| At present, the incidence of ischemic cerebrovascular disease is rising inthe whole world and the situation is increasingly grim. Over the years, manyresearchers have devoted to the endogenous protective mechanisms of brainischemic tolerance to increase the resistance to ischemic damage of neuronalcells, and make it remain alive after sufferring from severe ischemic insult,and still have normal physiological function after the restore of blood flow.In our previous work, it has been demonstrated that the limb ischemicpreconditioning (LIP) via3cycles of transient occlusion (10min) and openingof the bilateral femoral arteries immediately before global brain ischemicinsult for8min could alleviate the subsequent brain ischemia/reperfusioninjury and increase the survival of the pyramidal neurons in the CA1hippocampus. These results confirmed that the protective effect of remoteischemic preconditioning on brain. Although cerebral ischemia can’t bepredicted, but global brain ischemic injury is predictable in brain ischemiahigh risk surgery, such as cardiopulmonary bypass and so on. If interventionscan be given before damage, it undoubtedly has important practicalsignificance. Therefore, the studies of the mechanisms of LIP on brainprotection will provide a new view for the development of neuroprotectivedrugs and methods.MAPK is a kind of serine/threonine protein kinase in cells. The followingparallel MAPKs signaling pathways have been found in mammalian cells:ERK, JNK and p38MAPK pathways. Our previous studies have confirmedthat p38MAPK participated in the induction of brain ischemic tolerance byLIP in rats. It raised the expression of HSP70, inhibited apoptosis pathway,and changed the activity of superoxide dismutase (SOD). However, themechanisms of p38MAPK neuroprotection are not fully elucidated. Glutamate is a major excitatory neurotransmitter in the central nervoussystem. However excessive levels of glutamate are strong neurotoxicity. Brainischemic injury leads to significant increase of extracellular glutamate, whichresults in cell damage through the calcium overload and interfering energymetabolism. That excitatory amino acid transporter (EAATS) uptake glutamicacid is the main way of that extracellular glutamate is cleared. Fivehigh-affinity, sodium-dependent glutamate transporters have been cloned frommammalian tissue, including EAAT1(glutamate/aspartate transporter,GLAST), EAAT2(glail glutamate transporter-1, GLT-1）, EAAT3(excitatoryamino acid carrier1, EAAC1), EAAT4and EAAT5. Many studies havedemonsrated that GLT-1plays a more important role in preventing the toxicityof excitatory effect. Therefore, modulation of GLT-1to enhance theglutamate’s uptake in the extracellular fluid and prevent the occurrence ofreverse transportation is an effective method to prevent glutamateneurotoxicity. Our previous studies have found that brain ischemicpreconditioning inhibited the delayed neuronal death which caused by brainischemia, and increased the expression of GLT-1in CA1hippocampus. Butwhen the rats were applied to GLT-1specific inhibitors DHK or AS-ODNsbefore brain ischemic preconditioning, the cerebral ischemia preconditioningprotective effect could be blocked. The above results showed that GLT-1participated in the brain ischemic tolerance induced by brain ischemicpreconditioning.Based on the above data, we can suspect whether GLT-1participates inthe induction of brain ischemic tolerance by LIP? And whether there is therelationship between activation of p38MAPK and GLT-1? In fact, theprevious reports have showed that p38MAPK could modulate the expressionof GLT-1. In2009, Tsai found that the p38MAPK inhibitor SB203580attenuated the PTX-induced mechanical allodynia, thermal hyperalgesia,increased in spinal cerebrospinal fluid excitatory amino acids, anddownregulated the expression of GTs. And Matos etc in Alzheimer’s diseaseresearch confirmed that SB203580can reduce the GLT-1expression in astrocytes. In recent research, my lab also confirmed that p38MAPK throughmodulation GLT-1plays an important role in ischemic preconditioninginduced by cerebral ischemia tolerance.Therefore, the purpose of this study was to:①Investigate the differenttime windows of the effect