cerebral infarction. The mechanism may be related to ischemia-reperfusion injury induced by the recovery of rCBF in penumbra.     

Effects of ginkgolide B on glial fibrillary acidic protein expression after thrombotic cerebral ischemia in tree shrews

AIM: The present study was designed to examine the changes in glial fibrillary acidic protein (GFAP) expression during cerebral ischemia and the effects of ginkgolide B on GFAP expression. METHODS: The focal thrombotic cerebral ischemia was formed by photochemistry-induced in tree shrews. GFAP stained by ABC immunohistochemistry and absorbance were measured with image analyze system. RESULTS: GFAP expression in astrocytes increased significantly (P<0.01) at 24 h and kept in higher level at 72 h (P<0.01) within penumbra after focal cerebral ischemia. GFAP expression declined when the animals were given GB at 6 h after thrombotic cerebral ischemia. CONCLUSIONS: Neuronal necrosis resulted in GFAP expression in astrocytes after local cerebral ischemia and GB protected neurons by antagonizing PAF receptor and inhibiting GFAP expression.     

Inhibitory effect of ischemic postconditioning on autophagy induced by focal cerebral ischemia reperfusion in rats

AIM: To investigate the effect of ischemic postconditioning (IPC) on autophagy induced by focal cerebral ischemia reperfusion (I/R) in rats. METHODS: Healthy male SD rats were assigned randomly into sham-operation (sham) group, I/R group and IPC group with 10 rats in each group. The rats in sham group were only exposed the right common, internal and external carotid artery surgically. The rats in I/R group were subjected to right middle cerebral artery occlusion (MCAO) by the modified Longa suture method for 2 h followed by 24 h of reperfusion. The rats in IPC group were subjected to MCAO for 2 h followed by reperfusion of the ipsilateral common carotid artery occlusion for 10 s for 5 episodes, and then reperfusion for 24 h. Autophagy was obeserved by transmission electron microscopy (TEM). The protein levels of mammalian target of rapamycin (mTOR), p-mTOR and microtubule associated protein light chain 3 (LC3)-II in brain tissue of the rats were determined by Western blot. Pathological changes of brain tissue were observed by HE staining. RESULTS: The protein levels of mTOR and p-mTOR in IPC group were significantly higher than those in I/R group (P<0.05). The expression of LC3-II in IPC group was significantly lower than that in I/R group (P<0.01). The cerebral infarction area and brain water content in IPC group were significantly lower than those in I/R group (P<0.01). HE staining showed that neurons degeneration and necrosis in IPC group were significantly alleviated compared with I/R group. TEM observation showed that IPC revealed fewer autophagosomes, with much less severe cell damage than that in I/R group. CONCLUSION: IPC reduces brain ischemia reperfusion damage by decreasing autophagy of brain cells, which might be related to the activation of mTOR.     

Effect of Ginkgo biloba extract on cerebral ischemia during early stage of subarachnoid hemorrhage in rats

AIM: To investigate the effect of Ginkgo biloba extrac (GBE) on cerebral ischemia during early stage of subarachnoid hemorrhage (SAH). METHODS: Noncraniotomy models of SAH in Wistar rats were used and animals were divided into sham-operated group, SAH group and SAH+GBE group. Dynamic change of regional cerebral blood flow (rCBF) was detected. Brain endothelin-1(ET-1) and calcium contents were also determined at different time point during 24 hours after the operation. Pathological change of neurons of hippocampus CA1 region was observed. RESULTS: In SAH group, rCBF decreased immediately and persistently after induction of SAH. Values of brain ET-1 content and calcium content at 1 hour, 6 hours and 24 hours were significantly higher than those in sham-operated group. Neurons of hippocampus CA1 region were damaged severely 3 days after onset of SAH. Above abnormal changes in SAH+GBE group were much slighter than those in SAH group. CONCLUSION: GBE may relieve cerebral ischemic damage after SAH.     

