| BackgroundStroke,also known as “Cu Zhong” or cerebrovascular disease,is a set of sudden onset and is an acute cerebrovascular disease characterized by the rapid development of focal neurologic deficits,among which ischemic stroke is the most common.Stroke has the characteristics of the high morbidity,high mortality and high disability,it is currently the world's third leading cause of death and is the second cause of death within all cardio-cerebrovascular diseases.Although the mortality of stroke world-widely declined,survivors and disability adjusted life years increased significantly in development countries.In China,cerebrovascular disease has become the first cause of death threatened the residents,and the mortality of youth is on the rise.As the main characteristics of neurotoxic injury,glutamate excitoxicity has close relationship with ischemic progression,cell necrosis and neuron apoptosis.Ischemia-reperfusion injury has become a problem need to be solved.At present,studies reported that some neuro-protectives can reduce the release of glutamate,as well as low temperature.It is urgently needed to acknowledge the dynamic changes of glutamate after ischemia so as to know the severity of cell necrosis for theevaluation of pharmacotherapy to delay and prevent necrosis and apoptosis.Nowadays,though micro-dialysis has been used widely in neuroscience,electro-chemical micro-electrode sensor technology(FAST)has more advantages.Evidence showed brain tissue damage of micro-electrode is 70 ?M width,which less than the suborbicular damage with 250 ?M of micro-dialysis.Micro-dialysis is bound to cause more damages in deeper depth.In addition,the method of micro-dialysis are not unified which result in varied consequences,and disability to reach the second-class of dynamic measurement.FAST methods are consistent for neurotransmitters and metabolites,give rise to minor differences and sub-second rate(1-1000 Hz)importantly.Real-time dynamic measurement is truly realized.At present,this technology have been applied in putamen and cortex for non-human primates,and in striatum and cortex in rodents.However,researches of stroke with this technology to measure glutamate in ischemic cortex have not been reported.ObjectivesPurpose of this study is to establish ischemic model of cynomolgus monkeys with evaluations of neuro-imaging and neurological deficit score for clinical manifestation,to measure glutamate dynamic changes in ischemic cortex by using electro-chemistry microelectrode arrays.Employ these outcomes to discuss: 1.Reliability of cortex glutamate measurement with FAST.2.Dynamic changes of glutamate in ischemic cortex of cynomolgus monkey.3.The correlation between glutamate and dysneuria?infarction?blood indexes.In order to accrete real-time glutamate measurement as an evaluation of pre-clinical for creating a new technology to assess laboratory pharmacotherapies of cerebral ischemia.Methods1.Establishment of transient ischemic model of cynomolgus monkeys by transient occluding left middle cerebral artery M1 for 1 hour.2.Evaluation of magnetic resonance imaging for ischemic model of cynomolgus monkeys at three time points(pre-surgery,1st week and 2nd week of post-surgery).3.Evaluation of neurological deficit score for ischemic model of cynomolgus monkeys at six time points(pre-surgery,1st?3rd?5th?7th?14th day of post-surgery).4.Analysis of routine blood tests at three points(pre-surgery,1st week and 2nd week of glutamate measurement).5.Real-time measurement of glutamate in primary motor cortex of ischemic monkeys by applying Fast Analytic Sensing Technology,respectively,at five time points for MCAO monkeys(pre-surgery?MCA M1 occluding ?removal of occlusion?1st week and 2nd week post-surgery).6.Analysis of glutamate changes in cortex of ischemic monkeys and the relationship between glutamate and dysneuria or infarction,besides,glutamate and blood routine index.Results1.Establishment of ischemic stroke model by MCAOInfarction were successfully induced by MCAO in all three monkeys.Dysneuria was observed post-surgery in all three monkeys,such as right mild facial paralysis,ataxia,right hemiplegia,etc.MRI revealed high signal in left MCA territory in T2 WI and discontinuous development in proximal of left MCA M1 in MRA,which demonstrated infarction and blocked vessel.2.Neurological deficit scoreThe NDS is zero for all three monkeys pre-surgery,dysneuria mainly occurred within one week after operation and sustainably exacerbated,most Methodssevere partial paralysis was observed in 5th day of post-surgery.Falls of consciousness,inferior coordination of movement,sensorial deprivation and disorder of right