The Dynamic Detection Of NO In Rat Hippocampus During Cerebral Ischemia And Reperfusion In Vivo

Brain ischemic injury is a complex pathophysiologic progress and it causes theneurons ischemic injuries. It was considered that these injuries were a kind of celldeath because of the energy insufficiency in the cerebral cells after the cerebral bloodflow was interrupted. Recently many researches found that cerebral ischemia andreperfusion caused cell injuries through a series of at least 4 reactions, includingenergy barriers and excitatory amino acid toxicity, depolarization of infarct area,inflammation and program cell death. Abundant animal experiments and clinicalresearches approved that there was a complicated pathophysiologic progress incerebral ischemia and reperfusion. The reperfusion could save the dying cells. On theother hand it could aggravate the cell injury and cause cell death.Nitric oxide (NO) plays an important role in this process. As an importantmolecular messenger, NO works as a nerve mediator as well as a detrimental factor innervous system. In central and peripheral nervous systems NO participates in manyforms of synaptic transmission and pathological progress. Recently its dual effectbecomes a hot spot in experimental and clinical researches, especially under the statusof cerebral ischemia and reperfusion (I/R).In the present study, we measured NO levels in rat hippocampus using acarbon-fiber NO sensor during the process of global cerebral ischemia and the initialstage of reperfusion. Meanwhile, we separately observed the effects of two differentNOS inhibitors to the production of NO in this process. Based on the real-timemeasurement of NO concentration in rat hippocampus, the dynamic variance of NOconcentration in global cerebral ischemia and reperfusion has been revealed. Besidesthe animal experiments, we cultured hippocampal neurons in 20 min OGD andreperfusion to detect the changes of NO in OGD/reperfusion neurons. So we revealedthe dynamic changes of NO during ischemia and reperfusion, both in animal and celllevels.Although extensive research for I/R injury treatment has been performed in thepast several decades, few neuroprotectants and methods have been successfully translated from basic research into a clinical application. Recently ischemicpost-conditioning is a relatively novel concept. Unlike pre-conditioning,post-conditioning is composed of several repeated cycles of brief reperfusion andreocculusion of the artery applied at the onset of full reperfusion. Thie method hasbeen proved had powerful protective effects on ischemia, and may eventually lead tomore extensive clinical application. However, the underlying protective mechanism ofpost-conditioning remains illusive and in our experiment, we detected the dynamicchange of NO during the ischemic postconditioning against global cerebral I/R in vivo,and compared the effects of different numbers of cycles and periods forreperfusion/occlusion, to investigate the role of NO in ischemic postconditioning.Combining the cerebral blood flow (CBF) and the death ratio of neuron cells in rathippocampus, we could explore the mechanisms underlying the interruptingreperfusion. Furthermore, our results discover the best parameters that generate thestrongest protection and may provide important clues for cerebral I/R treatment.The results as follows:1. Alternation of NO levels in hippocampus resulted in a change of arterial bloodpressure and the electrochemical signal of NO could be recorded in real time.Intravenous administration of L-NAME (50mg/mL, 20μL-30μL) induced adecrease in NO concentration (4.5nM-6nM).2. In global cerebral ischemia and the initial stage of reperfusion, the whole NOconcentration profile could be divided into four stages as follows: the earlyischemia stage, during which there was a rapid and significant decrease of NOconcentration.; after the NO declined to the minimum, there was a platform withNO concentration keeping roughly unchanged; upon the onset of reperfusion, therewas an acute elevation stage lasted for 15 minutes; another steady stage appearedafter NO concentration reached the peak of reperfusion.3. We also observed the effects of two inhibitors of NOS on NO concentration.The two inhibitors (7-NI and 1400W) were respectively administratedintravenously at the onset of reperfusion and 1 h later. NO biosynthesis during the initial stage of reperfusion was mainly nNOS-dependent.4. We observed the dynamic changes of NO in hippocampal neurons inOGD/reperfusion. NO concentration was increased slowly and reached a platformafter 10-12 min.5. The speed of NO concentration increase was slowed down, but the extent onincrease was remarkably enlarged after ischemic post-conditioning, and CBF wasalso increased. We thought that post-conditioning induced a gentle and increasedNO synthesis, which inhibited the NO toxicity and strengthened the effect of NO toCBF, and finally decreased I/R lesion.6. Different numbers of cycles as well as different durations of reperfusion andocclusion had different effect to I/R injury. We found that 3 cycles of 30 sreperfusion and 30 s occlusion had the best protective effect.In conclusion, we detected the NO concentration in rat hippocampus in globalcerebral ischemia and the initial stage of reperfusion in vivo, and calculated thechange concentration of NO during this process. Meanwhile, we observed thechanges of NO in hippocampal neurons in 20 min OGD and reperfusion. So werevealed the dynamic changes of NO during ischemia and reperfusion, both inanimal and cell levels. It is very useful to reveal the behavior of NO concentrationin stroke and reperfusion. For post-conditioning, we detected the dynamic changeof NO during the ischemic postconditioning, and compared the effects of differentnumbers of cycles and periods for reperfusion/occlusion. We believed that NO alsoworked as an essential infactor in post-conditioning pathway.Our dynamic measurements of NO during this process would offer a timetherapeutic window for I/R treatment, which is very helpful for clinical therapies inthe future.