The Study On Mechanisms Of Sustained Tolerance Against Spinal Cord Ischemia Induced By Cyclic Short Episodes Of Hyperbaric Oxygen Preconditioning

During descending thoracic aortc aneurysm repair,the aorta was cross-clamped and unclamped subsequently which may result in spinal cord transient ischemia followed by reperfusion.Ischemia and reperfusion injury(IRI) of spinal cord after thoracoabdominal aortic aneurysm repair remains a devastating complication with an incidence of 11%～40%in postoperative paraplegia and paraparesis.First described in 1986,Murry and colleagues exposed open-chest dogs to a set of non-harmful ischemic insults consisted of four independent cyclic episodes of a 5-min coronary occulusion followed by 5-min reperfusion.This so called "ischemic preconditioning"(IPC) showed some beneficial effects to the pretreated heart against subsequent prolonged ischemia.Following the novel observation of Murry et al.,IPC has become an effective means of reducing harmful ischemic events.Two types of IPC,rapid and delayed,have been reported.Rapid ischemic tolerance occurs within a few minutes of the preconditioning ischemia and lasts for 1-2 hrs.Delayed ischemic tolerance starts at 12-24 hrs and will last for more than 3-4 days.The different molecular mechanisms in the mitochondria,which are involved in transient open of phosphorylated ATP sensitive potassium channel(mitoK_ATP) in the rapid tolerance and transcription of Bcl-2 family genes to inhibit the mitochondrial permeability transition,contribute to each type of tolerance.Both the instability of rapid tolerance and the followed non-protective effect window till the adventure of delayed ischemic tolerance greatly limit the application of IPC as preventive measures in against ischemia and reperfusion insults.Hyperbaric oxygenation(HBO) treatment is a mode of therapy for this serious complication with a established protocol of oxygen pressure not exceeding 3.0 atmospheres absolute(ATA) and a duration limited to a maximum of 2 hrs.HBO has been safely used for the patients with carbon monoxide poisoning,decompression sickness,and arterial gas embolism.It has also been used as an adjunctive therapy for reviving a patient with impaired oxygen delivery.Up to now,HBO has been reported as an effective method of preconditioning in preventing ischemia and reperfusion insults in the brain,heart and spinal cord.In most published experiments,the HBO preconditioning(HBO-PC) protocol comprises 3 or 5 consecutive cycles of 1-h HBO exposure(2.5 atmospheres absolute [ATA]) at a 1-day interval against ischemic attack 24 hrs later.The mechanism of late ischemic tolerance induced by HBO may be related to ROS generated in the preconditioning which upregulated endogenous antioxidant enzymes,hypoxia-inducible factor-1(HIF-1) and Bcl-2 to resist ischemia.Apparently this protocol is impractical in emergency scenarios for patients with thoracoabdominal aortic aneurysm.In 2005,Choi et al pretreated with HBO at 3 ATA for 20 mins,four cycles with 20-min intermission,and isolated hearts were subjected to 45-min ischemia followed by 90-min reperfusion.This protocol induced mRNA transcription of anti-apoptotic protein even in the traditionally non-protective window between the two stages of rapid and delayed tolerance.In the study of remote preconditioning performed in the ipsilateral hind limb against focal stroke,the results showed that rapid preconditioning with three cycles of 15-min occlusion followed with the same period of reperfuison of the left hind femoral artery could reduce infarct size of stroke that occurred 1-24 h after preconditioning.But at three cycles of 5 min,infarct was not reduced.However,whether cyclic short episodes of HBO-PC protects against spinal cord ischemia is not established.Moreover,few have addressed the sustained therapeutic time windows for HBO-PC.In the present study,the rats were preconditioned with repeated HBO of short interval and underwent spinal cord ischemia induced by occlusion of the descending thoracic aorta at different time point after HBO-PC.The purpose of this study was to analyze the relationship between different preconditiong paradigms and therapy time window,and examine the related mitochondrial mechanisms with its upstream transcription regulation.The main experimental procedures are as follows:1.Spinal cord ischemia was induced by occlusion of the descending thoracic aorta with 2F Fogarty catheter in combination with maintaining systemic hypotension(40 mmHg) during the procedure,and determined a suitable time of ischemia to establish basis for next experiments.2.The rats were divided into four HBO-PC and Control groups as follows:the rats in HBO-â… group were only pretreated with one cycle HBO of 20 min;the rats of HBO-â…¡group were preconditioned with four cycles HBO of 20 min with interval of 10 min and the rats in the HBO-â…¢and HBO-â…£groups underwent four episodes of 20 min with intermission of 30 min or 50 min respectively.Control group only underwent Sham preconditioning.3.At 0.5,2,4,8,12,24 and 48 h after HBO-PC,spinal cord ischemia was induced. Neurological scores were evaluated 12 and 24 h after reperfusion.After that,the spinal cords(L3-5) were harvested and sections were stained by Nissl and normal neruons in the anterior spinal cord were counted by means of light microscopy.4.Mitochondria in lumbar spinal cord were extracted at 0.5,2,4,8,12,24 and 48 h.Measurments of mitoK_ATP open extent was conducted by the decrease in light scattering closely paralles the percentage of the mitochondrial population undergoing permeability transition under the circumstance of Ca²⁺-induced mitochondrial swelling.Mitochondrial microstructure was observed by electron microscopy.Bcl-2 protein levels in the mitochondria were analyzed by Western blot using samples taken from spinal cord tissues in different time windows after HBO-PC.5.The changes of DNA-binding activity of nuclear factor-κB(NF-κB) after single HBO-PC were assessed by electrophoretic mobility shift assay(EMSA).The effects of NF-κB inhibitor pyrrolidine dithiocarbamate(PDTC) and antioxidant N-acetylcysteine (NAC) on HBO-PC and Bcl-2 expression were also evaluated.The main results are as follows: 1.Evaluation for rat model of ischemia and reperfusion injury.The rats underwent 9 min of spinal cord ischemia were stable in neurological scores during reperfusion and were susceptible to preconditionng.The rat model of spinal cord induced by occlusion of the descending thracic aorta is stable for study on mechanism of preconditioning.2.As displayed in neurological scores and pathological results,cyclic short episodes of HBO-PC could improve the rapid tolerance,and the sustained protective effect was induced if suitable interval of HBO-PC was used.But biphasical therapy window with an early and delay effect was presented again after HBO-PC of prolonged intermission.3.The extent of mitoK_ATP was increased after cyclic short episodes of HBO-PC compared with one cycle,and not be affected by the prolonged period of HBO-PC.4.Bcl-2 was expressed both in the early preconditioning and late preconditioning with 20-min or 50-min intermission of HBO-PC in HBO-â…¡andâ…£groups.Moreover,Bcl-2 also increased in the intermediate preconditioning between the conventional two time windows with 30-min interval in HBO-â…¢group.5.DNA-binding activity of nuclear factor-κB(NF-κB) peaked at 20-40 min and 24 h after single HBO-PC.PDTC and NAC could both abrogated the neuroprotective effect and Bcl-2 expression in HBO-â…¢group.In conclusion,cyclic short episodes of HBO-PC can induce sustained tolerance against spinal cord ischemia.This mechanism may be involved in anti-apoptotic protein expression of Bcl-2 in the early and late periods of preconditioning as well as in the intermediate time windows which may be related to peak of NF-κB DNA-binding activity induced by HBO-derived ROS.