The Studies On The Mechanisms Of Ischemic Tolerance Against Spinal Cord Ischemia Induced By Hyperbaric Oxygen Preconditioning In Rabbits

Objective To investigate the effect of hyperbaric oxygenation preconditioning on the activities of antioxidant enzymes in spinal cord after trasient spinal cord ischemia. Methods Clean male New Zealand white rabbits (2.2-2.5kg) were randomly assigned to one of the three groups (n=19 each): the hyperbaric oxygen (HBO), the hyperbaric air (HBA) and control groups. Animals in HBO group were exposed to lh of hyperbaric oxygen at 2.5 atmosphere absolute (ATA) in 100% oxygen each day for 5 days. Animals in HBA group underwent lh treatment in 21% O₂ at 2.5ATA each day for 5 days. Animals in the control group were placed in a chamber (21% O₂), which wasnot pressurized for sham pretreatments, on the same schedule as for the HBO group for 5 days. Four rabbits from each group were sacrificed at 24h after the last pretreatment as pre-ischemia control, others (n=15 in each group) were subjected to spinal cord ischemia for 20 min. Spinal cord ischemia was induced with clamping the aorta just below the renal artery. Animals were neurologically assessed according to the Tarlov criteria at 6h, 24h and 48h after reperfusion and then sacrificed (n=5 for each time point in each group). Spinal cord tissue of L5-7 level were sampled and frozen at -70℃ in freezer for the determination of antioxidant enzyme activities and MDA content. Results (1) The neurological outcome in HBO group was better than that of the control and HBA groups at 6h, 24h and 48h after reperfusion (p<0.05). (2) At 24h after the last pretreatment, activities of CAT and SOD of spinal cord tissue in HBO group were higher than that of the control group and HBA group statistically. A higher activity of CAT was seen in HBO group when compared with that in other two groups at 6h, 24h and 48h after reperfusion. The comparison of SOD activity among three groups revealed similar results as CAT at 24h and 48h after reperfusion. No significant difference was observed between control group and HBA group at corresponding time points. At 24h and 48h after reperfusion, CAT and SOD activities in three groups decreased compared with pre-ischemia (P<0.05). As for GSH-px activity, no difference was found among three groups at 24h after last pretreatment. The GSH-px activity in the HBO group was higher than that of other two groups at 48h after reperfusion. In control and HBA groups, GSH-px activity decreased at 48h after reperfusion when compared with pre-ischemia. In HBO group, no significant difference was observed among different time points. (3) MDA content in spinal cord tissue in HBO group was significantly lower at 24h and48h after reperfusion compared with that in control and HBA groups. In HBO group, MDA content increased at 48h after reperfusion. At 24h and 48h after reperfusion, MDA content in control and HBA groups increased significantly compared with pre-ischemia. Conclusion Repeated HBO preconditioning 5d (1ATA, 21%O₂, 1h per day) up-regulated activities of CAT and SOD in spinal cord tissue and higher activities of CAT and SOD was observed in HBO group than that in control and HBA groups at 24h and 48h after reperfusion following 20min spinal cord ischemia.Experiment 2 Effect of catalase inhibitor 3-amino-l,2,4-triazole on the ischemic tolerance against spinal cord ischemia induced by HBO preconditioningobjective To investigate the effect of catalase (CAT) inhibitor 3-amino-l,2,4-triazole (AT) on the ischemic tolerance against spinal cord ischemia induced by HBO preconditioning. Methods It consisted of two parts. The purpose of Part I was to determine the dose of AT to be given in the second part. Twenty male New Zealand White rabbits were randomly assigned into four groups. All the animals were exposed to lh HBO (2.5ATA, 100%O₂) a day for 5days. Animals in HBO group received saline, while animals in HB0+AT1, HBO+AT2 and HBO+AT3 groups received a catalase inhibitor 3-amino-l,2,4-triazole (AT) intraperitoneally at a dose of 0.5g/kg, 1g/kg and 1.5g/kg respectively at 23h after the last treatment of HBO. Spinal cord tissue were harvested lh after AT administration for the determination of CAT activity in spinal cord. In the second part of experiment 2, thirty-two rabbits were randomly assigned to one of four groups: control, AT, HBO andHBO+AT groups. Animals in HBO and HBO+AT group underwent HBO pretreatment as described in experiment 1. Those in control and AT groups received sham pretreatment. At 24h after the last pretreatment, all animals were subjected to spinal cord ischemia for 