| BACKGROUNDElevated concentration of potassium perfusate has protective effects on myocardial ischemia-reperfusion injury.But whether it has protective effect on cerebral ischemia-reperfusion injury is not clear.Previous studies have shown that potassium chloride can lead to sudden cardiac arrest,and it is easy to resuscitate.Clinically patients with cardiac arrest caused by hyperkalemia could exhibit mild neural defect post return of spontaneous circulation,no matter how long the CRP duration was.Our experiment studies showed that the rats with elevated serum potassium could tolerate longer ischemia duration.However it is not clear whether the elevated concentration of serum potassium by application of potassium chloride can alleviated the ischemia/reperfusion injury in the reperfusion phase.Using MCAO method and improved electrical stimulation method,we aimed to establish focal and global cerebral ischemia/reperfusion injury model to verify whether the elevated concentration of potassium perfusate could alleviated the cerebral ischemia/reperfusion injury.METHOD1.The focal cerebral injury model of rat was established by middle cerebral artery occlusion?MCAO?.After 90 minutes of occlusion,perfusion was resumed.Meanwhile 2.5%and 1.25%of potassium chloride?low-dose and high-dose?and saline?saline group?were pumped through the jugular vein with a micro pump at a dose of 3.2 ml/Kg.Two minutes after agents'administration,the serum potassium concentration?[K+]?was determined.After 24 hours of reperfusion.After 24 hours of reperfusion,all survivors were sacrificed and the brain tissue was taken.The area of cerebral infarct was detected by TTC staining,and the apoptosis index of brain tissue was detected by TUNEL staining.The number and distribution density of mitochondria in the infarcted area and the apoptosis of brain tissue were observed by transmission electron microscope?TEM?.Relative expression of Cleave caspase-3/Casepase-3,Bcl-2/BAX,p-CAMKII/CAMKII,reactive oxygen fluorescence intensity,malondialdehyde content,superoxide dismutase activity,mitochondrial Cytochrome cytase,calcium/potassium content in brain tissue,adenosine triphosphate,activity of Na+-K+-ATPase and Ca2+-ATPase activity were detected.We aimed to verify the effect of elevated potassium concentration perfusion on focal cerebral ischemia-reperfusion injury from the index of gross pathology and molecular biology.2.Cardiac arrest and cardiopulmonary resuscitation model was established by trans-esophagus electrical stimulation.The CPR was initiated at 7 minutes after cardiac arrest stimulation.Twenty seconds before the CPR initiation,0.0024ml/g of physiological saline,1.25%potassium chloride solution,and 2.5%potassium chloride solution were infused through the femoral vein.Animals were resuscitated with two options:chest compression+ventilation resuscitation and chest compression+ventilation+defibrillation.All the resuscitation outcome between different agents'dosages was compared.We aimed to explore the effect of elevated potassium concentration perfusion on cardioversion and resuscitation outcome of rat CA model of VF that could contribute to model stability in the further study of global cerebral ischemia-reperfusion injury.3.The CA/CPR model which were established using the rats of 8 weeks old were assigned to ventricular fibrillation group?VF group?or pulseless electrical activity group?PEA group?according to the electrocardiograph before CRP.With the improved molding method for CA,the PEA models were established using the rats of 8 weeks old and 24 weeks old and assigned to the young PEA group?PEA+Y group?and old PEA group?PEA+O group?respectively.The resuscitation success rate,blood pressure after resuscitation,and cerebral injury related indexes?nerve function score,neural specific enolase,active oxygen level and brain calcium content?and the content of troponin-I were recorded and compared in the four group.4.The CA/CPR model were established using improved CA molding of PEAmethod.Normal saline,1.25%KCL solution and 2.5%solution at dose of0.0024ml/g were administrated at onset of CPR initiation.The resuscitation success rate,neural defect score,neuron-specific enolase,the content of cerebral potassium,cerebral calcium and triphosadenine as well as the activity of Na+-K+-ATPase and Ca2+-ATPase.We aimed to verify the effect of elevated potassium