| Objective:The "rectangular method" was developed on the basis of the "2 o’clock method","diagonal method" and "parallel method",which are the current methods of taking ischemic penumbra in the rat model of transient middle cerebral artery occlusion(t MCAO),and proved to be equally representative as the traditional "2 o’clock method".Methods:The rat t MCAO model was established using the filament method.The regional cerebral blood flow(r CBF)was monitored by high-resolution laser Doppler to control the quality of modeling,and 2,3,5-triphenyl tetrazolium chloride(TTC)staining was used for cerebral infarction location assessment.The rats were randomly divided into 3groups: the control group,the traditional group,and the modified group.In the control and traditional groups,the ischemic penumbra was taken by the traditional method,namely the "2 o’clock method" : after TTC staining,the coronal section at the level of the striatum,i.e.the third brain slice,was selected to find the midline of the cerebral hemispheres on both sides,and then the ipsilateral side was cut longitudinally approximately 2 mm beside the midline,to remove the brain tissue supplied by the anterior cerebral artery.The ischemic penumbra was obtained by making a transverse diagonal cut at approximately the two o’clock position.In the modified group,the ischemic penumbra was taken by the modified method,namely the "rectangular method" : after TTC staining,the coronal section was taken at the level of the striatum,i.e.the third brain slice.First,the edge of the cortex was taken as the upper boundary,the border of the red-and white-dyed TTC staining was taken as the center line.Then the parallel lines were drawn with the distance of 1mm on each side away from the center line and 5mm long,forming a rectangle of 2×5mm,which was the ischemic penumbra.The expression of Superoxide Dismutase 2(SOD2),Cysteinyl aspartate specific proteinase 3(caspase3),Cysteinyl aspartate specific proteinase 9(caspase9)and Heat Shock Protein 70(HSP70)in each ischemic penumbra was measured by Western blot and the bands were quantified using Image J software.Data were analyzed using Graph Pad Prism 9.0 software.Quantitative data were expressed as mean ± standard deviation(?±s)and one-way analysis of variance was used for multiple comparisons,with P<0.05 being a significant difference between groups;SNK-q test was used for pairwise comparisons,with P<0.05 being a statistically significant difference.Result:The stable rat t MCAO model was successfully established using the filament method;r CBF was measured by high-resolution laser Doppler,after inserting the suture,r CBF decreased to <30% of the baseline blood flow,indicating successful modeling.Successful reperfusion was indicated by a recovery of CBF to >70% of the baseline blood flow after removal of the suture on the ipsilateral side.Signs of neurological deficits in the rats were: body falling to the right side,right limb dragging when walking,and right limb flexion and inability to extend when lifting the rat tail;TTC staining shows that infarcted brain tissue is white and normal brain tissue is pink.The expression of ischemic penumbra markers in rat brain tissues was measured by Western blot,and the comparison showed that the expression of SOD2,caspase3,caspase9 and HSP70 in the traditional and modified groups were significantly increased compared with the control group,and P<0.05;there was no significant difference in the expression of SOD2,caspase3,caspase9 and HSP70 between the traditional group and the modified group.Conclusion:The rectangular method proposed herein is based on the modification of the diagonal method and parallel line method,which could provide a directly observable infarct borderline and a sufficient sampling area for subsequent experimental operations regardless of the cerebral infarct location.In addition,by detecting the expression of ischemic penumbra markers in rat brain tissue,this study proved that the ischemic penumbra obtained by the "rectangle method" is as representative as the "2 o’clock method". |
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