Glibenclamide Extend The Therapeutic Time Window Of Hypothermia After Focal Ischemia-reperfusion In Rats

Ischemic stroke is a disease that causes high mortality and disability worldwide. Despite with the optimal medical treatment, the death cause by middle cerebral artery occlusion remains as high as 41-79%. The major causes of death are that vascular swelling, high intracranial pressure and brain hernia. At present, however, no clinically proven effective drug can reduce the BBB damage. The only therapy for the malignant edema is decompressive craniectomy (DC), which mainly reduce skull oppression to brain tissue, but has no effect on reducing cerebral edema. The principle of DC is that removing part of the skull to reduce intracranial pressure and relieve the oppression of the skull to brain tissue, which may cause side effects. The mortality of patients who received the surgery was as high as 30%, while 17%of the survivors were severely disabled and 22% eventually became vegetative state.Therefore, it is of great importance to find a neuroprotective agent which can effectively reduce the damage of blood brain barrier and brain edema. The pathogenesis of ischemic stroke has been studied for decades and the potential neuroprotective agent were screened, including sedation drugs, antiepileptic drugs, anti-inflammatory cytokines; however, all these drugs were proved clinically ineffective. The factor that contributed to the ineffectiveness of neuroprotective agents may be related to their single target and inability to penetrate the BBB.Therapeutic hypothermia, as a none pharmacological intervention, has an effect on the multiple targets in the ischemic cascade, which is considered to be one of the most effective treatment for ischemic stroke. Therefore, the neuroprotective effect of hypothermia has been widely concerned. The prognosis of patients with severe brain damage was significantly improved after hypothermia treatment. Studies showed that given the temperature of 32℃, the area of cerebral infarction was reduced from 211mm2 down to 17mm2. The feasibility and safety of combination of therapeutic hypothermia and intravenous thrombolytic drugs were confirmed by AID COOL clinical study. A large, multicenter, prospective randomized controlled study (EuroHYP-1) was carried out, which aims to further verify the value of hypothermia treatment for ischemic stroke, and compared with the treatment of hypothermia combined with optimal medical therapy and single medical therapy.Hypothermia is considered as one of the most promising therapy in the treatment of ischemic stroke, but its therapeutic effect is closely related to the time to cooling:the effect of hypothermia on ischemic stroke has therapeutic time window. At present, it is believed that hypothermia therapy should be initiated immediately after cerebral ischemia, however, in clinical setting it is impossible that patients can received cooling immediately after stroke. The main reason:1) hypothermia therapy requires complex instruments and strict bedside monitor; 2) patients often can not rushed to the hospital in a short time. Studies showed that patients from the onset of stroke to cooling needs at least 2-3 hours or more. Thus the time interval is inevitable, and this time interval will lead to reduced efficacy of hypothermia. Therefore, it is significant important to explore a way to extend the time window for treatment of low temperature.Sulfonylureas receptor 1-transient receptor potential M4 (SUR1-TRPM4) is an ion channel, which is composed of SUR1 receptor (regulatory subunit) and 4 TRPM4 subunits. Under normal physical conditions, the expression of the channel in brain tissue is very low. The expression of the channels will increase rapidly in neuronal cells, astrocytes, microglia and microvascular endothelial cells after cerebral ischemia. Depletion of intracellular ATP and increase of intracellular calcium can lead to the opening of SUR1-TRPM4 channels, resulting in a large influx of sodium, chloride and water. Excessive intracellular sodium and water cause cellular swelling and necrosis. Microvascular endothelial cells and astrocytes are the most important components of blood brain barrier, destruction of which may lead to the increase of blood brain barrier permeability and damage. In the case of ischemia and reperfusion, BBB damage leads to cerebral edema and even more serious condition----hemorrhage transformation. Preclinical experiments and clinical trials suggested that blockade of the SUR1-TRPM4 channel can relieve cerebral injury caused by ischemia and reperfusion, and has important role in the protection of brain function. The extent of swelling of the injured hemisphere in MCAO rats decreased (from 21% drop to 8%), reduced infarct size (40%), and reduced mortality by 24 hours (from 67% drop to 5%). In addition, glibenclamide improved the neurological function score and the protection of the cerebral cortex and white matter, which was superior to decompressive craniectomy. A retrospective analysis study found that a drop in NIHSS score and modified Rankin score was observed among type 2 diabetes mellitus patients with ischemic stroke taking sulfonylureas compared to those not