concentration perfusion on global cerebral ischemia-reperfusion injury.RESULTS1.Two minutes after the administration of Kcl and Nacl,there is no statistic difference in the concentration of serum potassium between the NS and Sham group.The concentration of serum potassium in LD and HD groups increased compared with the NS group?p<0.05?.There is no statistic difference in the concentration of heart rate between the NS and Sham group;compared with the NS group,the heart rate of LD and HD group decreased?p<0.05?.There is no statistic difference in the concentration of mean artery pressure between the NS and Sham group;compared with the NS group,the mean artery pressue of LD and HD group decreased 24 hours post reperfusion,the infarction volume of the NS groups increased compared with the Sham group.The infarction volume of the LD and HD groups decreased compared with the NS group?p<0.05?.C What is more the infarction volume of HD groups decreased compared with the LD group?p<0.05?.The apoptotic index?AI?of NS group increased significantly compared with the Sham group while that of LD and HD group decreased compared with the NS group?p<0.05?.What is more the HD group showed a decreased AI that that in the LD group?p<0.05?.In the detection of the relative expression of cleaved-caspase3/caspase3 in the infarction area,the NS group showed a significant increase compared with the Sham group?p<0.01?while that of the LD and HD group decreased compared with the NS group?p<0.05?.No statistic difference of the relative expression of cleaved-caspase3/caspase3was observed between LD and HD group.In the detection of the relative expression of BCL-2/BAX,the NS group showed a significant decreased compared with the Sham group?p<0.01?while that in the LD and HD group increased compared with the NS group?p<0.05?.No statistic difference of the relative expression of cleaved-caspase3/caspase3 was observed between LD and HD group.In the detection of the relative expression of p-CAMKII/CAMKII,the NS group increased compared with the Sham group?p<0.05?.Compared with the NS group,the HD group showed a decreased relative expression of p-CAMKII/CAMKII while a decreased trend was observed in the LD group without statistic difference.The level of ROS in NS group increased significantly compared with the Sham group while the LD and HD group showed a remarkable decrease than that in the NS group?p<0.01?.What is more,the level of ROS in HD group was lower compared with the LD group?p<0.05?.There were more vacuoles in the NS group compared with the Sham group in the observation of transmission electron microscope,the number of mitochondria decreased,and the distribution of endoplasmic reticulum was uneven.Compared with the NS group,the vacuoles in group LD and HD were reduced,the number of mitochondria increased and the distribution of endoplasmic reticulum was more uniform.The opening of MPTP in the NS group increased compared with the Sham group?p<0.05?while that in the LD and HD group decreased compared with the NS group?p<0.05?.What is more,the lower opening of MPTP was observed in the HD group compared with the LD group?p<0.05?.The cytochrome cytase?COX?activity in the NS group decreased significantly compared with the Sham group?p<0.01?while that in the LD and HD group showed remarkable increased increase compared with NS group?p<0.05?.What is more,the COX activity in the HD group was higher than that in LD group?p<0.05?.In the NS group,the cerebral potassium content of infarction area decreased compared with the Sham group?p<0.05?while that of LD and HD group increased compared with the NS group.There is no remarkable difference between LD and HD group.In the NS group,the cerebral calcium content of infarction area increased compared with the Sham group?p<0.05?while that of LD and HD group decreased compared with the NS group?p<0.05?.What is more,significantly lower cerebral calcium content of infarction area was observed in the HD group compared with the LD group?p<0.01?.The MDA content in the NS group increased obviously compared with the NS group?p<0.01?while that of LD and HD group decreased significantly compared with the NS group?p<0.05?.the SOD activity of NS group,LD group and HD group decreased compared with the Sham group?p<0.05?,however no statistic difference between the NS,LD and HD group.The activity of Na+-K+-ATPase decreased