Effect Of Neuroprotectant Single And Combined Therapy On Focal Cerebral Ischemia Volume And Neuron Apoptosis And The Expression Of The Antiapoptoic Protein Bcl-2 In Rats

Ischemic stroke is one of the major causes of death anddisability at present.But effective agent for ischemic stroke isuncertain till now.From the 1990th, Although the t-PAthrombolysis therapy have made great progress, it is still limitedby the short time window and serious complication likehemorrhage .Multiple research demonstrated that there arenecrosis and apoptosis in the acute phase of cerebral ischemia atthe same time, necrosis is the main way of cell death in the coreof cerebral ischemia and apoptosis is the main way of cell deathin the penumbaral region where cells are not so rapidly andseverely damaged. Apoptosis is the main way of cell death inthe delayed damage phase of cerebral ischemia. The penumbralregion will turn into a part of infarction consequently if theprocess of apoptosis do not be prevented,on the contary, It willshow the distinct effect of protection.Apoptosis is controlled by genes and lead cell to deaththrough physiological and biochemical reaction and theactivating of some enzymes that happened in cells.So it is calledphysiological or procedural cell death. Reserch of apoptosisrelated gene and protein is becoming advanced gradually. TheBcl-2 family protein plays an important role in the apoptosis ofneuron death ,including the antiapoptotic protein Bcl-2,Bcl-xL,Mcl-1 and the proapoptotic protein Bax,Bix,Bak,badetc.Among these protein ,Bcl-2 is an important antiapoptoticprotein,it is expressed in the neurons that be destined tosurvive.The Bcl-2 family numbers combined with itself or eachother then became dipolymer and the rate decided whetherapoptosis or not. Bcl-2 is an important antiapoptotic protein,it isprevent damage from gene and outer factors and theoverexpressed Bcl-2 increase the survival of many othersystems.The research of Isenmann demonstrated that the Bcl-2expressed distinctly in the neurons that be destined to survive inthe focal ischemia model.It is the ischemic injury cascade that decide the fate of theischemic neuron.These include involvement of overloadedintracellular calcium, production of oxygen free radicals(lipidperoxidation),release of excitatory amino acids(glutamate andaspartate),release of mediators of inflammation, failure ofenergy metabolism,and other mechanisms. The excitedtoxicity mediated by glutamate and the overload of intracellularcalcium are the main cause and the final common path of neurondeath.The medicine aimed at specific aspects of the injurycascade which have been called neuroprotectant have beenstudied in the past.There are many kinds of neuroprotectantsagainst for above the specific ischemic aspects.For example,freeradical scavengers, excitatory amino acid antagonists, calciumchannel blockers, anti-inflammatories ,energy metabolismssupporters and others have been investigated for years.But whatremains curious is that although many of the agents appear quiteeffective in preclinical studies with small-animal models ofischemia,nearly none of these have been proven conclusively tobe effective in humans.This fact limites the application and theeffect of neuroprotectant in clinical.The reason of thisphenomenon is complicated,besides the great differencebetween the ischemic animal model and the strokepatients,another main reason is that single neuroprotectant affectone of the pathological aspects of ischemia,but the cerebralinjury including all kinds of complicated pathologicalmechanisms and they affect each other,which combinedtogether to lead to the advance of cereberal injury.Themultiplicity of the mechanism and cascade effect of ischemiccerebral injury decided that there is no good effect withwhatever single agents.For that reason,combining severaleffective agents that act in different specific aspects and holdout the the mechanism of ischemia together and attain the aim ofprotect the brain is becoming one of the new way of theinvestigations of neuroprotection.There are two mainadvantages of combination neuprotectant: Firstly,A combinationof agents which individually inhibit different parts of theischemic injury cascade may provide much more protection thanany single agent.This goal is much significant since no singlestrategy has showed an universally protective in brain ischemiaof human.Secondly,by combining agents it might be possible touse agents at lower dose than required in single use .So it ispossible to avoid dose-related toxic effect.The purpose of this study was to investigate the synergisticeffects of combinations agents.We use the MCAO model onrats to test the benefits of several proposed neuroprotectionagents , including antagonism calcium ion receptor with NDP,antagonism of NMDA receptors with MgSO4 and MK-801,scavenging free radicals with GSH,anti-inflammatory withCyclophosphamide. With the use of MCAO model we studiedthe effect of single and combined neuroprotectant on infarct sizewith TTC stain ,apoptosis of neurons with TUNEL andexpression of antiapoptotic protein Bcl-2 withimmunohistochemistry.Part 1 Effect of Neuroprotectant single and combinedtherapy on focal Cerebral ischemia volume in ratsObjective: To study the effect of single and combinedneuroprotectant on the ischemic volume of focal ischemia inrats. In order to investigate whether combination ofneuroprotectants have more effective protect than any singleagent.Methods: Male Wistar rats were 4 to 5 months old andweighing 300-320g.All animals were randomly assigned beforesurgery to one of the following groups: NDP-treated group,MgSO₄-treated group,GSH-treated group, MK-801-treatedgroup, Cyclophosphamide-treated group,combined group andcontrol group. Each group had 10 animals and equally