of LIP in brain ischemic reperfusion. And observethe effect of GLT-1specific inhibitors DHK on the ischemic tolerance inducedby LIP by inhibiting the expression of GLT-1;②Observe and compare time ofthe expression of p38MAPK and GLT-1in CA1hippocampus after LIP;③Observe the change of the expression of GLT-1in CA1hippocampus afterapplying p38MAPK inhibitor SB203580. Through the above studies, thispaper will discuss that whether p38MAPK involve in the brain ischemictolerance by LIP by modulating GLT-1, and will provide a new view ofpreventing brain ischemic diseases.1LIP reduced delayed neuronal death in CA1hippocampus of rats induced bybrain ischemia/reperfusion and the role of GLT-1in this process1.1Investigate the different time window of the effect of LIP in brainischemia/reperfusion50Wistar rats whose bilateral vertebral arteries were occludedpermanently were randomly divided into four groups:①brain ischemic insultsham group（n=5）: bilateral common carotid arteries were exposed for8min,but without blocking the blood flow.②brain ischemia insult group(n=5):bilateral common carotid arteries were clamped for8min.③LIP+brainischemic insult group (n=35): bilateral femoral arteries were occluded for10min,3times, at10min intervals. Bilateral common carotid arteries wereclamped for8min after LIP. The LIP+brain ischemic insult group was furtherdivided into LIP0h,1h,3h,6h,12h,1d and2d groups according to thetime of reperfusion after LIP.④LIP sham+brain ischemic insult group(n=5):According to the results of③, brain ischemic insult for8min was underwentimmediately after bilateral femoral arteries were exposed but not clipped for50min. The animals were sacrificed by decapitation at7d after the shamoperation or the last time of ischemia. Two sets of brain sections were made, one set for neuropathological evaluation by thionin staining, and another onefor HE staining. Histological changes of the hippocampus were evaluatedusing thionin staining by histological degree (HG) and neuronal density (ND).HG was divided into four grades:0: no neuron death;: scattered singleneuron death;Ⅱ: mass neuron death; Ⅲ: almost all neuron death. The ND ofthe hippocampal CA1subfield was determined by counting the number ofsurviving pyramidal neurons with intact cell membrane, full nucleus, and clearnucleolus within1mm liner length of the CA1. The average number ofpyramidal neurons in3areas of the CA1hippocampal was calculated toestablish the ND value. HE staining was used to observe the changes of gliacells.The thionin staining showed that the pyamidal neurons in hippocampalCA1subfield were unchanged in the brain ischemic insult sham group, thestructure of neurons was arranged in order, the outline of the neurons wasintact, no significant delayed neuronal death (DND) was observed, the HGwas0, and the ND was167±5.21. Obvious DND in hippocampal CA1subfield was observed in brain ischemic insult group and LIP sham+brainischemia insult group. The pyamidal neurons in hippocampal CA1subfieldwere changed significantly. The HG was III, and the ND was36±9.28and35±5.93. Compared with the brain ischemic insult sham group, brain ischemiainduced significant damage of the neurons, HG was significantly increased(P<0.01) and ND was significantly decreased (P<0.01). Pyramidal neuronswere not seen obvious damage in LIP0h,1h,3h and6h prior to brainischemia. Especially in LIP0h group, the structure of neurons was arrangedin order. HG was decreased (0～) and ND was increased (154±5.93)comparing with the LIP sham+brain ischemic insult group (P＜0.05).However, significant DND was observed in LIP12h, LIP1d and LIP2dgroups. There was no significant difference compared with the LIP sham+brain ischemic insult group. The HG was II～III, and the ND was46±7.66,40±7.69and42±10.80respectively. These results suggested that LIP onimmediate (0h),1h,3h and6h after brain ischemia/reperfusion injury has a protective effect, in whose LIP immediately before brain ischemia is mostobvious. Thus LIP could reduce the DND of the hippocampus CA1subfieldinduced by cerebral ischemia reperfusion injury.The HE staining indicated that Pyramidal cell is compact and neat rowsfor2-3layers in the hippocampal CA1subfield of the brain ischemic insultsham