Mitochondrial ATP Sensitive Potassium Channel Mediates The Neuroprotective Effect Of Limb Ischemic Post-conditioning On Rat Brain Ischemia And Reperfusion Injury

Limb ischemic preconditioning, defined as one or several brief episodes of transient limb ischemia and reperfusion is believed to provide protection from the detrimental consequences of prolonged ischemia and reperfusion. The protection can occurred locally in the preconditioned tissue, or the tissue remote from the preconditioned one. The later, i.e., the prior transient ischemia in an organ remote from another target organ was called remote ischemic preconditioning. A number of studies provided evidence that pretreatment with transient ischemia of limb, small intestine or kidney could reduce subsequent myocardial ischemia and reperfusion injury.Limb ischemic post-conditioning (LIPC) is similar with limb ischemic preconditioning. The only one differentia is that, the brief episodes of limb ischemia and reperfusion in LIPC are at the right beginning of the reperfusion of the target organ. However, there are no any studies about the protection of LIPC against brain ischemia and reperfusion injury. It should be realized that LIPC is feasible in the clinical thrombolytic therapy of brain ischemic infarction with simple manipulation and minor injury to the human body.Activation of mitochondrial ATP sensitive potassium channel (mitoKATp) has been widely reported to protect rats against local brain ischemia and reperfusion injury, however, the downstream signal transduction of mitoKATP is not clear yet. The purpose of the present study was to investigate the neuroprotective effect of LIPC on the local brain ischemia and reperfusion injury in rat, and to elucidate whether p was involved in neuroprotection and its underlying mechanism.Objectives:1. To observe the neuroprotective effect of LIPC on local brain ischemia and reperfusion injury in rat, and to investigate whether mitoKATP was involved in the neuroprotection;2. To clarify the role of mitoKArp in local brain ischemia and reperfusion injury, and to explore whether mitochondrial calcium and mitochondrial permeability transition pore participate in the pathway of mitoKATP activation.Methods:1. LIPC modelThe unilateral or bilateral hindlimbs underwent ischemia by clamping the femoral artery for 10 min and then reperfused for 10 min at the right beginning of rat brain reperfusion for three cycles.2. Determination of plasma levels of dynorphin and enkephalin by radioimmunoassayAt 5 min, 15 min, 30 min, 1 h, 2 h, 12 h and 24 h after LIPC, the rat plasma was collected to determine the dynorphin level;and at 5 min, 15 min, 30 min, 2 h and 24 h after LIPC, the rat plasma was obtained to determine the enkephalin level.3. Local brain ischemia and reperfusion model in ratA silicon-coated nylon monofilament with the tip rounded by flame-heating wascarefully inserted from the lumen of the external carotid artery to that of the internal carotid artery to occlude the origin of middle cerebral artery, and 90 min later the monofilament was withdrawn to allow reperfusion.4. Intracerebro ventricular injection5. Evaluation of neurological score6. Determination of the brain infarct volume by 2,3,5-triphenyl tetrazolium chloride7. Isolation of rat brain mitochondria8. Mitochondrial protein determination9. Observation of mitochondrial microstructure by electron microscopy10. Mitochondrial swelling measurementsOpening of the mitochondrial permeability transition pore was determined by Ca2+-induced mitochondrial swelling. The decrease in light scattering closely parallels the percentage of the mitochondrial population undergoing permeability transition. The absorbance at 520 nm (A520) was measured by spectrophotometer (UV-4802, UNICO).11. Measurements of mitochondrial calcium fluorescenceIsolated brain mitochondria were loaded with 5 umol/L rhod-2 acetoxymethylester. The rhod-2 fluorescence intensity was measured in 96-well microplates using SpectraMax M2 (Molecular Devices, USA). The excitation wavelength was 540 nm and the emission wavelength was 590 nm.Results:Part I: The neuroprotective effect of LIPC on local brain ischemia and reperfusion injury in rat and the role of mitoKATp1. Unilateral LIPC partially improved the neurological score after local brainischemia and reperfusion injury in rat (P<0.05), and decreased the infarct volume compared with the untreated group undergoing brain ischemia and reperfusion (P<0.01);bilateral LIPC significantly improved the neurological score after local brain ischemia and reperfusion injury (PO.01), and decreased the infarct volume (PO.01). The effect of bilateral LIPC was more significant compared with that of unilateral LIPC (PO.05).2. Five min, 15 min, 30 min, lh and 2h after bilateral LIPC, the plasma levels of dynorphin were significantly increased (PO.01), however, it deceased to the normal level 12 h and 24 h after bilateral LIPC. The plasma level of enkephalin showed no obvious change after bilateral LIPC (P>0.05).3. The antagonist of K-opioid receptor, nor-binaltorphimine, abolished the effect of bilateral LIPC (PO.01).4. The mitoK.ATP blocker, 5-hydroxydecanoate (5-HD), also abolished the effect of bilateral LIPC (PO.01).Part II: The neuroprotection of mitoKATP activation in local brain ischemia and reperfusion injury in rat and its downstream mechanism1. Intracerebroventricular injection of 1 umol/L cyclosporine A, the blocker of mitochondrial permeability transition pore, 15 min before reperfusion significantly increased neurological score and reduced the infarct volume compared with the untreated group undergoing brain ischemia and reperfusion (P<0.01). Subsequent injection of 2 mmol/L atractyloside, the mitochondrial permeability transition pore opener, 10 min before reperfusion attenuated the effect of cyclosporine A (P<0.01). Compared with the untreated group undergoing brain ischemia and reperfusion, the mitoKATP opener, diazoxide, at 2 mmol/L significantly increased neurological score and reduced the infarct volume (P<0.01). Subsequent injection of 2 mmol/L 5-HD and atractyloside 10min before reperfusion attenuated the effect of diazoxide (PO.01), while 5-HD or atractyloside alone had no effect (P>0.05). However, diazoxide at the lower concentration (0.2 mmol/L) had no effect.2. In isolated brain mitochondria, 200 umol/L Ca2+ significantly decreased A520 compared with control (PO.01). Pretreatment with 0.5 or 1 umol/L cyclosporine A 2 min before Ca2+ administration significantly attenuated the decrease of A520 induced by high Ca2+ (P<0.05), and the attenuation was abolished by 100 umol/L atractyloside given together with Ca2+ (P<0.05). However, 5 umol/L cyclosporine A or 100 umol/L atractyloside alone had no effect (P>0.05). Pretreatment with 30 umol/L diazoxide 2 min before Ca2+ administration significantly attenuated the decrease of A520 induced by high Ca2+ (P<0.01), while 100 umol/L 5-HD given immediately after diazoxide, as with Atr, abolished the effect (P<0.05). 5-HD alone had no effect (P>0.05).3. In isolated brain mitochondria, diazoxide at 100 umol/L significantly reduced the rhod-2 fluorescence intensity, and pretreatment with 100 umol/L 5-HD or 100 umol/L atractyloside 30 min before diazoxide administration attenuated its effect (PO.01). Similarly, treatment with 1 umol/L cyclosporine A showed a marked reduction in rhod-2 fluorescence intensity, which was abolished by pretreatment with 100 umol/L atractyloside 30 min before cyclosporine A administration (P<0.01).Conclusion:1. LIPC protects rat from local brain ischemia and reperfusion injury. MUoKatp may be involved in the neuroprotection, and K-opioid receptor may also participate in the protective effect.2. Activation of mitoKATP maybe attenuate the mitochondrial calcium overload and thus inhibit mitochondrial permeability transition, eventually protect neuronsagainst ischemia and reperfusion injury.