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.     

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.     

Effects of hyperglycemia and cerebral ischemia on VEGF expression in different subfield of cerebral cortex in tree shrews

AIM: To observe the changes of VEGF expression in different subfield of brain in tree shrews during hyperglycemia and focal cerebral ischemia, in order to explore the relationship between cerebral ischemia, hyperglycemia and VEGF. METHODS: High blood glucose in tree shrews was induced by intraperitoneal injection of streptozotoctin. Focal cortical thrombotic cerebral ischemia was induced by photochemical method in tree shrews. At 4 h, 24 h and 72 h after cerebral ischemia, the histopathological changes and hippocampal neuronal density were examined. VEGF expressions in the ischemic core, penumbra and contralateral cerebral cortex were detected by immunohistochemistry technique at different times after cerebral ischemia. RESULTS: The results of histopathological study showed that there was infarction zone in the exposured cerebral cortex at 4 h after photochemical reaction, and the damage was most severe at 24 h, subsequently accompanied with the glia multiplication and rehab reaction at 72 h. The animals in hyperglycemic ischemic group suffered from greater neurological lesion than the normoglycemic stroke animals, especially at 24 h (P<0.01) and 72 h (P<0.05) after cerebral ischemia. Immunohistochemical analyses of VEGF expression revealed that it started to increase at 4 h after brain ischemia in the penumbra, reached a peak at 24 h, and weakened at 72 h. The stimulated VEGF production was also observed in hyperglycemic only group. When hyperglycemia and brain ischemia were combined, the VEGF expression was higher than that in hyperglycemic only group (P<0.05). Compared to normoglycemic ischemic group, no additivity of the effects of hyperglycemia combined with brain ischemia was observed. CONCLUSION: (1) The model of experimental hyperglycemia and cerebral ischemia is replicated successfully by applying the method combined in vivo injection of streptozotocin in the lower primate tree shrew with thrombotic focal cerebral ischemia. (2) This study shows that hyperglycemia aggravates the focal cerebral ischemia damage. (3) Cerebral ischemia and hyperglycemia both can independently up-regulate VEGF expression, but there is no additional increase in VEGF expression when hyperglycemia combined with brain ischemia is applied.     

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.     

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.     

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.     

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.     

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.     

Effect of apigenin on expression of VEGF in cerebral ischemia and reperfusion rats

AIM: To investigate the effect of apigenin on the expression of vascular endothelial growth factor (VEGF) in the rats under the condition of cerebral ischemia and reperfusion. METHODS: Ninety-one male SD rats were randomly divided into 13 groups: sham operation group (S), model groups (group M6 h, group M24 h, group M72 h, group M7 d), apigenin treatment groups (group A6 h, group A24 h, group A72 h, group A7 d) and dexamethasone treatment groups (group D6 h, group D24 h, group D72 h, group D7 d). The acute transient focal cerebral ischemia reperfusion model was established by modified method of inserting the nylon thread into middle cerebral artery, staying for 2 h and then withdrawing from the artery. In the experiment groups, the TTC staining of brain slices were performed and the neurological behavior scores were determined. The expression of VEGF by immunohistochemistry (ICH) was semi-quantitatively analyzed by measuring the integrated absorbance(IA). RESULTS: Abnormal neurological behaviors were observed in the animals of M groups, A groups and D groups, but the neurological behaviors of the rats in A7 d group were better than that in the other groups (P<0.05). Typical cortical infarct lesions in M groups, A groups and D groups were found by TTC staining, mainly in cerebral cortex and striatum. The immunnohistochemical results showed that the expression of VEGF was significantly higher in M, A and D groups than that in S group (P<0.05). Moreover,the expression of VEGF in A groups(A24 h and A72 h)was higher than that in M groups (M 24 h and M72 h,respectively)(P<0.01).The expression of VEGF in D72 h group was higher than that in M72 h group (P<0.05), and that in A7 d group was obviously higher than that in D7 d group (P<0.01).CONCLUSION: Apigenin promotes the expression of VEGF in the model of acute transient focal cerebral ischemia-reperfusion injury, improves the process of brain injury and recovers the brain functions in rats.     

