Effect of phosphodiesterase inhibitor IBMX on cGMP production in gerbil hippocampus

AIM: To observe the effects of IBMX on cGMP production in gerbil hippocampus after recirculation following ischemia. METHODS: Bilateral occlusion of common carotid arteries and immunofluorescent staining methods in gerbil hippocampal tissue slice were used. RESULTS: Recirculation following ischemia caused a rise in hippocampus cGMP concentration. IBMX increased cGMP production. cGMP positive cells mainly distributed in rediatum layer and molecular layer in the CA1 subfield. CONCLUSION: IBMX increased cGMP production in gerbil hippocampus after recirculation following ischemia.     

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.     

Protective roles of hypobaric hypoxic pretreatment on hippocampus neurons in mice

AIM: This study was designed to investigate the protective role of hypobaric hypoxic pretreatment (HHPT) on hippocampal neurons in Babl/c inbred mice. METHODS: HHPT was produced by simulating a 7 000 m high altitude 2.5 h/d for 3 d. At 36 h after last time decompression, three subgroups consisted of both the control and pretreatment mice were subjected to the 12 000 m high altitude hypobaric hypoxia for 4 h, the severe ischemia produced by bilateral common carotid artery occlusion for 18 min, and the severe ischemia/hypoxia produced by permanently ligation of right lateral common carotid artery followed by a 8 000 m high altitude hypobaric hypoxia for 4 h, respectively. The extents of protection to hippocampal CA1 neurons by HHPT were evaluated by accounting the number of intact neurons between HHPT and control group. RESULTS: The results indicated that HHPT protected hippocampal neurons against severe hypobaric hypoxia, severe ischemia, and ischemia combined with hypobaric hypoxia. CONCLUSION: Hypobaric hypoxic pretreatment induces a delayed protection to hippocampal neurons against hypoxic and ischemic injuries.     

OX1R expression in chronic ischemic brain tissue of rats

AIM: To study the orexin-1 receptor (OX1R) expression in chronic ischemic brain tissue of rats and the change following the ischemic process.METHODS: The cerebral ischemic model was established by ligating double carotid arteries in rats. The behavior of the models was evaluated by water maze. The OX1R expression was determined by immunohistochemical technique, and the location of OX1R expression was further confirmed by double immunofluorescent staining.RESULTS: The intelligence of rats 15 d after ligating double carotid arteries was impaired obviously, and improved in 1 month and 2 months compared with the model in 15 d. At the same time, the OX1R expression increased obviously from acute phase until 15 d of cerebral ischemia and decreased notably in 1 month compared to the model in 15 d, and then increased significantly for the second time in 2 months of model. From the histology, partial neurons of 15 d model rats got atrophy, and most neurons in 1 month model rats got cytomorphosis and atrophy, however partial neurons in 2-month model rats recovered normally. The OX1R expression was confirmed in neurons definitely by double immunofluorescent staining.CONCLUSION: During the pathologic process of chronic ischemic injury, orexin system has two-way regulatory functions through.     

Protective effect of ischemic preconditioning on rat retina with sustaining ischemia

AIM: To observe the effect of ischemic preconditioning (IPC) on the subsequent permanent ischemic rat retina. METHODS: A model of two-vessel occlusion (2VO) was used to give an ischemic insult to rat retina. Bilateral carotid arteries were occluded directly in the simple ischemic groups. A procedure of double 2 min ischemia- 3 min reperfusion was applied to IPC groups before their carotid arteries were occluded. Experimental control groups received the same operation except that their exposed vessels were not occluded. Other 6 normal rats served as immunohistochemical controls. Eyeballs were taken out after being subjected to 1, 3 and 7 days of ischemia. The morphometry of retina was measured by stereologic methods. The apoptosis during the retinal ischemia was detected by TUNEL. Immunohistochemistry staining was used for the localization of bcl-2 in retina. RESULTS: In IPC groups, the thickness of retina was thinner than that in simple ischemic groups. The apoptosis was less when compared with the simple ischemic groups at the same ischemic time. The apoptotic cells show only in INL and the apoptosis of RGCs was not appeared until 7 days postischemia. There was no significant difference of the numerical density of RGCs at the different time after the operation. The bcl-2 immunoreactivity was weaker than that of the simple ischemic groups too. CONCLUSION: IPC reduced the injury caused by ischemia in retina, showing a protective effect.     

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.     

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.     

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 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.     

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.     

