The Neural Pathway In Brain Protection And Up-regulation Of P38MAPK Induced By Limb Ischemic Preconditioning

Objective: Cerebral ischemic diseases, as a common disease andfrequently-occurring disease, are harmful to human health. Neurons areextremely sensitive to the ischemic injury and dead suffering a certain time ofischemia. However, neurons are also hard to regenerate and have many serioussequelae to the patients after restoring the blood supplies. Therefore, it is animportant strategy to improve the resistance of neurons to the ischemicdamage, reduce the damage and keep alive after the severe ischemia in suchdiseases treatment, which ensure normal physiological function after bloodsupply.Kitagawa, a Japanese scholar, firstly discovered the phenomenon that thetransient ischemic attack preconditioning can induce tolerance of brainischemia. After that, many scholars were engaged in the research, andconfirmed the protective effect of ischemic preconditioning in the same organ.Although ischemic preconditioning on central organs is a powerfulendogenous protection inducing the tolerance to cerebral ischemia, it isdifficult to be directly applied to clinic. As the further studies of the ways andmethods in ischemic preconditioning, it have been found that transientischemic attack happened in the remote organs, such as limb, kidney andintestine, played protective effect to ischemia-reperfusion injury ofmyocardium. This phenomenon of the protections through different organsischemic preconditioning called the remote ischemic preconditioning (RIPC).In our previous studies, 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 CA1 hippocampus of rats. These results confirmed that the protective effect ofremote ischemic preconditioning on brain. In the further studies, we found thatLIP played an important role of brain protection by raising the expression ofp38MAPK in the CA1hippocampus. However, it is so far away between theischemic preconditioning limb and the brain suffering ischemia. How does theLIP protect the brain, and by what ways raising the expression of p38MAPKin hippocampus is not fully clear. Zhao et al demonstrated that the bilateralfemoral nerves resection before LIP inhibited the protection of LIP againstbrain ischemia, which suggested neural pathway was involved in the brainischemic tolerance induced by LIP. Many foreign scholars proved remotedouble lower limbs with fatal ischemic preconditioning induced brainischemic tolerance within24h, and the protection might be mediated byneurogenic way.Based on the above data, the purpose of the study was to:①Observingthe effect on brain ischemic tolerance induced by LIP on rats by resectting thebilateral femoral nerves and/or sciatic nerves;②Observing the effect ofresectting the bilateral femoral nerves and/or sciatic nerves on the expressionof p38MAPK in CA1subfield of hippocampus in the brain protection by LIP.Through these studies, the aim of this paper is to explore the effect ofresectting the bilateral femoral nerves and/or the sciatic nerves on the brainprotection and up-regulation of p38MAPK induced by LIP. The solution ofthese problems may provide new ideas of increasing tolerance of neurons toischemic damage and preventing and treating the cerebral ischemic diseases.1The role of femoral nerve and/or sciatic nerve in brain protection induced byLIP99Wistar rats whose bilateral vertebral arteries were occludedpermanently were randomly divided into nine groups:①brain ischemia shamgroup (n=11): bilateral common carotid arteries were exposed for8min, butwithout blocking the blood flow.②brainischemia (BI) group (n=11):bilateral common carotid arteries were clamped for8min.③LIP+brainischemia group (n=11): bilateral femoral arteries were occluded for10min,3 times, at10min intervals. Bilateral common carotid arteries were clamped for8min after LIP.④FNS (femoral nerve resection)+BI group (n=11): resectthe bilateral femoral nerves. Bilateral common carotid arteries were clampedfor8min immediately after FNS.⑤FNS+LIP+BI group (n=11): resect thebilateral femoral nerves before LIP, and then occlude the bilateral commoncarotid arteries at once.⑥SNS (sciatic nerve resection)+BI group (n=11):resect the bilateral sciatic nerves. Bilateral common carotid arteries wereclamped for8min immediately after SNS.⑦SNS+LIP+BI group (n=11):resect the bilateral sciatic nerves before LIP, and then occlude the bilateralcommon carotid arteries at once.