Effects of flavonoids isolated from Scutellaria stem and leaf on expression of NMDAR and VEGF in chronic cerebral ischemia rats

AIM: To study the effects of flavonoids isolated from Scutellaria stem and leaf (SSF) on the expression of N-methyl-D-aspartate receptor (NMDAR) and vascular endothelial growth factor (VEGF) in chronic cerebral ischemia rats. METHODS: The model of chronic cerebral ischemia was established by bilateral carotid artery occlusion for 2 months in female SD rats. The effects of SSF on mRNA expression of NMDAR in hippocampus and VEGF in cerebral cortex were evaluated by the method of RT-PCR. RESULTS: Compared with the sham group, the expression of NMDAR1, NMDAR2A and NMDAR2B in hippocampus and VEGF in cerebral cortex were significantly increased (P<0.01). However, the cerebral ischemia rats daily and orally administered with SSF at doses of 17.5 mg·kg^-1·d^-1, 35 mg·kg^-1·d^-1 and 70 mg·kg^-1·d^-1 for 38 days appeared that the mRNA expression of NMDAR1, NMDAR2A and NMDAR2B in hippocampus was obviously reduced (P<0.05), and the mRNA content of VEGF in the cortex (P<0.05) was increased. CONCLUSION: SSF decreases the expression of NMDAR in hippocampus, increases the expression of VEGF in cerebral cortex of cerebral ischemia rats, suggesting that the neuroprotective effect of SSF may be exerted by influencing the production of NMDAR and VEGF in the brain.     

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.     

Neuroprotective effect of wortmannin on epileptic rats by inhibiting autophagy

AIM: To investigate the role of autophagy in hippocampus injury induced by seizures and to observe the neuroprotective effects of autophagy inhibitor wortmannin(WM) on epileptic rats.METHODS: The Wistar rats were randomly divided into control group, model groups at 2 h, 8 h, 16 h, 24 h or 72 h after seizure induction by pilocarpine, and WM pretreatment group. The methods of HE and Nissl staining were used to evaluate the hippocampus injury. The expression of microtubule-associated protein 1 light chain 3(LC3) was detected by Western blotting. The ratio of LC3II to LC3I was calculated and used to represent the activity of autophagy. RESULTS: The significant increase in the ratio of LC3II to LC3I began at 2 h, peaked at 24 h, and maintained at high level at least to 72 h after seizure induction. Obvious neural injury and neuron depletion were observed in hippocampus CA1 area at 24 h after seizure induction. The number of surviving neurons at 24 h was sharply decreased in rats with seizures(75.50±5.92) as compared to the controls(110.67±18.56, P＜0.01). WM significantly decreased the neuron depletion induced by seizures(100.88±18.73, P＜0.05). Moreover, WM significantly decreased the ratio of LC3II to LC3I in rats with seizures at 24 h(P＜0.05). CONCLUSION: Autophagy was activated in hippocampus injury induced by seizures. WM reduces the transformation of LC3II to LC3I to inhibit the autophagy activated by seizures. WM has neuroprotective effect on epileptic rats by increasing the surviving neurons in hippocampus CA1 area.     

Effect of Chinese herbs 9602 on the alterations of evoking population spike and morphology in hippocampal CA1 region after ischemia/reperfusion in rats

AIM:To explore the relationship between dynamic changes of population spike (PS) and morphologic alterations in hippocampal CA1 region and morphology after transient ischemia/reperfusion and the improving effects of Chinese herbs 9602.METHODS:Changes of evoking population spike ware investigated by electrical stimulating Schaf er collateral in CA1 region of hippocampal slice after ischemia/reperfusion in vivo.Apoptosis and morphologic alterations at different time points after cerebral ischemia/reperfusion were detected by using TUNEL and Nissl staining.RESULTS:The threshold voltage of CA1 region in evoking population spike increased markedly as compared with sham control. The enhancement of wave amplitude was reduced significantly after tetanic stimulation. The duration of enhancement in amplitude decreased with the passage of reperfusion. Above all were observed from 8 h after ischemia/reperfusion. They became remarkable and got to its top at 7 day after ischemia/reperfusion treatment. TUNEL positive cells were observed in hippocampal CA1 region at 8 h, got to the top at 24 h and then gradually reduced after ischemia/reperfusion. A lot of abnormal cells in CA1 region was found, and the number of pyramidal cell reduced progressively by Nissl staining after ischemia/reperfusion. Chinese herbs 9602 reduced the threshold voltage of CA1 region in evoking population spike remarkably, enhanced the wave amplitude and prolonged the duration of PS enhancement; decreased the number of TUNEL positive cell, prevented the reduction of pyramidal cell in CA1 region. CONCLUSIONS:The excitability and reactivity were decreased and there was a gradual functional disturbance of synaptic transmission in CA1 pyramidal cell and most notable changes happened at 7 d ischemia/reperfusion, suggesting that was partly due to delayed neuronal death induced by ischemia/reperfusion. Apoptosis plays an important role in the functional deficiency of CA1 region of hippocampus induced by cerebral ischemia/reperfusion. The effects of 9602 on ameliorating the excitability and reactivity of CA1 pyramidal cells relate to inhibiting apoptosis, attanuating delayed neuron death induced by ischemia/reperfusion.     