Effect of limb ischemic postconditioning on brain focal ischemia and reperfusion injury in mice

AIM: To establish the mouse model in which the limbic ischemic postconditionning (LIPostC) enhances the tolerance against brain ischemia, and to investigate the effects of LIPostC on the ischemic extent and roles of heat shock protein 70 (HSP70) in ischemia and reperfusion injury. METHODS: The male Kunming mice were used in the study. The brain ischemia reperfusion (I/R) model was made by middle cerebral artery occlusion (MCAO). In the first test, the male mice were randomly divided into 9 groups (n=10): sham group, ischemia/reperfusion (I/R) groups (with ischemia for 0.5 h, 1 h,1.5 h and 2 h) and LIPostC+I/R groups (0.5 h+LIPostC,1 h+LIPostC,1.5 h+LIPostC,2 h+LIPostC). The reperfusion was performed after LIPostC for 24 h. After the neurologic deficit scores were evaluated, the brains were taken out to measure the infarct volume with TTC staining and to observe the pathological changes of cerebral cortex with HE staining. The neuronal apoptosis was determined by TUNEL. In the second test, the male mice were randomized into 4 groups (n=6): sham group, I/R group, LIPostC+I/R group and LIPostC+I/R+quercetin group (2 h ischemia). The neurological deficit scores were evaluated at 24 h after operation. The expression of HSP70 was determined by Western blotting.RESULTS: The duration of brain ischemia was related to the motor behavior and degree of brain injury. The longer the ischemic duration of the brain was performed, the more severe the pathological and behavioral changes were observed. The brain injury in 2 h MCAO mice was more severe than that in 1 h and 1.5 h MCAO mice (P<0.05). Compared to I/R group, each LIPostC group showed lower neurological score, less infarct volume and TUNEL positive neuron. The expression of HSP70 protein was increased and neurological functions were improved significantly in the mice with LIPostC. However, the neuroprotective role of LIPostC was attenuated by treating with quercetin, an inhibitor of HSP70.CONCLUSION: LIPostC promotes the expression of HSP 70, improves the neurological functions and attenuates the ischemia and reperfusion injury in MCAO mice. HSP70 produces a marked effect on the ischemic tolerance induced by LIPostC in MCAO mice.     

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.     

The mechanism of neuronal injury and repair after focal cerebral ischemia

AIM:To explore the mechanism of neuronal injury and repair by investigating the expression of caspase-3 and apurinic/apyrimidinic endonuclease (APE/Ref-1) after focal cerebral ischemia. METHODS:A model of middle cerebral artery occlusion in rats was performed. The expression of caspase-3P₂₀ and APE/Ref-1 was examined by immunohistochemistry staining, TUNEL was applied to detected DNA damage, and double labeling with TUNEL and APE/Ref-1 was used to determine the relationship between APE/Ref-1 and DNA damage. RESULTS:The active subunit P₂₀ of caspase-3 was predominantly expressed within ischemic penumbra. The peak time of caspase-3P₂₀ positive cells preceded the appearance of TUNEL. With aggravation of cerebral ischemia, APE/Ref-1 immunoreactive cells in penumbra were significantly decreased. CONCLUSION:The activation of caspase enzymatic cascade following cerebral ischemia leads to degradation in DNA, meanwhile, decrease in DNA repair molecules or the failure of DNA repair may deteriorate the course.     

Acute cerebral ischemia activates EphB2/ephrin-B1/NMDA receptor signaling pathway to promote hippocampal neurogenesis in mice

AIM To investigate the effect of acute cerebral ischemia on hippocampal neurogenesis in mice and its possible mechanism involving EphB2/ephrin-B1/NMDA receptor signaling pathway. METHODS C57BL/6 mice (n=52) were randomly divided into sham group and acute cerebral ischemia group (model group). The model of acute cerebral ischemia in mice was established by bilateral common carotid artery occlusion. The pathological changes of the hippocampal CA1 region in mice were observed by HE staining. The learning and memory functions of the mice were assessed by Morris water maze. The BrdU positive cells and doublecortin (DCX) protein expression were observed by immunofluorescence staining for detecting hippocampal neurogenesis. The mRNA and protein expression levels of EphB2, ephrin-B1, reelin, microtubule-associated protein-2 (MAP-2) and NMDA receptor subunits NR2A and NR2B in the hippocampus were determined by RT-qPCR and Western blot. RESULTS The neuronal damage in the hippocampal CA1 region was significant (P<0.01), and the learning and memory functions were significantly decreased in the cerebral ischemia mice(P<0.01), suggesting that the cerebral ischemia model was successfully established. The BrdU positive cells and DCX protein expression were increased significantly (P<0.01), indicating that neurogenesis occurred in the hippocampus after cerebral ischemia. At the same time, the mRNA and protein expression levels of EphB2, ephrin-B1, reelin, MAP-2, NR2A and NR2B in the hippocampus were also significantly up-regulated (P<0.05). CONCLUSION Acute cerebral ischemia promotes the proliferation of hippocampal neural stem cells and endogenous neurogenesis, which may be related to the activation of EphB2/ephrin-B1/NMDA receptor signaling pathway.     

