| Background:Ischemia postconditioning is defined as alternate brief episodes of reperfusion and interruptions of reperfusion applied after a longer period of ischemia and just before the onset of reperfusion.Ischemia remote-postconditioning is defined as alternate brief episodes of ischemia/reperfusion applied to distant organ.For the past few years,the organ protective effects against ischemia-reperfusion injury of ischemia postconditioning and ischemia remote-postconditioning have been confirmed by Chinese and overseas investigators.Naloxone is an opioid receptor antagonist commonly used in clinical practice,its neuroprotive effect had been confirmed long ago,but it still remains unknown whether naloxone can provide neuroprotective effect against ischemia-reperfusion injury by postconditioning.In view of this,we conducted the present study to investigate whether ischemia postconditioning,ischemia remote-postconditioning and naloxone postconditioning can provide neuroprotective effects and to determine the interaction among these postconditioning procedures.Furthermore,the correlations between the neuroprotective effects of postconditioning and the expression of Microtubule-associated protein 2(MAP 2) were estimated by western blot analysis. The alterations of cerebral microvascular structure under postconditioning were also further observed by Laser scanning confocal microscopy(LSCM).Methods:One hundred and thirty-four adult male SD rats weighing 270～330g were randomly divided into 8 groups(groupâ… -â…¡,n=19;groupâ…¢-â…¦,n=16).The detailed experimental protocols were as follows:groupâ… was sham operation group and the rest groups received transient focal cerebral ischemia induced by occluding the right middle cerebral artery(MCA) for 90min followed by 24 h reperfusion;groupâ…¡was ischemia control group;groupâ…¢was ischemia postconditioning group:after the onset of reperfusion,ischemia postconditioning was established through three cycles of 30s reperfusion followed by 30s occlusion;groupâ…£was ischemia remotepostconditioning group:ischemia remote-postconditioning was performed via a transient right femoral artery ischemia 5 min before reperfusion;groupâ…¤was ischemia combined with ischemia remote-postconditioning group,which received both ischemia postconditioning as described in groupâ…¢and remote-postconditioning as described in groupâ…£;groupâ…¥was low-dose naloxone postconditoning group; groupâ…¦was high-dose naloxone postconditoning group and groupâ…¦was ischemia combined with ischemia remote and high-dose naloxone postconditoning group.At the starting of reperfusion,the rats were injected intraperitoneally either naloxone 10 ml(1 mg/kg in groupâ…¥and 10 mg/kg in groupsâ…¦andâ…§) or equal volume of saline (in groupsâ… ,â…¡,â…¢,â…£andâ…¤).The rat model with the transient focal cerebral ischemia-reperfusion was established by suture occlusion of the middle cerebral artery(MCAO).Nylon suture was inserted from common carotid artery(CCA) 1 cm beneath the cross of CCA into internal carotid artery(ICA).When an obvious resistance was met during advancement of suture,it was implied that the tip of suture had been inserted into the initial segment of MCA.MCAO induced focal cerebral ischemia was then initiated, 90 min later,the suture was removed and reperfusion was started.Ischemia remote postconditioning was induced by single lower limb ischemia model.Right femoral artery was clipped 5 min before reperfusion and clamp was removed at the onset of reperfusion.Exclusion criteria included:hemoglobin(HBG) less than 80g/L;SO2 less than 90%;vagus nerve injury;failure of procedure;MAP less than 60mmHg; subarachnoid hemorrhage;failure of nylon suture insertion or vessel injury; postoperative infection or death within 24h.Heart rate(HR),mean arterial pressure(MAP),and a leadâ…¡electrocardiogram were continuously monitored during ischemia reperfusion process.At 1 h after the starting of reperfusion,arterial blood sample was obtained for blood