Progress in brain protection with ischemic postconditioning

Cerebrocardiovascular diseases and stroke are serious threat to human health. More attentions focus on cerebral ischemia and the publications suggest that ischemic preconditioning prevents ischemic myocardium from ischemia/reperfusion injury, soon followed by preconditioning against cerebral ischemic injury. However, in clinical management, ischemia is often unpredictable. In recent years, researchers found that ischemic postconditioning and ischemic preconditioning obtained the similar effects on brain protection after cerebral ischemia. The endogenous protective mechanisms play a key role in protecting against brain ischemic injury. As a novel manner to protect against brain injury, postconditioning attenuates the infarct volume following brain ischemia/reperfusion and prompts the neurological recovery by prolonging the therapeutic time window. This article reviews the processes of ischemic, hypoxia, hypothermia and pharmacologic postconditioning, and the possible brain protection mechanisms activated by the signal transduction of serine/threonine kinase (Akt), phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and ATP-sensitive potassium channel (K_ATP) pathways.     

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.     

Relation between astroglial activation state and ischemic tolerance in the gerbil hippocampus

AIM:To observe the relation between astroglial activation state and ischemic tolerance in the gerbil hippocampus. METHOD: Bilateral occlusion of common carotid arteries and immunofluorescent methods in the gerbil hippocampal tissue. Slices were used. The morphological changes of the neurons were observed by light microscopy.RESULTS:Pretreatment with 2 minute bilateral carotid artery occlusion produced protective effects of CA₁ neurons. Glial fibrillary acidic protein (GFAP) staining showed weak positive cells in control group. Most of GFAP positive cells showed weak and middle positive cells after recirculation 1d and 2d following ischemic 3.5 min and preconditioning of the brain with sublethal ischemia respectively.CONCLUSION:Astroglial played an important role in cerebral ischemia. It is possible that state of astroglial activation related to neuronal survival in ischemic tolerance.     

Effects of hyperglycemia on ionic homeostasis in hippocampal microenvironment and secondary neuronal injury after focal ischemia in cerebral cortex of tree shrews

AIM：To observe the effects of hyperglycemia on the ionic homeostasis in hippocampal microenvironment after thrombotic cortical ischemia in tree shrews, and to explore the action and mechanisms of hyperglycemia in secondary neuronal injury after ischemia. METHODS：High blood glucose in tree shrews was induced by intraperitoneal injection of streptozocin. Focal thrombotic cortical ischemia was induced by photochemical method in tree shrews. At 4, 24 and 72 h after ischemia, the changes of pH, K+, Na+, Ca2+ and Cl- in the ipsilateral ischemic hippocampal microenvironment were tested by a single-pumped push-pull microdialysis system and an ion analyzer. The histopathological changes and hippocampal neuronal density were also examined. RESULTS：After cortical ischemia in tree shrews, the pH and the concentrations of Na+, Ca2+ and Cl- in the hippocampal microenvironment decreased, while the concentration of K+ increased. These differences were the most significant at 4 h, the second at 24 h and insignificant at 72 h. Combination of hyperglycemia and cerebral ischemia worsened the turbulence of ionic homeostasis. Compared with the normoglycemic ischemic animals, the changes of pH, K+ and Ca2+ concentrations at 4 h as well as pH and Na+ at 24 h in the hyperglycemic ischemic animals were more significant (P<0.05). The results of histopathological examination showed that there was ischemic neuronal damage in the exposed cerebral cortex and the ipsilateral hippocampal CA1 region at 4 h after photochemical reaction, and the damage was the most severe at 24 h, subsequently accompanied with glial proliferation at 72 h. The hyperglycemic ischemic animals suffered from greater neuronal injury in the cortex and hippocampus than the normoglycemic ischemic animals, especially at 24 h (P<0.01) and 72 h (P<0.05). CONCLUSION: The disturbance of acid-base equilibrium and ionic homeostasis in hippocampal microenvironment, following the spreading of the microenvironment in ischemic core, may be an important reason for secondary neuronal injury in the hippocampus after thrombotic cortical ischemia in tree shrews. Hyperglycemia aggravates the turbulence of ischemic ionic microenvironment.     