Influence of murine cytomegalovirus infection on the differentiation and the differentiation genes expression in neural stem cells

AIM: The influence of MCMV infection on differentiation and differentiation gene expression in neural stem cells(NSCs) in vitro were investigated for studying the mechanisms of brain abnormalities caused by congenital cytomegalovirus infection.METHODS: NSCs were separated from fetal BALB/C mouse, and cultured and identified in vitro. The differentiation potency of NSCs was observed by immunofluorescence. The NSCs infected by MCMV at dosage of MOI(multiplicity of infection) equaled to 5, 1 and 0.1,respectively, were cultured in differentiation medium. The morphological changes of infected cells were observed under inverted microscope. The ratios of NSCs and its differentiated cells were detected by flow cytometry. The expressions of nestin, GFAP and NSE, markers of NSCs and its differentiated cells, were studied by immunofluorescence(MOI=1). The expression of early antigen(EA) of MCMV was detected to observe the infection process. Real-time RT-PCR method was employed to measure the expression levels of the key genes Neurog2, Myc and Ccnd1 in Wnt signal pathway of NSCs at early stage of differentiation culture.RESULTS: NSCs isolated from embryonic mouse brains proliferated to form neurospheres, strongly expressed nestin and differentiated into NF-200 positive neurons or GFAP positive astrocytes. The infected NSCs did not adhere to the wall and appeared differentiation growths, but showed swollen gradually after differentiation culture. The nestin expression in the infected cells downregulated slowly and was higher than that in control groups(P<0.05). The GFAP and NSE expressions of the infected cells were lower than those in control groups(P<0.05). The early antigen(EA) of MCMV was always detected in the cells in infected groups. The ratios of nestin positive cells in infected groups were higher than those in control groups, but the ratios of GFAP and NSE positive cells of former were lower than that of the latter from 3rd to 9th d after differentiation culture(P<0.05). The levels of Neurog2 mRNA and Myc mRNA in infected groups were markedly lower than those in normal control groups on 1st d and from 1st to 4th d after differentiation culture, respectively(P<0.05). The levels of Ccnd1 mRNA of infected groups were obviously lower than those in normal control groups from 12th h to 1st d(P<0.05). These changes in infected groups became more obvious as MCMV MOI increased.CONCLUSION: MCMV significantly inhibits differentiation of NSCs to neurons and astrocytes, and leads to the decrease in differentiated cells. MCMV inhibits or interferes with the gene expression of Neurog2, Myc and Ccnd1 in Wnt signal pathway of NSCs. The effect that MCMV inhibits the expressions of differentiation and the differentiation genes in NSCs shows dose-dependent with MCMV MOI. The inhibitory effect of MCMV on the differentiation of NSCs might be induced by interfering with the expression of differentiation gene in NSCs, which is possibly the one of primary causes of brain development disorders induced by congenital CMV infection.     

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.     

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.     

Effect of cerebral ischemic preconditioning on NOS activity and NO content in the CA1 region of the hippocampus in rats

AIM: To explore the role of NO in the induction of brain ischemic tolerance (BIT) by observing changes of NOS activity and NO2-/NO3- content following a transient cerebral ischemia. METHODS: The rat 4-vessel occluding brain ischemic model was used. 140 male Wistar rats were divided into sham, cerebral ischemic preconditioning (CIP), ischemic insult and CIP+ischemic insult groups. An occlusion of the 4 vessels for 3 min was normally used as CIP, and a relative long one for 10 min was used as ischemic insult. When CIP was followed by ischemic insult, the interval between them was 3 d. The CA1 region of the hippocampus of rats was dissected out at 0 h, 2 h, 16 h, 24 h, 36 h, 72 h and 7 d after the last time of ischemia to assay its NOS activity and NO2-/NO3- content. RESULTS: The NOS activity and NO2-/NO3- content began to increase at 16 h, peaked at 24 h and decreased to basal level at 36 h of reperfusion after CIP. The duration of the up-regulation of NOS activity and NO2-/NO3- content was much shorter than that of BIT, which usually takes place 1-7 d after CIP. The pattern of upregulation of the NOS activity and NO2-/NO3- content was similar to the CIP group, but the maximum (24 h) was much more than that in CIP group (P<0.05). In the CIP＋ischemic insult group, the NOS activity and NO2-/NO3- content increased at 2 h of reperfusion, but the maximum (24 h) were much lower than that in ischemic insult group (P<0.05). CONCLUSION: A moderate increase in NOS activity and NO production after CIP might participate in the induction of BIT by triggering a series of cellular signal transduction. In addition, inhibiting effect of CIP on over-production of NO caused by ischemic insult might be another way to induce BIT.     

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.