group rats, whose cell morphology was normal and the nucleolus wasclear, the nucleus was dyed ling blue, and the cytoplasmic was dyed pink. Thevast majority of microglial cells were deactivated, the cells were elongated,and the nucleus was a triangle. In brain ischemia insult group and LIP sham+brain ischemic insult group, significant neurons damage was observed in theCA1subfield. The microglia cell was proliferation. Cells shape became into acircular. A large number of apoptotic cells of the nucleus were color deepened.No significant DND was detected in the CA1hippocampus of the LIP+brainischemic insult0h,1h,3h and6h groups, whose cell morphology wasnormal and dyeing was uniformly, microglia did not be activated, nuclearmembrane was clear and staining was shallow. However, in LIP+brainischemic insult12h,1d and2d groups, the pyramidal neurons were edemaand nuclei had different degrees of fragmentation and dissolution. Aboveresults indicated that the inflammation appeared in the process of brainischemia. LIP in0h,1h,3h and6h before brain ischemia could slow downthe effect of inflammation before brain ischemic insult, in which the effectwas most obvious in LIP performed instantly brain ischemia.1.2The effect of dihydrokainate (DHK) on the induction of brain ischemictolerance induced by LIP50Wistar rats whose bilateral vertebral arteries were occludedpermanently were randomly divided into four groups:①brain ischemia insultsham group (n=5): bilateral common carotid arteries were exposed for8min,but without blocking the blood flow.②brain ischemia insult group (n=5）:bilateral common carotid arteries were clamped for8min.③LIP sham+brain ischemic insult group (n=5): bilateral common carotid arteries wereclamped for8min after bilateral femoral arteries were exposed for50min, but without blocking the blood flow.④LIP+brain ischemic insult group (n=5):bilateral femoral arteries were occluded for10min,3times, at10minintervals. Bilateral common carotid arteries were clamped for8min after LIPimmediately.⑤ACSF group (n=5):20μl ACSF was administered into theright lateral ventricle at30min before LIP. Others were the same as LIP+brain ischemic insult group.⑥DHK+LIP+brain ischemic insult group(n=20): DHK solution (20μl) was injected into the right lateral ventricle at30min before LIP. Others were the same as LIP+brain ischemic insult group.According to the dose of DHK, this group was divided into10nmol,100nmol,200nmol and400nmol groups.⑦DHK group (n=5): DHK solution (20μl)were injected into the right lateral ventricle at30min before LIP. According tothe result of⑥group, the best dose of DHK was selected. All rats weredecapitated at7d after the sham operation or the last time of ischemia. Themethods for pathological evaluation of CA1hippocampus were the same asthose in the part of1.1.The thionin stainning results showed that no significant DND wasdetected in the CA1hippocampus of the brain ischemic insult sham group rats,whose HG was0and ND was181±8.29. The rats in brain ischemic insultgroup appeared obvious DND in the CA1subfield. Compared with the brainischemic insult sham group, HG (Ⅲ) was significantly incresed, while the ND(27±8.64) was significantly decreased (P<0.01). The rats in LIP+brainischemic insult group did not appear obvious DND in the CA1subfield. HG(Ⅲ) was significantly decreased, while the ND (27±8.64) was significantlyincresed comparing with brain ischemic insult sham group (P<0.01). Theseresults indicated that LIP protected the neurons against the DND induced bybrain ischemic insult. Intraventricular administration of ACSF30min beforeLIP had no obvious effect on the CA1subfield compared with the LIP+brainischemic insult group. After administration of DHK，the CA1subfield showedsignificant DND. As dose increased, the damage was aggravated in DHK+LIP+brain ischemic insult group, whose HG was significantly incresed andND were121±10.91,82±10.35,16±5.71and8±6.04. However, HG was 0-Ⅰand ND was158±11.31in DHK group. No significant DND wasdetected in the CA1hippocampus. These results suggested DHK has noobvious neuronal toxic effect to neurons. These results indicated that DHKdose-dependently blocked the neuroprotective effect of LIP.2Whether activation of p38MAPK participats in the induction of brainischemic tolerance induced by LIP by up-regulating GLT-12.1The effect of LIP on the expression of p38MAPK and GLT-1in the CA1subfield of hippocampus of rats110Wistar rats whose bilateral vertebral arteries were occludedpermanently expect control group were randomly divided into ten groups:①Control group (n=10): Don’t do any operation.