⑧FNS+SNS+BI group (n=11): resect thebilateral femoral nerves and the bilateral sciatic nerves before LIP, and thenocclude the bilateral common carotid arteries immediately.⑨FNS+SNS+LIP+BI group (n=11): resect the bilateral femoral nerves and the bilateralsciatic nerves before LIP, and then occlude the bilateral common carotidarteries immediately.6animals of each group were sacrificed by decapitationat7d after the sham operation or the last time of ischemia for thionin staining.Histological changes of the hippocampus were evaluated using thioninstaining by histological degree (HG) and neuronal density (ND). HG wasdivided into four grades:0: no neuron death;: scattered single neuron death;II: mass neuron death; III: almost all neuron death. The ND of thehippocampal 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 CA1hippocampus was calculated toestablish the ND.5animals of each group were sacrificed by decapitation at3d after the sham operation or the last time of ischemia for TUNEL staining tocounting the number of neuronal apoptosis. The number of the positive cellswithin1mm linear length of the CA1hippocampus was accounted forquantification of TUNEL staining density. The average number of the positivecells in3areas of the CA1hippocampus was calculated to establish theneuronal apoptosis. The thionin staining results showed that the pyamidal neurons inhippocampal CA1subfield in the brain ischemic insult sham group werearranged in order. The outline of the neurons was intact, and no significantdelayed neuronal death (DND) was observed. The HG was0, and the ND was223±20.23. The brain ischemia group appeared obvious DND in the CA1subfield of the hippocampus. Compared with the brain ischemia sham group,HG (Ⅲ) was significantly increased, while the ND (46±12.52) wassignificantly decreased (P<0.01). However, LIP+brain ischemia group didnot appear obvious DND in the CA1subfield, compared with the brainischemia group, HG (0～I) was significantly reduced, ND (198±19.49) wassignificantly increased (P＜0.01). In FNS+LIP+BI and SNS+LIP+BIgroups, the pyamidal neurons in hippocampal CA1subfield were changedsignificantly. Compared with the LIP+brain ischemia group, HG (I～II) wassignificantly increased, and ND (165.83±21.33and173±13.59) wassignificantly reduced (P＜0.01). Furthermore, it appeared obvious DND in theCA1subfield of hippocampus in FNS+SNS+LIP+BI group. Comparedwith the LIP+brain ischemia group, HG (II～III) was significantly increased(P＜0.01) and ND (93±8.17) was significantly reduced (P＜0.01). Theseresults suggested that LIP could obviously reduce the DND of the rathippocampal CA1subfield induced by brain ischemic injury, and resecttingthe bilateral femoral nerve and sciatic nerve respectively or jointly couldpartly block the role of LIP.TUNEL staining results showed that the CA1area of hippocampus inbrain ischemia of sham group occasionally has hyperchromatic TUNELpositive cells, neurons are smaller, nucleus pycnosis, triangular, circular orfan-shaped, nuclear chromatin is crescent, block or bar near the nucleus. Inbrain ischemia group, there are a lot of TUNEL positive cells in the CA1areaof hippocampus. Compared with the sham group, the number of cellsincreased significantly (P<0.01), which suggested the brain ischemia of8minutes is fateful to the neurons in CA1region of hippocampus and causedapoptosis. The number of TUNEL positive neurons in LIP+brain ischemia group decreased significantly (P<0.01) compared to brain ischemia group,which suggested that the LIP inhibited the cells apoptosis in CA1area ofhippocampus induced by ischemia. Compared with LIP+brain ischemiagroup, the numbers of TUNEL positive cells in the CA1subfield wereobviously increased in the FNS+LIP+BI group, the SNS+LIP+BI groupand FNS+SNS+LIP+BI group, suggesting resectting the bilateral femoralnerves and sciatic nerves respectively or jointly could be against the protectiverole of LIP.2The role of femoral nerve and/or sciatic nerve on the up-regulation of p38MAPK of hippocampal CA1subfield induced by LIP90Wistar rats whose bilateral vertebral arteries were occludedpermanently were randomly divided into nine groups:①brain ischemia shamgroup (n=10): bilateral common carotid arteries were exposed for8min, butwithout blocking the blood flow.