obviously in the NS group?p<0.05?compared with the Sham group while that in the LD and HD group increased significantly?p<0.01?.No statistic difference between LD and HD group.The activity of Ca2+-ATP decreased in the NS group compared with the Sham group?p<0.05?while that in the LD and HD group increased compared with the NS group?p<0.05?.No statistic difference between LD and HD group.2.In the 1st program of CPR,the cardioversion rate from VF to PEA in the NS group was 0/18,and the LD group 0/18,MD group 8/18 as well as HD group 18/18.The trend of termination rate among all group is HD>MD>LD>NS,which was in a dose-dependent manner.The successful rates of CPR in each group are 8/18?44%?in the NS group,10/18?55%?in the LD group,17/18?94.4%?in the MD group and the 8/18?44%?in the HD group.The MD group was divided into 2 sub-group,MD-VF and MD-PEA,according to the electrocardiograph post KCL administration.Compared with the NS group,the MD,MD-PEA and MD-VF group presented shorter CPR duration?p<0.05?.Compared with the LD group,the MD,MD-PEA and MD-VF group presented shorter CPR duration?p<0.05?.Compared with the MD-PEA sub-group,the MD-VF sub-group presented shorter CPR duration?p<0.05?.The mean artery pressure?MAP?trend among 4 groups post ROSC was LD>MD>HD>NS.Compared with the MD-PEA sub-group,the MD-VF presented an elevated trend in MAP.There is no statistic difference of VF amplitude among 3 groups.Compared with the NS group,LD group showed larger VF amplitude in 7th minute?p<0.05?and elevated trend in 3rd and 5th minute without statistic difference,while the MD-VF group exhibited larger VF amplitude at each observation point(p>0.05.In the 2nd program of CPR,the HD group was excluded,and the rats receiving middle-dose KCL presented VF after KCL administration were defined as MD-VF-2 group.The rats maintained VF after receiving NS or low-dose KCL that were defined as NS-2 group and LD-2 group respectively.The NS-2,LD-2 and MD-VF-2 group were included in the 2nd program of CPR.Among 3 group,the successful rate of defibrillation among 3group are MD-VF-2>LD-2>NS-2,which was in a dose-dependent manner.Compared with the NS-2 group,the MD-VF-2 group needed lower defibrillation energy?p<0.05?.The lower trend of defibrillation energy was observed in the LD group compared with the NS group,however there is no statistic difference between LD and NS group.3.The successful rate of resuscitation among all groups were 11/17?64.7%,NS group?,11/13?84.6%,PEA group?,13/15?86.7%,PEA+Y group?and12/20?60%,PEA+O,group?.Compared with NS group,PEA group showed shorter stimulation duration and CPR duration?p<0.05?.Compared with the PEA group,the PEA+O group showed longer CPR duration?p<0.05?while no difference was found in the PEA+Y group.The MAP trend among all groups was PEA+Y>PEA>VF>PEA+O.compared with the Sham group,the VF,PEA,PEA+Y and PEA+O group presented decreased NDS?p<0.05?.Compared with the NS group,the NDS in PEA and PEA+O group increased?p<0.05?.Compared with the PEA+Y group,the NDS in PEA+O decreased?p<0.05?.No statistic difference was observed between PEA and PEA+Y group.Compared with the Sham group,the neuron-specific enolase increased significantly in NS,PEA,PEA+Y and PEA+O group?p<0.01?and no statistic difference was observed among the 4 groups.Compared with the Sham group,the ROS level increased significantly in NS,PEA,PEA+Y and PEA+O group?p<0.01?.What is more,PEA,PEA+Y and PEA+O group presented lower level of ROS compared with the NS group?p<0.05?and the ROS level in the PEA+Y group was lower than that in the PEA+O group?p<0.05?.Compared with the Sham group,the calcium content in cerebral tissue increased significantly in NS,PEA,PEA+Y and PEA+O group?p<0.01?while there is no statistic difference was observed among the 4 groups.The troponin of all group 24 hours post ROSC are 0.001±0.008ng/ml?Sham group?,0.57±0.12 ng/ml?VF group?,0.31±0.15 ng/ml?PEA group?,0.29±0.11 ng/ml?PEA+Y group?,0.37±0.09ng/ml?PEA+O group?respectively.Compared with the Sham group,the VF,PEA,PEA+Y and PEA+O group presented higher level of troponin?p<0.05?.Compared with the VF group,the PEA+Y group showed increased level of troponin while the PEA and PEA+O group presented a decreased trend without statistic difference.4.The successful rate of resuscitation in the 3 groups were 11/15?73.3%,NS group?,12/15?80%,LD group?,14/15?93.3%,MD-group?.Compared