dividedinto 6h group and 24h group(n=5). The rats were then weighedand intraperitoneally anesthetized with10%chloralhydrate(40mg/kg). Focal ischemia was induced bythe suture occlusion technique based on Longa's threadmethod.NDP were purchased from Germany Bell Co. MgSO₄were purchased from Yangzhou zhongbao pharmaceutical Co.GSH were purchased from Beijing jingkehongda biology andtechnique Co.MK-801 were purchased from sigmaCo .Cyclophosphamide were purchased from Shanghai hualianpharmaceutical Co. Tirty minutes after vessel occlusion, NDP(1mg/kg), MgSO4(100mg/kg), GSH(1g/kg), MK-801(1mg/kg),Cyclophosphamide(63mg/kg) were singly and combined(NDP,MgSO4,GSH,Cyclophosphamide) infused intraperitoneally.Thecontrol group received 1.5ml normal saline intraperitonealinfusions of.After 6 hours or 24 hours the animals were weighedand neurologically assessed(rating scale:0=no deficit to 4=loseconsciousness).The animals scaled 0 or 4 point were deletedand suppled randomly. After 6h or 24h of ischemic animals wereto undergo TTC staining, then they were reanesthetized withchloralhydrate(40mg/kg)and decaptitated.The brains wereremoved and then coronally sectioned into six 2-mm coronalslices,incubated for 30 minutes in a 2%(wt/vol)solution of TTCat 37oC,and fixed by immersion in10%(wt/vol)phosphate-buffered paraformaldehyde.TTC stainsviable brain tissue red,while infracted tissue remainsunstained.TTC-stained brain sections were photographed with acomputer camera(Sony corporation).The infarct volumes werecalculated with the investigators blinded to the treatment.Theinfarct size in each of the 6 slices was quantified with the use ofan image processing software(Image Center Of PeikingNavigate University).Then the infarct areas on each slice wereadded together and multiplied by slice thickness to get theinfarct volumes.Results: After 6 hours of focal cerebral ischemia, the meanvolume of infarction was149.31±9.37 mm3 in the control group,All treated group decreased the infarct volume(P<0.05 Ftest).But the combined group decreased the infarct volumedistinctly than every singly-treated group(P<0.05 F test).Thesmallest volume of ischemia is the MK-801 group in all singlegroups,but there is no distinct difference compared with othersingle groups(P>0.05 F test). After 24 hours of focal cerebralischemia ,the infarct volume increased in each group. The meanvolume of infarction was190.00±22.32mm3 in the control group,All treated groups decreased the infarct volume(P<0.05 Ftest).But the combined group decreased the infarct volumedistinctly than every singly-treated group(P<0.05 F test).Thesmallest volume of ischemia is the MK-801 group in allsingle-treated groups,but there is no distinct differencecompared with other single groups(P>0.05).Conclusion: The results showed that whenever after 6hor 24h of cerebral ischemia the combined group decreased theinfarct volume more distinctly than any single-treated group. Itproved that combined neuroprotectant includingantagonism calcium ion receptor NDP, antagonism of NMDAreceptors MgSO4 and scavenging free radicals GSH ,anti-inflammatory Cyclophosphamide decreased the infarctvolume distinctly in different ischemic time point than everysingly-treated group(P<0.05 F test).Part 2 Study on Neuroprotectant single and combinedtherapy on apoptosis of neurons and the expression ofantiapoptotic protein Bcl-2 of focal ischemia in ratsObjective: In order to study the synergistic effects ofdifferent neuroprotectants aimed at specific aspect of theischemic injury cascade.To investigate the effect of combinedneuroprotectants on apoptosis of neurons and the expression ofantiapoptotic proteins Bcl-2 of focal ischemia in rats.Methods: All animals were randomly assigned to sevengroups and induced to focal ischemia and neurologicallyassessed and then treated as the same as part 1. After 6h or 24hof ischemic animals were transcardially perfusion fixed with 4%paraformaldehyde in 5-10ml/min phosphate buffer.The brainswere removed from the skull,postfixed 12 hours in the samefixative at 25℃and then sectioned into 2-mm coronal sliceanterior to optic chiasma.After paraffin embedding,5-цm-tricksections were cut and used for HE staining and TUNEL forapoptosis and immunohistochemical analysis for Bcl-2.Results: HE stain: After 6 hours of focal cerebralischemia,in the core of the ischemic lots of neuronsdisappeared ,some red cells and ghost-shadow neuronsappeared,at the same time few reversible damaged neuronsexsisted.At the edge of ischemic most of the cells were normaland reversible damaged neurons and some leucocytes infiltratedgently. After 24 hours of focal cerebral,in the control groupnecrosis of ischemic appeared distinctly,the core of ischemicdistinctly increased than that in 6h group ,at the same timeleucocytes infiltrated distinctly.Compared with the controlgroup,each treated group decreased the range of necrosis and thedegree of the neuron damage. Most of all the combined grouphad the most effective protection. Apoptosis of neuron: After 6hours of focal cerebral ischemia the apoptotic cells mainlyappeared at the edge of the ischemic basipodite ,the count ofapoptotic cells of control group was 12.07±1.31. After 24 hoursof focal cerebral ischemia,the range of ischemia increased andthe count of apoptotic increased to 19.91±2.82(P<0.05).whenever after 6h or 24h of ischemia, all treated groupsdistinctly decreased the count of apoptotic cells(P<0.05). At thesame time the combined group decreased the count of apoptoticcells more distinctly than that of other single-treatedgroups(P<0.05). .The smallest count of apoptotic cells is theMK-801 group in all single-treated groups,but it is no distinctcompare with other single groups(P>0.05). Bcl-2 expression:After 6 hours of focal cerebral ischemia,the antiapoptosis...