②brain ischemia (BI) group (n=10):bilateral common carotid arteries were clamped for8min.③LIP+brainischemia group (n=10): bilateral femoral arteries were occluded for10min,3times, at10min intervals. Bilateral common carotid arteries were clamped for8min after LIP.④FNS (femoral nerve resection)+BI group (n=10): resectthe bilateral femoral nerves. Bilateral common carotid arteries were clampedfor8min immediately after FNS.⑤FNS+LIP+BI group (n=10): resect thebilateral femoral nerves before LIP, and then occlude the bilateral commoncarotid arteries at once.⑥SNS (sciatic nerve resection)+BI group (n=10):resect the bilateral sciatic nerves. Bilateral common carotid arteries wereclamped for8min immediately after SNS.⑦SNS+LIP+BI group (n=10):resect the bilateral sciatic nerves before LIP, and then occlude the bilateralcommon carotid arteries at once.⑧FNS+SNS+BI group (n=10): resect thebilateral femoral nerves and the bilateral sciatic nerves before LIP, and thenocclude the bilateral common carotid arteries immediately.⑨FNS+SNS+LIP+BI group (n=10): resect the bilateral femoral nerves and the bilateralsciatic nerves before LIP, and then occlude the bilateral common carotidarteries immediately. The rats of each group were sacrificed by decapitation at 12h after the sham operation or the last time of ischemia. In each group,5ratswere used for immunohistochemical staining. The other5rats were used forWestern blotting analysis.Immunohistochemical results showed that it was a low level of theexpression of p-p38MAPK in CA1subfield of hippocampus in the brainischemia of sham group and brain ischemia group, and the positive cellnucleus coloring dark brown, the optical density were3.37±0.12and3.30±0.03, and the total area were6056.23±522.3and6106.62±458.43(μm2, thesame as following). The expression of p-p38MAPK in CA1hippocampus inLIP+brain ischemia group significantly increased. The optical density andtotal area of the positive cells were11.90±0.27and12036.60±693.91,which was significantly increased compared with the brain ischemia group(P<0.01). It showed that LIP up-regulated the expression of p-p38MAPK inCA1hippocampus compared with the brain ischemia group. Compared withLIP+BI group, the expression of p-p38MAPK in CA1hippocampus in FNS+LIP+BI group is less, lighter nuclei, with statistically significant difference(P<0.01), the optical density and the total area of the positive cells were4.63±0.07and6561.17±301.5. The expression of p-p38MAPK in CA1hippocampus in SNS+LIP+BI group significantly decreased. Comparedwith the LIP+BI group, the optical density and the total area of the positivecells were5.07±0.13and6835.91±401.47, it had significant statisticaldifference (P<0.01). The expression of p-p38MAPK in CA1hippocampus inFNS+SNS+LIP+BI group was in a low level, the optical density and thetotal area of the positive cells were3.15±0.22and6361.81±210.41,compared with LIP+BI group, with significant statistical difference (P<0.01).These results demonstrated that separately or jointly resectting the bilateralfemoral nerves and sciatic nerves could block the up-regulation of p38MAPKin CA1hippocampus induced by LIP.The results of Western blot showed that the expression of p-p38MAPK inCA1hippocampus both in brain ischemia of sham group and brain ischemiagroup were very low. LIP prior to brain ischemia increased the expression of p-p38MAPK, with the statistical difference (P<0.01). However, Theexpression of p-p38MAPK was less in FNS+LIP+BI group, SNS+LIP+BI group and FNS+SNS+LIP+BI group, IOD was significantly decreasedcompared to the brain ischemia group (P<0.01). These results showed that LIPincreased the expression of p38MAPK in CA1hippocampus of rats withbrain ischemia. The brain protective effect of LIP could be inhibited byresectting the bilateral femoral nerves and/or sciatic nerves.Conclusions:(1) Resectting the bilateral femoral nerves and/or sciatic nerves inhibitedthe brain protection of LIP partially. It suggested that neural pathway wasinvolved in the brain protective role of LIP.(2) Resectting the bilateral femoral nerves and/or sciatic nerves blockedthe inhibition of LIP to the neurons apoptosis happened in CA1area ofhippocampus with brain ischemia.(3) Resectting the bilateral femoral nerves and/or sciatic nerves blockedthe up-regulation of p38MAPK in CA1hippocampus by LIP, whichsuggested that LIP could up-regulate the expression of p38MAPK throughneural pathway.