extremity,dyskinesia of right extremity(especially right upper limb),these symptoms embodied in all monkeys after operation.For MCAO monkeys,from pre-operation(0)to 5th day of post-operation(35.3±11.4),the score has increased significantly(P<0.05).Scoring change from 7th(35.5±18.4)to 14th(25.5±12.6)after surgery was not significant(P>0.05),which manifested steady neurologic function performed during this time.Capability of eating,drinking and grooming independently revealed from 1st to 2nd day of post-operation.Less of locomotor activity,eating,drinking and grooming was observed from 3rd to 4th day of post-operation,while recovered since 7th day.3.Magnetic Resonance Imaging(MRI)Pre-operative MRI of MCAO monkeys showed normal cerebral morphological structure and continuous development of left MCA M1 in Magnetic Resonance Angiography(MRA),no obvious abnormal signals have been observed in the bilateral cerebral hemisphere.At 1st week of post-surgery,T2 Weighted Imaging(T2WI)revealed contrast enhancement in the left MCA territory,discontinuous development in proximal of left MCA M1 was observed in MRA.At 2nd week of post-surgery,T2 WI showed that there is still high signal in the left MCA territory and MRI demonstrated continuous development of left MCA M1.Volumes of ischemic edema were calculated in terms of enhancement in Diffusion Weighted Imaging(DWI),what indicated ischemic edema of 1st week after operation(6.58±1.43 cm3)has increased comparing with pre-operation(0cm3)P<0.05).Compared with 1st week after operation(6.58±1.43cm3),edema of 2nd week after surgery(4.89±1.02 cm3)declined but no statistical significance(P>0.05).4.Routine blood testsRoutine indexes of routine blood tests in three time points varied of MCAO monkeys.WBC raised but no significance(P>0.05).There is a significant decline(P<0.05)between 2nd week(4.7±2.0 x1012/L)and 1st week(5.3±0.2x1012/L)after surgery for RBC,but not significant(P>0.05)compared 1st week with pre-surgery.Analysis of HGB had the same consequences with RBC.A significant decline(P<0.05)between 2nd week(101±8 g/L)and 1st week(113±11g/L),but not significant(P>0.05)compared 1st week with pre-surgery.PLT ascended significantly(P<0.05)compared with 1st week of post-surgery(489±68x109/L)and pre-surgery(319±48 x109/L),but no statistical change(P>0.05)in comparison of 2nd week and 1st week after surgery.5.Glutamate changes during ischemiaConcentrations of glutamate in monkeys' primary motor cortex as below,pre-surgery:(7.7±6.1)?M,occluding MCA M1:(37.8±25.2)?M,removal of the clip:(38.3±25.1)?M;1st week post-surgery:(12.2±9.5)?M;2nd week post-surgery:(23.7±26.1)?M ? Compared with pre-operative basic level,glutamate in cortex of MCAO monkeys has increased at the time of MCA M1 occluding and removal of the clip(all P<0.01).There are no significant changes between removal and occluding(P>0.05).Level declined at the time of 1st week after surgery,compared with MCA M1 occluding and removal of the clip(all P<0.01).No statistical changes of glutamate in the comparison of 2nd week and1 st week of post-surgery(P>0.05).6.Correlation between NDS and glutamateOutcomes showed no correlation between NDS and glutamate at 1st week post-surgery(P>0.05),and a positive correlation at 2nd week post-surgery(P<0.01).7.Correlation between ischemic edema and glutamateStatistics revealed no correlation between ischemic edema and glutamate at1 st week post-surgery(P>0.05),and a positive correlation at 2nd week post-surgery(P<0.01).8.Correlation between blood routine indexes and glutamateAt 1st week post-surgery,results demonstrated positive correlations between WBC/RBC and glutamate(all P<0.01),a negative correlation between PLT and glutamate(P<0.01),and no correlation between HGB and glutamate(P>0.05).At 2nd week post-surgery,statistics declared a positive correlation between PLT and glutamate(P<0.01),but no correlations between WBC/RBC/HGB and glutamate(all P>0.05).ConclusionsIschemic stroke model of cynomolgus monkeys can be established by MCAO.FAST is stable and reliable to measure glutamate in ischemic cortex,and measurement is available at a certain timing during ischemia.Results revealed cerebral ischemia may potentially accelerate extra-cellular glutamate release in cortex,but reperfusion may ameliorate or balance off glutamate release.There are corresponding changes of glutamate in reperfusion and severity of ischemia,likewise,glutamate has correlation with dysneuria extents and infarction.Therefore,glutamate can be used as an objective index of ischemia and provide a new feasible approach for mechanism and therapeutics of stroke. |
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