taking the drug. Recently a clinical study reported that glibenclamide can effectively reduce cerebral edema and cerebral infarction area. Subsequently, Simard and other studies found that the effective time window of glibenclamide was 10 hours after stroke. Another study by Kimberly confirmed that glibenclamide is capable of reducing vascular edema after cerebral ischemia.Combination of drug and hypothermia therapy is a feasible and effective way to improve the hypothermia effect. Hypothermia may not be induced immediately after cerebral ischemia, and neuroprotective agents are likely to extend therapeutic time window of hypothermia. Green et al. showed that NMDA antagonists (MK-801) in combination with delayed hypothermia for the treatment of cerebral ischemia has a long-term protective effect on neurological function. Dietrich et al. reported that anti-inflammatory cytokine (interleukin 10, IL-10) combined with delayed hypothermia also has a long-term protective effect. Liu et al. found that topiramate can prolong the time window of hypothermia in neonatal rats with hypoxic-ischemic encephalopathy. In addition, Lee et al. found that atorvastatin can prolong the therapeutic time window of hypothermia on focal cerebral ischemia reperfusion injury in rats.Glibenclamide and therapeutic hypothermia could alleviate cerebral ischemia reperfusion injury, and both mechanisms are in complement:1) glibenclamide can block the opening of SUR1 receptor so as to reduce brain edema and inhibition of swelling and necrosis of brain cells and inhibit the expression of TNF-a after ischemia; 2) hypothermia can reduce the depletion of intracellular ATP, thereby reducing the opening of SUR1 channel, and in addition, hypothermia inhibited inflammatory factors release and oxidative stress injury. Therefore, it provides a theoretical basis for the combination of glibenclamide and hypothermia.Our previous study found that the protective effect of hypothermia on cerebral ischemia in rats could be improved by glibenclamide. Therefore, we put forward the following hypothesis:glibenclamide could extend the therapeutic time window of hypothermia on focal cerebral ischemia in rats.In order to verify the hypothesis, the following experiments were carried out in our research.Ⅰ. Verification of the therapeutic window of hypothermia on cerebral ischemia1. Background and purpose:hypothermia is a promising therapy in treatment of cerebral ischemia, which can reduce cerebral edema, infarct volume and improve neurological score.2. Materials and methods:(1) 3 h of MCAO rat model was established; (2) hypothermia intervention method:hypothermia was induced at 0,2,4, and 6 hours after ischmia. Anesthetized rats were placed at a cold room (4 ℃, about 15 minutes, rectal temperature fell to 33℃), then were placed on a heat-mat for monitoring temperature. Hypothermia was maintained about 2 h. Slow rewarming was started after hypothermia (0.5/h℃). (3) the hemispheric swelling index and infarct volume were compared between the control group and the hypothermia group (at different time points).3. Results:Brain swelling index:ischemic control group was 11.199%, delay 0 h hypothermia group was 4.304%(P<0.001), delayed 2 h hypothermia was 5.109% (P<0.001), delayed 4 h hypothermia was 7.799%(P<0.05). The brain edema index in these three hypothermia groups were significantly less than the ischemia group. In comparison with the simple ischemia group, the brain edema index was 11.298% (P>0.05) in the delayed 6h hypothermi group, with no significant statistical significance. Hypothermia can reduce brain edema in MCAO rats. Infarct volume: ischemic group was 40.434%, delayed 0 h hypothermia group was 27.451% (P<0.001), delayed 2 h hypothermia was 32.048%(P<0.001). Infarct volume in these two groups were significantly less than the ischemia group. Compared with the ischemia group, the delayed 4 h and 6 h hypothermia group was 38.546%(P>0.05) and 41.131%(P>0.05).4. Conclusion:hypothermia can effectively reduce ischemic brain edema and infarct volume. Hypothermia attenuates ischemic brain edema and reduce infarct volume within a certain therapeutic time window:in our experimental model, hypothermia can alleviate cerebral swelling within 4 h after ischemia, and reduce the infarct volume within 2 h after ischemia.Ⅱ. Glibenclamide extend the therapeutic window of hypotheemia on cerebral ischemia reperfusion injury1. Background and purpose:hypothermia is one of the most effective therapy in treating cerebral ischemia, however, therapeutic hypothermia has a short time window. We confirmed that the therapeutic time window of hypothermia does exist in treatment of MCAO rats. Thus, this study aims to further confirm whether glibenclamide could enhance the effect of hypothermia as well as prolong its therapeutic window.2. Materials and methods:(1) 3 h of MCAO rat model was established; (2) hypothermia intervention method:hypothermia was induced at 6,8 and 10 h after ischmia. Anesthetized rats were placed at a cold room (4℃, about 15 minutes, rectal temperature fell to 33℃), then were placed on a heat-mat for monitoring temperature. Hypothermia was maintained about 2 h. Slow rewarming was started after hypothermia (0.5/h℃). (3) Drug