20min. Rabbits in the control and HBO groups received saline administration, and AT was administered intraperinoneally according to the result of part I in AT and HBO+AT groups 1h before ischemia. At 48h after reperfusion, neurological and histopathological evaluation were performed. Results (1) Increased catalase activity in spinal cord induced by HBO preconditioning was reversed by administration of AT 1g/kg (intraperitoneally) at 23h after the last treatment. Therefore, rabbits in AT and HBO+AT groups received AT 1g/kg lh before ischemia. (2) At 48h after reperfusion, animals in HBO group showed higher hind-limb motor function scores than those in control group and AT group (P<0.01). Scores of animals in HBO+AT group revealed no significant difference when compared with other three groups. (3) At 48h after reperfusion, more normal motor neurons in the anterior spinal cord were seen in HBO group than in other three groups (P<0.05). The number of normal motor neurons in HBO+AT group revealed no significant difference compared with AT and control groups. No difference was observed between control and AT groups. Conclusion The ischemic tolerance against spinal cord ischemia induced by HBO preconditioning was reversed by administration of CAT inhibitor AT lh before ischemia.Experiment 3 Effect of dimethylthiourea on the antioxidant enzymes activities after HBO preconditioningObjective To investigate the effect of dimethylthiourea (DMTU) on the antioxidant enzymes activities after HBO preconditioning. Methods Twenty-four rabbits were assigned into four groups (n=6 each): control, HBO, HBO+DMTU and DMTU groups. In control and HBO groups, the saline 5ml/kg was administered to rabbits 1h before each session of pretreatment. In DMTU and HBO+DMTU groups 10% DMTU saline solution 5ml/kg were given at the same time point. The conditions of sham pretreatment and HBO preconditioning were the same as experiment 1. At 24h after the last pretreatment, spinal cord SOD activity, CAT activity and the content of MDA were measured. Results (1) Free radical scavenger DMTU abolished the upregulation of CAT and SOD activities in spinal cord induced by HBO preconditioning. (2) MDA content in spinal cord was lower in DMTU and HBO+DMTU groups than control and HBO groups respectively. Conclusion DMTU administration before each session of HBO treatment completely abolished the increase of CAT and SOD activities after HBO preconditioning.Therefore, we concluded that ROS generated in HBO preconditioning activates endogenous antioxidant enzymes, which then scavenge the ROS produced during ischemia-reperfusion and protect the spinal cord from ROS-mediated injury.Part â…¡ The changes of mitochondrial structure and ATPase activity during spianl cord ischemic tolerance induced by repeated hyperbaricoxygenation in rabbits.objective The present study was designed to investigate the changes of mitochondrial structure and ATPase activity during spianl cord ischemic tolerance induced by repeated hyperbaric oxygenation in rabbits. Methods Fifty male New Zealand rabbits were divided into two groups: Control group in which the animals were subjected to spinal cord ischemia for 20min, and HBO group in which animals were preconditioned with hyperbaric oxygenation (100%O2,2.5ATA,lh/d,5d) and were subjected to spinal cord ischemia for 20 min at 24h after the last HBO treatment. Spinal cord ischemia was induced by claming infrarenal aorta. The motor function of hind-limbs were scored at 6h, 24h and 48h after reperfusion. The mitochondrial ATPase activity was measured and compared between two groups before ischemia, at 6h, 24h and 48h after reperfusion. The ultrastructure of spinal cord mitochondria was observed by electron microscopy. Results The scores of hind-limbs motor function in HBO group are higher than those in control group at each time point (P<0.01). The activities of mitochondrial ATPase in both groups decreased after ischemia. Compared with HBO group, the mitochondrial Na+, k+-ATPase activity in control group was lower than that in HBO group at 6h,24h and 48h after reperfusion and lower activity of mitochondrial Ca2+,Mg2+-ATPase was observed at 6h and 24h after reperfusion in control group(p<0.01). The structures of mitochondria in control group were seriously destroyed while rnitochondrias of spinal cord tissue in HBO group were less damaged at 48h after reperfusion. Conclusion The mechanism of ischemic tolerance induced by hyperbaric oxygenation pretreatment may partly be related to the attenuation of mitochondria ATPase activity decrease after ischemia, thus protected the function of mitochondria.