②LIP+brain ischemic insultgroup (n=100): bilateral femoral arteries were occluded for10min,3times, at10min intervals. Bilateral common carotid arteries were clamped for8minafter LIP immediately. Rats were decapitated at0h,30min,1h,3h,6h,12h,1d,3d,5d and7d after brain ischemia. In each group,5rats were used forimmunohistochemical staining. The other5rats were used for Westernblotting analysis.Immunohistochemical staining of GLT-1showed that there was noobvious expression in hippocampal CA1subfields of control group. It’s dyedlighter. The number of optical density and total area in the CA1hippocampuswere2.66±0.71and5035.02±1087(μm2, the same as following). TheGLT-1expression was up-regulated from0h to7day in LIP+brain ischemicinsult group. The immunoparticles tightly surrounded the pyramidal neuronsand made the pyramidal layer characteristically look like a “shape grid”. Thenumber of optical density and total area had no difference from0h to6h inLIP+brain ischemic insult group compared with the control group (P＞0.05).However, the expression of GLT-1was significantly up-regulatd from12h to3day and reached peak at1day. Strongly labeled dots and strings wereobserved in astrocyte somata and bumps, the number of optical density andtotal area were10.81±0.93and21351.19±2025, and there was obviousdifference compared with the control group (P<0.01). The expression of GLT-1was significantly decreased from5d to7d and had no differencecompared with the control group (P＞0.05). Immunohistochemical staining ofp-p38MAPK showed that there was no obvious expression in hippocampalCA1subfields of the control group. It’s dyed lighter. The number of opticaldensity and total area in the CA1hippocampus were1.65±0.28and5486.19±3596(μm2, the same as following). The p-p38MAPK expression wasup-regulated from0h to7d in LIP+brain ischemic insult group. The numberof optical density and total area had no difference from0h to3h in LIP+brain ischemic insult group compared with the control group (P＞0.05).However, the expression of p-p38MAPK was significantly up-regulatd from6h to1d and reached peak at12h. The number of optical density and total areawere9.22±0.87and49762.16±7799, and there was obvious differencecompared with the control group (P<0.01). The expression of p-p38MAPKwas significantly decreased from3d to7d and had no difference comparedwith the control group (P＞0.05).Western blot of GLT-1showed that there was no obvious expression inhippocampal CA1subfields of the control group. The GLT-1expression wasup-regulated from0h to7d in LIP+brain ischemic insult group. The GLT-1expression had no difference from0h to6h in LIP+brain ischemic insultgroup compared with the control group (P＞0.05). However, the expression ofGLT-1was significantly up-regulatd from12h to3d and reached peak at1d,and there was obvious difference compared with the control group (P<0.01).The expression of GLT-1was significantly decreased from5d to7d and hadno difference compared with the control group (P＞0.05). Western blot ofp-p38MAPK showed that there was no obvious expression in hippocampalCA1subfields of the control group. The p-p38MAPK expression wasup-regulated from0h to7d in LIP+brain ischemic insult group. Theexpression of p-p38MAPK had no difference from0h to3h in LIP+brainischemic insult group compared with the control group (P＞0.05). However,the expression of p-p38MAPK was significantly up-regulatd from6h to1dand reached peak at12h and there was obvious difference compared with the control group (P<0.01). The expression of p-p38MAPK was significantlydecreased from3d to7d and had no difference compared with the controlgroup (P＞0.05).The above results indicated that LIP up-regulated the expression of p-p38MAPK and GLT-1in hippocampal CA1subfields of rats and the appearanceand up-regulation of p-p38MAPK was earlier than those of GLT-1.2.2The effect of SB203580on the expression of GLT-1during the inductionof brain ischemic tolerance induced by LIP40Wistar rats whose bilateral vertebral arteries were occludedpermanently were randomly divided into eight groups:①LIP+brainischemic insult group (n=5): bilateral femoral arteries were occluded for10min,3times, at10min intervals. Bilateral common carotid arteries wereclamped for8min after LIP immediately.