| The concept of preconditioning was first proposed by Murry in 1986. It is an interventional strategy that is given before ischemia to prevent the following ischemic injury. The internal environment changes caused by preconditioning could accommodate the organism to the ischemic insult and alleviate the damage. This phenomenon is known as ischemic tolerance. After it has been successfully applied to protect the heart from ischemic injury, various studies have confirmed its protective effect in the central nervous system. And the way of preconditioning has been expanded from moderate ischemia to hypoxia, hyperbaric oxygen (HBO), inhaled anesthetics, electric acupunture, hypothermia and several drugs. And the mechanisms involved in this protective effect include inhibiting excitatory toxicity, oxidative stress and inflammation during ischemia, affecting signal transduction and neurovascular pathophysiology, and preventing cell death and gene damage. HBO has been applied clinically treating many neurological diseases. It has been proved that HBO treatment could reduce ischemia reperfusion injury in the central nervous system. And we have reported that 2.5-3.0 ATA, 100% O2, 1h·d-1, 3-5 d of HBO preconditioning could induce ischemic tolerance in rat's brain or rabbit's spinal cord. However, the effect of long term repeated HBO preconditioning on ischemia reperfusion injury in the central nervous system has never been evaluated before. Therefore, the following two experiments are designed to investigate whether long term repeated preconditioning could induce more potent ischemic tolerance in the rabbit spinal cord, and to investigate the relationship between the neuroprotective effect produced by HBO preconditioning and neuronal apoptosis/degeneration. This research may provide essential information for protocol design of HBO preconditioning when coming to clinical use.Experiment 1 The effect of long term repeated hyperbaric oxygen preconditioning on the spinal cord ischemia reperfusion injury in rabbitsObjective: To investigate the effect of long term repeated HBO preconditioning on spinal cord ischemia reperfusion injury in rabbits.Methods: 45 New Zealand rabbits were randomly divided into 5 groups. Sham group (SHAM, n=5): open the abdominal wall and dissect the abdominal aorta without cross clamping. Control group (CON, n=10): animals received no pretreatment. The abdominal aorta was cross clamped for 20 min to produce spinal cord ischemia reperfusion injury. Hyperbaric oxygen preconditioning groups (HBO5, HBO10 and HBO20 group, n=10 respectively): animals in each group received 2.5 ATA, 100% O2, 1 h·d-1 of HBO preconditioning for consecutive 5 d, 10 d or 20 d, respectively. And spinal cord ischemia reperfusion injury was induced at 24 h after the last session of HBO preconditioning. Neurologic function was evaluated at 4, 8, 12, 24 and 48 h after reperfusion according to modified Tarlov score criteria. The spinal cords were collected after the last neurologic function evaluation and stained with hematoxilin and eosin. The number of normal neurons in the anterior horn of the lumber spinal cord was calculated.Results: Animals in HBO5 and HBO10 groups scored higher than the animals in CON group (P<0.05 and P<0.05, respectively) in the neurologic function evaluation. But the modified Tarlov score of animals in HBO20 group is similar to that of animals in CON group. All groups but HBO5 group had fewer normal neurons in the anterior horn of the lumber spinal cord as compared to SHAM group (P<0.05). But more normal neurons were preserved in HBO5 group and HBO10 group (P<0.05, P<0.05) compare to CON group, whereas no significant difference in this evaluation was found between HBO20 group and CON group. And the number of normal neurons in the spinal cord of animals in HBO20 group was less than that in HBO5 group.Conclusion: 5 d or 10 d of HBO preconditioning induce ischemic tolerance whereas 20 d of HBO preconditioning fails to protect the spinal cord from ischemia reperfusion injury. 5 d of HBO preconditioning is the most effective protocol in this research.Experiment 2 The effect of long term repeated hyperbaric oxygen preconditioning on neuronal apoptosis and degeneration in the spinal cord of rabbits after ischemia reperfusion injuryObjective: To investigate the effect of long term repeated HBO preconditioning on neuronal apoptosis and degeneration in the spinal cord of rabbits after ischemia reperfusion injury.Methods: 25 New Zealand rabbits were randomly divided into 5 groups. Sham group (SHAM, n=5): open the abdominal wall and dissect the abdominal aorta without cross clamping. Control group (CON, n=5): animals received no pretreatment. The abdominal aorta was cross clamped for 20 min to produce spinal cord ischemia reperfusion injury. Hyperbaric oxygen preconditioning groups (HBO5, HBO10 and HBO20 group, n=5, respectively): animals in each group received 2.5 ATA, 100% O2, 1 h·d-1 of HBO preconditioning for consecutive 5 d, 10 d or 20 d respectively. And the spinal cord ischemia reperfusion injury was induced at 24 h after the last session of HBO preconditioning. Spinal cords were collected at 48 h after reperfusion and stained with TUNEL or Fluoro-Jade B. The number of TUNEL positive neurons and Fluoro-Jade B positive neurons in the anterior horn of the lumber spinal cords were calculated and analyzed.Results: TUNEL positive neurons and Fluoro-Jade B positive neurons in the spinal cords of animals in HBO5 group and HBO10 group were fewer than that in CON group (P<0.05 and P<0.05, respectively). But no significant difference was found between HBO20 group and CON group in these two evaluations. And the number of Fluoro-Jade B positive neurons in HBO20 group was more than that in HBO5 group or HBO10 group (P<0.05 and P<0.05, respectively). Conclusion: 5 d or 10 d of HBO preconditioning prevented apoptosis and degeneration of neurons in the ischemic spinal cord. But 20 d of HBO preconditioning had no protective effect on these patterns of neuronal death, which might be a major cause for the inability of HBO preconditioning to induce neuroprotective effect when prolonged to consecutive 20 d.
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