Ischemic Postconditioning Protects Against Global Cerebral Ischemia-reperfusion Induced Neuronal Injury

BackgroundFollowing myocardial infarction and cancer, stroke is the third leading cause of morbidity and mortality in the developed world. The high mobility and mortality of stroke has made a high economical and psychological load on patients and their family, therefore it becomes an urgent medical problem needed to be solved. Rapid initiation of reperfusion is the most effective treatment to reduce infarct area and behavioral deficits, which caused by ischemia. Timely recovery of cerebral blood flow could rescue some reverse injured neurons. However, reperfusion has the potential to introduce additional injury; which was called reperfusion injury. Many studies have shown that overproduction of reactive oxygen species and overloading of calcium occurs in the early reperfusion period.Mechanisms of ischemia and reperfusion (I/R) injury are very intricate and they compose a negative feedback web with each other. Extensive research has aimed at finding effective strategies and drugs to ameliorate or prevent brain I/R injury. However, few have been successfully applied in clinical practice, although several strategies and drugs have shown neuroprotective effect against I/R injury in animalmodels. Another endogenous protective strategy, termed "ischemic postconditioning", has been recently reported, in which several repeated cycles of brief reperfusion and reocclusion of the coronary artery were applied at the onset of full reperfusion. Since ischemia events cannot be predicted and happen suddenly, ischemic postconditioning, which could be applied after ischemia, is attracting considerable attention. Recently, it was reported that ischemic postconditioning has neuroprotective effect: it can reduce infarct size after focal brain ischemia and reduce neuronal injury after spinal cord ischemia. In clinical emergencies, many accidents lead to global ischemia, such as drowning, cardiac arrest, and marked hypotension during cardiopulmonary surgery, and brain is intrinsically more vulnerable to ischemia than other organs. However, whether postconditioning has neuroprotective effects against global cerebral I/R injury is unknown. The elucidation of this hypothesis may bring another clinical selection to cure stroke.In this paper, we tested the hypothesis that ischemic postconditioning (interrupting reperfusion) protects against neuronal loss and behavioral deficits after global cerebral I/R, and the possible protective mechanisms involved.Objectives1. To investigate the protective effect of ischemic postconditioning against global cerebral I/R induced neuronal cell death of hippocampal CA1 layers and cortexes.2. To observe the role of ischemic postconditioning in protecting against global cerebral I/R induced behavior deficient.3. To observe the change of cerebral blood flow (CBF) in different processes of operation: before ischemia, in the process of ischemia, postconditioning and full reperfusion.4. To investigate the role of ischemic postconditioning in the release of cytochrome c from mitochondrial into cytosol by immunohistochemistry.Methods1. Animals and Surgical Procedures: 10 min transient cerebral ischemia wasinduced by four-vessel occlusion (4-VO) technique.2. Postconditioning Protocols: Rats were assigned to 6 groups. All groups were subjected to 10 min of global cerebral ischemia except the sham group. Sham: rats were subjected to the same procedures except for occlusion of the common carotids. Control: rats were subjected to 10 min ischemia only, without any further interruption of reperfusion. Post-15/15: rats were subjected to 3 cycles of 15 s/15 s reperfusion/reocclusion after 10 min of ischemia. Post-30/30: as for Post-15/15, but with 3 cycles of 30 s/30 s reperfusion/reocclusion. Post-60/15: as for Post-15/15, but with 3 cycles of 60 s/15 s reperfusion/reocclusion. Post-45-15/15: as for Post-15/15, but with 3 cycles of 15 s/15 s reperfusion/reocclusion applied after 45 s reperfusion3. Neuron Counts: 5 μm coronal sections at the level of the bregma were cut and stained with Hematoxylin/Eosin. The number of surviving neurons in the hippocampal CA1 layer per 1 mm length, and in the parietal cortex adjacent to both hippocampus and striatum per 1 mm~2, were counted as neuronal density.4. Behavioral Assessment: Seven days after ischemia, spatial learning and memory were tested in Morris water maze. Escape latency and swim speed in the training trials, and time spent in the former platform quadrant in the probe trial were quantified.5. Cerebral Blood Flow Measurement: Cerebral blood flow in different processes of operation was measured by laser Doppler flowmeter.6. Cytochrome C Immunohistochemistry: Cytochrome c immunoreactivity was determined by two-step method (EnVision+? system) and cytochrome-c-positive neurons were counted under microscope.Results1. Global Ischemia-Induced Neuronal Death and Neuroprotective Effect of IschemicPostconditioningTreatment with 3 cycles of 15 s/15 s, 30 s/30 s and 15s/15s (applied after 45 s reperfusion) reperfusion/reocclusion significantly reduced neuronal cell death after 7d or 3 weeks reperfusion, while 60 s/15 s reperfusion/reocclusion failed to provide neuroprotection.2. Ischemic Postconditioning Improved Behavioral DeficitRats in the control group required more time to find the platform and spent significantly less time in the quadrant where the platform had been than those in the sham and ischemic postconditioning groups (Post-15/15, Post-30/30 and Post-45-15/15). There was no significant difference between Post-60/15 group and control group.3. Ischemic Postconditioning Improved the Disturbance of Cerebral Blood Flow after IschemiaAfter reperfusion a stretch of hyperperfusion and then followed by a stretch of hypoperfusion was observed in all animals. The hyperperfusion time was shortened from 30 min to 20 min and the hyperperfusion value was decreased and the hypoperfusion value was increased by three cycles of reperfusion/reocclusion (except Post-60/15 group).4. Ischemic Postconditioning Decreased the Number of Neurons Immunoreactive for Cytochrome CTreatment with 3 cycles of 15 s/15 s or 30 s/30 s reperfusion/reocclusion significantly decreased the number of immunoreactive neurons both in the hippocampal CA1 layer and in the parietal cortex, while 60 s/15 s reperfusion/reocclusion did not.Conclusions1. Appropriate ischemic postconditioning strategies have neuroprotective effect against global cerebral I/R induced injury.2. The underlying protective mechanisms of postconditioning may involve improving the disturbance of cerebral blood flow and reducing the release of cytochrome c from mitochondria into cytosol.