intervention:glibenclamide administration immediately after cerebral ischemia (an initial dosage of 10 g/kg, continuous 3 times per 6 h); (4) hemispheric swelling index, infarct volume, neurological score and Evans Blue dye leakage were compared among control group, hypothermia group (at different time points), glibenclamide group and combination group.3. Results:There was no significant difference in cerebral ischemia between ischemia control group (11.281%) and delayed 6 h hypothermia group (11.315%). The cerebral edema indexs were 7.685%(P<0.001) in glibenclamide group and 4.085% in combination group. It was obvious that the effect of combination of glibenclamide and hypothermia in the brain edema was significantly enhanced, and it was better than that of single use of glibenclamide (P<0.001) and hypothermia group (P<0.001). There was no significant difference in infarct volume between ischemia control group (41.102%) and delayed 6 h hypothermia group (41.814%). The infarct volumes were 39.200% in glibenclamide group and 29.970% in combination group. It was obvious that the effect of glibenclamide and hypothermia in infarct volume was significantly enhanced, and it was better than that of single use of glibenclamide (P<0.001) and hypothermia group (P<0.001). Neurological function score:vehicle group:the median of the neurological score was 10, the average was 12.65; the delayed hypothermia group was 10, the average was 12.72. The glibenclaimde group was 8, with an average of 9.57. The median of neurological function score was 6 in the combination group, with an average of 6.83. were There are statistical significance (P< 0.05) among combination therapy, vehicle, delayed hypothermia and glibenclamide groups. Blue Evans leakage detection:the two hemispheres of the sham operation group was almost close to 1. Evans Blue leakage ratio (ipsilateral/contralateral) in cortex:vehicle group,4.463 times, delayed hypothermia group 4.438 times, glibenclamide group 2.919 times, and combination therapy group 1.333 times (P<0.05). Striatal Evans Blue leakage ratio ipsilateral/contralateral):vehicle group 4.202 times, delayed hypothermia group 4.174 times, glibenclamide group 3.589 times, and combined therapy group 2.375 times (P<0.05).4. Conclusion:Glibenclamide enhanced the effect of delayed hypothermia in brain swelling and infarct volume; glibenclamide prolonged the therapeutic time window of hypothermia. Combination of glibenclamide and hypothermia improved the neurological outcome and effectively alleviate BBB damage after MCAO.Ⅲ. Combination of glibenclamide and delayed hypothermia reduced the release of pro-inflammatory factors1. Background and purpose:SUR1 channel blocker prolonged the therapeutic time window of hypothermia after cerebral ischemia, and their combination can improve neurological function and protect BBB integrity. The aim of this study was to investigate the change of SUR1 expression after MCAO insult and the effects of the combination of glibenclamide and hypothermia in reducing the release of pro-inflammatory factors.2. Materials and methods:The expression of SUR1 protein was determined by immunohistochemistry and western blot, and the levels of pro-inflammatory factors were determined by western blot.3. Results:A very low amount of SUR1 was expressed in normal tissues, while a large amount of SUR1 expressed in ischemic tissues. The results suggest that the expression of SUR1 was increased after cerebral ischemia. A low amount of pro-inflammatory factors was detected in normal tissue, while a large amount of pro-inflammatory factors was detected in ischemic tissue.4. Conclusion:The expression of SUR1 protein in infarct areas increased after MCAO, with no significant difference among each therapy group; the levels of pro-inflammatory factors in combination group was significantly decreased.Summary1. We established 3 hours of local cerebral ischemia model in rats, hypothermia was induced at 0,2,4,6 hours after ischemia onset, which aimed to explore the time window of hypothermia therapy for ischemic stroke. Results showed that hypothermia reduced brain oedema at 0,2,4 hours and infarct volume at 0,2 hours after ischemia onset. It indicated that there was a time window of hypothermia therapy in treating cerebral ischemia.2.Based on the time window of hypothermia treatment previously verified, we combined glibenclamide immediately after ischemiaand and hypothermia at 6,8,10 hours after ischemia onset, which aimed to explore whether glibenclamide can enchance and prolong the time window of hypothermia. Results showed that the combination group could effectively reduce cerebral edema and cerebral infarction volume as well as improve neurological function outcome and alleviate Evans Blue leakage. It indicated that glibenclamide could enhance the hypothermic effect and prolong its time window.3. We further investigated the expression of SUR1 protein and inflammation factors. Results showed that the SUR1 receptors were not significantly changed; glibenclamide combined with hypothermia can effectively reduce the release of inflammation factors. It indicated that combination treatment might be associated with reduction of inflammatory factors.