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.     

Age-related changes of expression of Bcl-2, Bax and caspase-3 activity after cerebral ischemia/reperfusion in aged rats

AIM: To study age-related changes of expression of Bcl-2, Bax and caspase-3 activity after focal cerebral ischemia/reperfusion (I/R) in aged rats. METHODS: The aged SD rats (20-21 months) and the young animals (4-5 months ) were subjected to 3 h of middle cerebral artery occulsion with the intraluminal filament technique, followed by 3 h, 6 h, 12 h, 24 h and 72 h of referfusion. Expression of Bcl-2, Bax and caspase-3 activity of the young and the aged rats were examined. RESULTS: Cerebral infarct zone increased in the aged at ischemia 3 h and I/R 12 h than that in the young. With I/R time longer, increase in neuron apoptosis showed early and lasted longer in the aged. The Bcl-2 expression increased in the young with I/R time longer, but was not obvious in the aged. Bax expressd obviously and early, and kept on longer in the aged during I/R than that in the young. The enhanced activity of caspase-3 showed early in the aged than that in the young during I/R.　CONCLUSION: The mechanisms of serious cerebral injury and neuron apoptosis might be related to the increase in Bax expression and caspase-3 activity.     

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 maternal limb ischemic preconditioning on structural and functional changes of mitochondria in fetal hippocampal neurons induced by intrauterine distress-reoxygenation in rats

AIM: To investigate the effects of maternal limb ischemic preconditioning (LIP) on the mitochondrial structures and functions of the hippocampal neurons induced by reoxygenation in the intrauterine distress fetal rats. METHODS: Pregnant rats (n=40) were randomly divided into 4 groups: sham (S) group, LIP group, fetal distress (FD) group and LIP+FD group. Intrauterine ischemia model was established through the experimental design. The ultrastructure of the mitochondria in CA1 area of the hippocampus was observed. The mitochondrial membrane potential and reactive oxygen species (ROS) were measured. The content of ATP and MDA in the hippocampus tissue was detected. The activity of Mn-SOD was observed. RESULTS: Compared with sham group, the ultrastructure of mitochondria in CA1 area of the hippocampus was damaged in FD group and LIP+FD group. The mitochondrial membrane potential, the content of ATP and the activity of Mn-SOD were decreased. However, the content of ROS and MDA was increased. Compared with FD group, the ultrastructure of mitochondria in CA1 area of the hippocampus was intact in LIP+FD group. Furthermore, the reduced mitochondrial membrane potential and ATP content were inhibited. The activity of Mn-SOD was increased, but the content of ROS and MDA was decreased in LIP+FD group. CONCLUSION: Limb ischemia preconditioning inhibits the damage the mitochondria of fetal hippocampal neurons induced by reoxygenation in the intrauterine distress fetal rats.     