gas analysis.At 2 h and 24 h after reperfusion,the neurologic deficit scores(NDS) were evaluated using a four-point scale:0,no apparent neurologic deficits;1,slight focal neurologic deficits,failure to extend left forepaw fully if pulled by the tail;2,moderate focal neurologic deficits,circling or walking to the left;3,severe focal neurologic deficits, walking fall down to the left;4,in a state of unconsciousness,without spontaneous movement.At the end of 24 h reperfusion,the rats were anesthetized and sacrificed by decapitation.The brains were rapidly removed for measuring the cerebral infarct size (n=10) and the expression of MAP2(n=3).The cerebral infarct size was expressed as a percentage of the area at ipsilateral hemisphere.For other 3 rats each group,at the end of 24 h reperfusion,FICT-dextran 1 ml(50 mg/ml) was administered intravenously 1 min before decapitation for LSCM.For another 3 rats in groupsâ… andâ…¡,at the initiation of reperfusion,FICT-dextran 1 ml was also administered intravenously 1 min before decapitation for LSCM.Results:there were no significant differences among all groups in HBG,SO2 and the baseline values of HR and MAP(P＞0.05).There were also no statistical significance among all groups in HR and MAP at each time points during ischemia-reperfusion (P＞0.05).Except for groupâ… ,there were no significant differences between the NDS values at 2 h and 24 h after reperfusion each group(P＞0.05 ).As compared with groupâ…¡,NDS values showed a significant decrease in groupsâ… ,â…¢,â…¤,â…¦andâ…§at 2 h after reperfusion and in groupsâ… ,â…¢,â…£,â…¤,â…¦andâ…§at 24 h after reperfusion.As compared with groupâ…§,NDS values in groupsâ…¡,â…¢,â…£,â…¥andâ…¦increased significantly at 24 h after reperfusion(P＜0.05).The cerebral infarct size was greater in the groupâ…¡than in groupsâ… ,â…¢,â…£,â…¤,â…¦andâ…§(P＜0.05),but less in the groupâ…§than in groupsâ…¡,â…¢,â…£,â…¤,â…¥andâ…¦(P＜0.05).The expression of MAP2 in ipsilateral hemisphere increased significantly in groupsâ… ,â…¢,â…£,â…¤,â…¦andâ…§compared with groupâ…¡(P＜0.05),but decreased significantly in groupsâ…¡,â…¢,â…£,â…¤,â…¥andâ…¦compared with groupâ…§(P＜0.05).At the onset of reperfusion,the volumes of cerebral microvascular plasma and microvessel diameters in ipsilateral hemisphere of MCAO group(groupâ…¡) reduced significantly compared to its contralateral hemisphere and ipsilateral hemisphere of sham group(groupâ… )(P＜0.05).The microvessel diameters in contralateral hemisphere of MCAO group(groupâ…¡) also decreased significantly compared to both hemispheres of sham group(groupâ… )(P＜0.05).At 24 h after reperfusion,the volumes of cerebral microvascular plasma, microvessel diameters and segment length of microvessel increased significantly in groupsâ… ,â…¢,â…£,â…¤,â…¦andâ…§compared with groupâ…¡(P＜0.05),but decreased significantly in groupsâ…¡,â…¢,â…£,â…¥andâ…¦compared with groupâ…§(P＜0.05).There was no significant difference in all observed values between groupâ…¤and groupâ…¢orâ…£.Conclusions:On the basis of the results from this experiment,the following conclusions can be drawn:1.In the rats with a local ischemia-reperfusion injury,ischemia postconditioning, remote-postconditioning and high-dose naloxone postconditioning each has provided significant neuroprotective effects,with reduced cerebral infarct size and improved nerve dysfunction.2.A combination of both ischemia and ischemia remote-postconditioning can not offer additional neruoprotection against local ischemia-reperfusion injury.3.The neuroprotective effects of naloxone postconditoning is only achieved at a high-dose(10 mg/kg).4.The combined use of ischemia,ischemia remote and naloxone postconditioning can improve neuroprotective effect of postconditioning.5.The postconditioning significantly increases the blood plasma perfusion in ischemia brain tissue.Also the neuroprotective effects of the postconditioning highly correlate with the increased expression of MAP2.
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