with the Sham group,the NDS of NS decreased?p<0.05?.Compared with the NS group,higher NDS were observed in both LD and HD group?p<0.05?.There is no statistic difference between LD and HD group.the neuron specific enolase?NSE?in all group were 0.42±0.04ng/ml?Sham group?,5.2±1.4 ng/ml?NS group?,3.9±0.4 ng/ml?LD group?;3.0±0.2 ng/ml?MD group?.The NSE of NS was increased compared with the Sham group?p<0.05?.Compared with the NS group,LD and HD group showed higher level of NSE?p<0.05?and the NSE of HD group was lower than that in LD group?p<0.05?.The cerebral potassium content at 24h post ROSC were 0.96±0.10 mmol/gprot?Sham group?,0.6±0.1mmol/gprot?NS group?,0.68±0.02 mmol/gprot?LD group?,0.74±0.04mmol/gprot?MD group??The cerebral potassium content of NS group decreased compared with the Sham group.Compared with the NS group,the cerebral potassium content of MD group was higher?p<0.05?while that of LD group showed an increased trend without statistic difference.And the cerebral potassium content of MD group was higher than that of LD group.The cerebral calcium content at 24h post ROSC were 0.08±0.01mmol/mgprot?Sham group?,0.63±0.12 mmol/mgprot?NS group?,0.60±0.11 mmol/mgprot?LD group?,0.55±0.09 mmol/mgprot?MD group?.The cerebral calcium content of NS group decreased compared with the Sham group?p<0.05?.Compared with the NS group,the cerebral calcium content of MD group was higher while that of LD group showed an increased trend without statistic difference.And the cerebral potassium content of MD group showed an increased trend without statistic difference compared with the LD group.The cerebral ATP content at 24h post ROSC were 410±14.0umol/gprot?Sham group?,281±20.1 umol/gprot?NS group?,309±29.3 umol/gprot?LD group?,342±14.5 umol/gprot?MD group?.The cerebral ATP content of NS group decreased significantly compared with the Sham group?p<0.01?.Compared with the NS group,the cerebral ATP content of MD group was higher?p<0.05?while that of LD group showed an increased trend without statistic difference.No difference was observed between LD and MD group.The cerebral Na+-K+-ATPase activity at 24h post ROSC were3.95±0.12 U/mgprot?Sham group?,2.1±0.2 U/mgprot?NS group?,2.3±0.31U/mgprot?LD group?,2.7±0.1 U/mgprot?MD group?respectively.The cerebral Na+-K+-ATPase activity of NS group decreased significantly compared with the Sham group?p<0.01?.Compared with the NS group,the cerebral Na+-K+-ATPase activity of MD group was higher?p<0.05?while that of LD group showed an increased trend without statistic difference.And the cerebral Na+-K+-ATPase activity of MD group was higher than that of LD group.The cerebral Ca2+-ATPase activity at 24h post ROSC were 5±0.3U/mgprot?Sham group?,3±0.13 mgprot?NS group?,3.1±0.4 U/mgprot?LD group?,3.5±0.2U/mgprot?MD group??The cerebral Ca2+-ATPase activity of NS group decreased significantly compared with the Sham group?p<0.01?.Compared with the NS group,the cerebral Ca2+-ATPase activity of MD group was higher?p<0.05?while that of LD group showed an increased trend without statistic difference.And the cerebral Na+-K+-ATPase activity of MD group was higher than that of LD group?p<0.05?.Conclusion1.In the MCAO model,high potassium perfusion can reduce the 24 hour focal ischemia reperfusion injury in rats.The mechanism is related to maintaining the homeostasis of potassium ions in the cell,restoring the function of mitochondria,reducing the overload of calcium and oxidative stress injury.2.In the rat ventricular fibrillation cardiopulmonary resuscitation model,the appropriate concentration of high potassium perfusion can increase the amplitude of ventricular fibrillation,promote the conversion of ventricular fibrillation,reduce the energy required for defibrillation,and improve the success rate of cardiopulmonary resuscitation in animals.3.The method of electric stimulation plus chest compression was used to establish the model of cerebral ischemia/reperfusion injury after cardiac arrest/cardiopulmonary resuscitation in rats,which was simple,reproducible,and of less interfering factors.It is worth to be propagated4.Elevated concentration of potassium perfusate can reduce the global cerebral ischemia-reperfusion injury in rats,and its mechanism is related to the recovery of energy metabolism and the reduction of intracellular calcium overload. |
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