②DMSO+LIP+brain ischemicinsult group (n=5): DMSO solution (25μl) was injected into the right lateralventricle at30min before LIP. Bilateral common carotid arteries wereclamped for8min after LIP immediately.③SB203580+LIP+brainischemic insult group (n=20): SB203580solution (25μl) was injected into theright lateral ventricle at30min before LIP. Bilateral common carotid arterieswere clamped for8min after LIP immediately. According to the dose of SB203580, this group was divided into50μmol,100μmol,200μmol and400μmol groups.④SB203580sham group (n=10): SB203580solution (25μl)was injected into the right lateral ventricle. Thirty minutes later, bilateralfemoral arteries were exposed for50min and bilateral common carotidarteries were exposed for8min. According to the result of②group, the bestdose of SB203580was selected.⑤brain ischemic insult sham group (n=5):bilateral common carotid arteries were exposed for8min, but withoutblocking the blood flow. All rats were decapitated at1day (According to theresults of2.1, the peak of GLT-1expression was selected) after the shamoperation or the last time of ischemia. Sections were used for immunohisto-chemical staining.The results of immunohistochemical staining of GLT-1showed that there was a few expression in hippocampal CA1subfields of brain ischemic insultsham group, the staining were shallow, the distribution was diffuse and thenumber of optical density and total area in the CA1hippocampus were6.17±0.6and11394.17±1225(μm2, the same as following). The GLT-1expressionwas less in the SB203580sham group and had no significant differencecompared with the brain ischemic insult sham group (P＞0.05）. The numberof optical density and total area in the CA1hippocampus were5.89±0.9and10984.5±1185. These results suggested that SB203580has no obviousneuronal toxic effect to neurons. The expression of GLT-1was up-regulated inLIP+brain ischemic insult group. Strongly labeled dots and strings wereobserved in astrocyte somata and bumps. Dyeing was deeper. The number ofoptical density and total area were10.74±0.85and19951.19±1508.Compared with the LIP+brain ischemic insult sham group, the expression ofGLT-1was significantly down-regulated in SB203580+LIP+brain ischemicinsult group (P<0.01). As dose increased，the decreased was more obvious.The number of optical density was4.03±0.55,2.84±0.54,2.46±0.5and2.38±0.31respectively. The number of total area was9645.17±1140,8032.16±1197,6844.36±929and5780.43±939respectively. The expression ofGLT-1was up-regulated in DMSO+LIP+brain ischemic insult group. Therewas no obvious difference compared with the LIP+brain ischemic insultgroup (P＞0.05). The number of optical density and total area in the CA1hippocampus were9.04±0.96and18339.8±1103. The results indicated thatSB203580, an inhibitor of p38MAPK, dose-dependently inhibited theexpression of GLT-1in CA1region during the induction of brain ischemictolerance induced by LIP.Conclusions：1The different time windows of LIP reduced DND of the CA1hippocampus induced by brain ischemic insult, in which LIP immediatelybefore brain ischemia presented most obvious role.DHK, an inhibitor of GLT-1, dose-dependently blocked theneuroprotective effect of LIP, suggesting that GLT-1promoted the brain ischemic tolerance induced by LIP.2LIP up-regulated the expression of p-p38MAPK and GLT-1inhippocampal CA1subfields of rats and the time of beginning and peak ofp-p38MAPK was earlier than those of GLT-1.SB203580, an inhibitor of p38MAPK, dose-dependently inhibited theexpression of GLT-1in CA1region of hippocampus during the induction ofbrain ischemic tolerance induced by LIP. These results suggested that p38MAPK participated in the neurprotection of LIP by up-regulating theexpression of GLT-1in the CA1hippocampus. |
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