Study on changes of neurological function and neuron apoptosis after intravenous administration of bone marrow stromal stem cells for treating permanent focal cerebral ischemia in rats

AIM: To explore the survivorship and the mechanism of the intravenous administration of bone marrow stromal stem cells (BMSCs) for treating permanent focal cerebral ischemia in rats. METHODS: After purified, proliferated, and marked with BrdU, the BMSCs were injected intravenously into rats 1 d after focal cerebral ischemia.Modified neurological severity score (mNSS) was evaluated before and following 1, 7, 14 and 28 d after middle cerebral artery occlusion (MCAO). Rats were executed at 1, 7, 14 and 28 d after MCAO. Brain sections were stained with hematoxylin and eosin (HE) for determining the infarct volume. Slides were stained by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) and immunostaining for cleaved caspase-3 method for apoptosis detection and mechanism exploration in situ.RESULTS: mNSS in BMSCs-transplanted group at 14th day and 28th day of MCAO was significantly lower than that in control group(P<0.05). TUNEL-positive cells in the hippocampus and thalamus area of BMSCs-transplanted rats were significantly fewer than those in control rats at 14th day and 28th day of MCAO(P<0.05). Double immunostaining showed that small grafted BMSCs and small endogenous neural cell apoptosis depended on the capase-3 in hippocampus.CONCLUSION: The intravenous administration of BMSCs promotes the recovery of neurological function of rats with focal cerebral ischemia. The therapeutic effect of BMSCs on rats with focal cerebral ischemia may be derived from the reduction of apoptosis and the mobility and migration of endogenous neural stem cells in the ischemic boundary zone.     

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.     

Changes of dendritic morphology and spine density in hippocampal CA1 pyramidal cells of chronic cerebral ischemic rats

AIM: To investigate the changes of dendritic morphology and spine density in hippocampal CA1 pyramidal cells of the chronic cerebral ischemic rats. METHODS: The model of chronic cerebral ischemia was established by permanent occlusion of the bilateral common carotid arteries (2VO) in rats. Two weeks, 4 weeks or 8 weeks later, the behavior of the rats in each group was evaluated through the Morris water maze to select the successful modeling, and the brains were collected for processing Golgi staining. The changes in dendritic branch and length, and spine density in hippocampal CA1 pyramidal cells were observed under optical microscope. RESULTS: Compared with sham-operated group, dendritic branch and length in model group was significantly reduced in 4-week group and 8-week group (P<0.01), and spine density in model group were significantly reduced in 2-week, 4-week and 8-week groups (P<0.01). With prolonged ischemia, dendritic branch and length, and spine density in model group were all significantly reduced (P<0.05). CONCLUSION: Chronic cerebral ischemia leads to traumatic changes in dendrites and spines in hippocampal CA1 pyramidal cells, which constitutes the pathophysiological basis in the progressive cognitive dysfunction.     

Ischemic preconditioning relieves the ischemia/reperfusion injury of neurons in hippocampus by inhibiting the expression of p53

AIM: To examine whether ischemic preconditioning (IPC) can protect against apoptosis in CA1 subfield of hippocampus following reperfusion of a lethal ischemia in rats and explore the role of IPC by inhibiting the expression of p53 in this process. METHODS: Wistar rats were used in the experiment. A global ischemia/reperfusion model was induced by 4-vessel occlusion. The rats were divided into the following three groups randomly: (1) ischemic preconditioning group (IPC group); (2) ischemia/reperfusion group (IR group); (3) control group. The histopathological changes, the percentage of apoptosis and the expression of p53 gene in CA1 region of rat hippocampus were examined by HE staining, FCM, RT-PCR and immunohistochemistry techniques. RESULTS: The neuronal density of CA1 region in IPC group ［(217±9)/0.72 mm²］ was significantly higher than that in IR group ［(29±5)/0.72 mm², P<0.01］. The percentage of apoptotic neurons in IPC group (2.07%±0.21%) was lower than that in IR group (4.26%±0.08%), P<0.01. Compared with IR group, the expression of p53 gene in IPC group was significantly weakened. CONCLUSION: Ischemic preconditioning protects the ischemic neurons in CA1 region of rat hippocampus by inhibiting the expression of p53 gene.