The Animal Study Of The Protective Effect On Long-term Functional Outcome After Ischemic Stroke By Tissue Plasminogen Activator Mutant

Part I Role for tPA mutant after middle cerebral artery occlusion in miceAim:Using the model of middle cerebral artery occlusion(MCAO),we apply multiple doses of tissue plasminogen activator mutant(tPAm)in mice after surgery.Depending on the outcome of short-term behavior tests,we will pick an optimal dose in order to study the long-term behavioral/cognition function of mice by intranasal supply of tPAm.Methods:1.8-12 weeks old C57/BL6 male mice were randomly divided into 5 groups,four different doses(0.25?g/g,0.5?g/g,1.0?g/g,2.0?g/g)were selected.In the drug group and the PBS control group,middle cerebral artery embolization(MCAO)was performed on all the experimental mice.The behavior tests were examined at various time points after MCAO,and all the mice were sacrificed on the 14th day to quantify the infarct volume.2.8-12 weeks old C57/BL6 male mice were randomly divided into two groups,as the treatment group and the PBS control group.After the MCAO were established,a series of behavioral tests were performed at various time points and all the mice were sacrificed on the 35th day to quantify the volume of brain atrophy.Results:1.In the 14 days short term behavior tests after MCAO,the drug groups turned out to be dose-depended comparing to the vehicle group.The outcome in the group using 0.5?g/g was significantly better than the vehicle group and end up in a smaller brain tissue loss.2.In the 35 days long term behavior tests after MCAO,tPAm treatment improved the neurofunctional outcome and reduced the brain atrophy volume comparing to the vehicle group.Part ? Neuroprotective effect of tPAm after MCAO regarding white matter integrityAim:We want to find out the relationship between the neuroprotective effect of tPAm after ischemic injure and white matter integrity.Based on the preliminary data,we established the condition knock out(CKO)mice,and in the further experiment we will study the long-term functional outcome of CKO mice,and if possible,the effect of tPAm on CKO mice.Methods:1,Using brain tissues from part? as treatment group and control group,we will apply immunohistology staining to all the slices such as SMI32/MBP and APC/Brdu.2,8-12 weeks old C57/BL6 male mice were randomly divided into two groups,as the treatment group and the PBS control group for 6 mice per group.We will use diffusion tensor image(DTI)to evaluate the white matter integrity.Two days after the surgery,we will first engage the T2WI scanning for every animal to confirm the stability of operation.Each animal will then be scanned at 2,3weeks in vivo and 5 weeks ex vivo after tMCAO.3,8-12 weeks old C57/BL6 male mice were randomly divided into two groups,as the treatment group and the PBS control group for 6 mice per group.We will evaluate compound action potentials(CAPs)in each animal after tMCAO.4,8-12 weeks old C57/BL6 male mice were randomly divided into two groups,as the CKO group and the WT group for 10 mice per group.After the MCAO were established,a series of behavioral tests were performed at various time points and all the mice were sacrificed on the 35th day to quantify the volume of brain atrophy.Results:1.At 35 days after MCAO,the fluorescence ratio of SMI32/MBP in the treatment group was significantly lower than that in the control group.2.In terms of the images we collected from the T2WI scanning at 2 days after tMCAO,there is no significant difference between the volum of brain edema in treatment group and control group.In the DTI scanning,the treatment group shows higher FA values both 3 weeks and 5 weeks after tMCAO.In the ex vivo scanning,RD value decreased after tPAm intervention.Throughout the assessment,AD value shows no difference between these two groups.3.In the electrophysiological examination at 35 days after tMCAO,the CAPs response of the treatment group was much better than that of the control group after electrical stimulation.4,In the long-term behavior test after tMCAO,CKO mice showed significantly worse neurobehavioral deficits compared to WT mice both in sensorimotor and cognitive function.In the MAP2 stainning of coronal sections at 35 days after tMCAO,the CKO mice showed an increase in the atrophy volume compared to WT mice.Conslusion:Intranasal supply of tPA mutants after tMCAO can significantly ameliorate white matter damage,which is conducive to preserve the structural and functional integrity of white matter.In the CKO mice,after knocking out the relevant receptor gene of endogenous tPA,the mice showed Significantly worsened functional recovery after brain injury.