Preliminary study on pyroptosis involved in cerebral ischemia-reperfusion injury

AIM:To investigate the changes of pyroptosis in hippocampus and cortex at different time points after cerebral ischemia-reperfusion, and to explore its mechanism from NLRP3-mediated classical pyroptosis pathway, and to analyze the role of pyroptosis in different parts of cerebral injury. METHODS:SD rats were randomly divided into sham operation group (sham group) and model group (MCAO/R group). The rats in model group was further divided into cerebral ischemia-reperfusion 6 h group (MCAO/R 6 h group), 12 h group (MCAO/R 12h group)and 24 h group (MCAO/R 24 h group). The rat model was established on rats by middle cerebral artery occlusion and reperfusion (MCAO/R) induced by modified right-side thread method. Neurologic function score, 2, 3, 5-triphenyltetrazolium chloride (TTC) staining and morphological observation were used to evaluate the degree of nervous cell injury. TUNEL and caspase-1 immunofluorescence double staining were used to detect pyroptosis. The protein expression of NLRP3, cleaved caspase-1, pro-caspase-1 and interleukin-1β (IL-1β) was determined by Western blot. RESULTS:Neurological damage occurred at different times after cerebral ischemia-reperfusion. TTC staining showed that the volume of cerebral infarction gradually increased with the prolongation of reperfusion time (P<0.05). The hippocampal CA1 area and cortical area showed typical morphological features such as loose tissue structure, interstitial edema, disordered arrangement of nerve cells, deepening of nucleus staining, nuclear fragmentation and decreased cell number. Immunofluorescence double staining showed that there was a phenomenon of pyroptosis at different time after cerebral ischemia-reperfusion. The pyroptosis of hippocampal CA1 and cortical area was most obvious at 12 h and 24 h after reperfusion (P<0.05). Western blot analysis showed that the expression of NLRP3, cleaved caspase-1, pro-caspase-1 and IL-1β in NLRP3-mediated classic pyroptosis pathway was regulated in different degrees after cerebral ischemia-reperfusion. The protein expression of NLRP3 in hippocampus was significantly increased at 12 h and 24 h after reperfusion (P<0.05), and the protein expression of NLRP3 in cortex was significantly increased at 6 h after reperfusion (P<0.05). The protein expression of pro-caspase-1 in hippocampus was significantly increased at each time points of reperfusion (P<0.05), and the protein expression of pro-caspase-1 in the cortex was significantly increased at 24 h after reperfusion (P<0.05). The protein expression of cleaved caspase-1 in the hippocampus was significantly increased at 12 h after reperfusion (P<0.05), and increased in the cortex at 24 h after reperfusion (P<0.05). The protein expression of IL-1β in the hippocampus was significantly increased at 24 h after reperfusion (P<0.05), and increased in the cortex at 6 h after reperfusion (P<0.05). CONCLUSION:Pyroptosis is involved in neuronal injury after cerebral ischemia-reperfusion. The classic pyroptosis pathway plays an important regulatory role in hippocampus and cortex, especially in hippocampus, suggesting that hippocampus is the main part of secondary nerve impairment induced by pyroptosis and inflammation after cerebral ischemia-reperfusion.     

Effect of peurarin pretreatment on endothelial nitric oxide synthase in rat brain tissues at the early stage of cerebral ischemia in rats

AIM: To investigate the neuroprotective effect of puerarin on the expression of endothelial nitric oxide synthase (eNOS) in rat brain tissues at the early stage of cerebral ischemia.METHODS: Forty-five rats were randomized into 3 groups: 5 in sham-operated group (S group), 20 in cerebral ischemia group (M group) and 20 in puerarin pretreatment group (P group).The rats in M group and P group were further divided into 4 subgroups to apply cerebral ischemia for 0.5 h, 1 h, 2 h and 4 h,respectively.The rats were subject to middle cerebral artery occlusion (MCAO) except those in S group.Puerarin was administered with intraperitoneal injection (100 mg/kg, ip) in P group 10 min before MCAO.The equal volume of the vehicle was administered in M groups and S group at the same time.Neurological deficit scores were determined to evaluate the functional changes of the central nervous system.The pathological changes of the brain tissues were observed under microscope with neuron nissl body staining.The protein expression and distribution of eNOS in the brain tissues were evaluated by the methods of immunohistochemistry and Western blotting.RESULTS: Neurological deficit scores of the rats in all subgroups of P groups were significantly lower than those in the corresponding subgroups of M groups (P<0.05).The dissolution extent of neuron nissl body in P groups was lower than that in M groups.The protein expression of eNOS in the brain tissues in all subgroups of P groups was higher than that in the corresponding subgroups of M groups.CONCLUSION: Pretreatment with puerarin protects brain tissues from injury of cerebral ischemia at the early stage by up-regulating the protein expression of eNOS in the brain tissues.