| PART 1: Establishment of an acute thromboembolic stroke model in rat suitable for delayed thromolytic therapyObjective: To evaluate the feasibility of an acute thromboembolic stroke model in Sprague-Dawley (SD) rats suitable for delayed thrombolytic therapy, with the aim to improve the repeatability and controllability of the model.Materials and Methods: An evaluation of a new thromboembolic stroke model was performed on male SD rats (n=55),which were randomly assigned before surgery into three group: untreated group (groupⅠ; n=30) ,delayed thrombolytic group (groupⅡ; n=20) and control group (n=5) .Ten to twelve fibrin-rich autologous blood clots (0.8-1.0 mm in length) were injected into the internal carotid artery of rat to establish a thromboembolic stroke model. Lesion size, location, regional cerebral blood volume (rNEI) at different time, and intracranial hemorrhage after occlusion were evaluated with MRI and HE respectively, and the survival of the rats were observed simultaneously.Results: There is no abnormity in the control group on MRI or HE.98% (49/50) of the rats were detected definite lesions after injection of clots in group I and groupⅡ. 91.84% (45/49) was located in ipsilateral hemisphere ,and 89.80%(44/49)was localized in ipsilateral parietal cortex and basal ganglion. The mean infarction size at 24 h or death was 48.29±18.57% of the ipsilateral hemisphere in group I. The rNEI at 3h,6h,9h after clots injected were 32.40±20.67%,39.43±33.50%,51.60±29.10% respectively, and there were no significant differences among them,which indicated no recanalization occurred at least in 9h. The hemorrhage rate is 3.33%(1/30) in group I and 20%(4/20) in groupⅡ. 2 rats(6.67%) in groupⅠand 10 rats(50%) in groupⅡdied in 24h after occlusion.Conclusion: The modified thromboembolic stroke model in rat had a good repeatability and controllability, and the infarction size was appropriate. It was suitable for delayed thrombolytic therapy.PART 2: Evolution of DWI and PWI in acute thromboembolic stroke model in ratObjective: To investigate the evolution of DWI and PWI after acute stroke at the center and margin of the cerebral infarction, and determined the specific criterion of MRI for assessment of penumbra in acute cerebral ischemia model.Materials and Methods: Male SD rats (n=30), weighting 300-450g, were established to acute stroke model as part 1. MRI was performed at different time with the aim to explore the evolution of rADC, rNEI, rTTM and rMSI. HE, electron microscope, immuno-hstochemistry and laser scanning confocal microscope (LSCM) were performed after finishing MRI scan.Results: The rADC at centre and margin of the cerebral infarction both declined after occlusion, and rADC at centre is significant than that at margin. There was no significant difference of rADC at the centre within 24 hours after stroke onset, then it ascended gradually and it showed sham normalization in 3d to 4d, and continued to increase to 270% at 14d. rADC at margin descended in a slow fashion , reached minimum at 10h , subsequently started to increase gradually and peaked at 5d, then resumed at 7d. There was significant different(P<0.05) of rADC at center with that at margin in 24h.The evolution of rNEI over time at centre and margin of the cerebral infarction showed a significantly difference(P<0.05), and shared the same curve diagram. rNEI grew in a slow fashion over time, started to increase quickly at 24 h, peaked at 4d, and decreased gradually later. The evolution of rTTM over time at centre and margin showed no significant difference(P>0.05), and decreased slowly. The evolution of rMSI at centre and margin show no significant difference(P>0.05), and shared the same curve diagram. Score of rMSI descend in 3h, then ascended after that. In combination of the evolution of rADC and rNEI with the changes of pathology, we could see that the changes of rADC could reflect the degree of the injured neuron and astrocyte within 24h after stroke, and rADC less than 70% of the contralateral degree indicated that the cerebral tissue was irreversible.Conclusion: With observation on the evolution of DWI and PWI, we can understand the micro blood circulation and pathology in acute cerebral ischemia, and distinguish the penumbra from ischemic necrosis, which can provide a strong theoretical basis for evaluation of the therapeutic efficacy after pharmaceutical intervention or therapy.PART 3: Evolution of DWI and PWI in acute thromboembolic stroke model in rat after pharmaceutical interventionObjective: To evaluate the neuroprotective effects of thrombosis with recombinant tissue plasminogen activator (rt-PA) combined with human albumin and magnesium sulfate in thromboembolic stroke model, with the aim to explore the possibility of extention of the thrombolytic window.Materials and Methods: A new thromboembolic stroke model was performed on male SD rats (n=90), which were randomly assigned before surgery into three groups. A group: thrombolysis with rt-PA, B group: thrombolysis with rt-PA combined with human albumin, C group: thrombolysis with rt-PA combined with human albumin and magnesium sulfate. The three groups were subsequently divided into nine subgroups according to different time after ischemia. Animals in A group received an infusion of 10mg/kg rt-PA over a period of one hour at 3h, 6h and 9h after occlusion; an infusion of rt-PA (10mg/kg) and 20% human albumin (2.5g/kg) was given i.v. alternatively in animals in B group over a period of two hours at 3h, 6h and 9h after occlusion; a dose of 500mg/kg MgSO4 was given intraperitoneal injection in C group at 3 h after surgery, and subsequently an infusion of rt-PA (10mg/kg) and 20% human albumin (2.5g/kg) was administered i.v. alternatively over a period of two hours at 3h, 6h and 9h after occlusion. An additional dose of 500mg/kg MgSO4 was given intraperitoneal injection in C group 12 hours after thrombosis. MRI was performed in each animal at prethrombolysis, 24 h, 7 d and 14 d after thrombosis. After that the animals were killed under anesthesia. Pathologic examination (including electron microscope, light microscope, immuno- histochemistry,) was performed in each animal, and LSCM were performed in two animals in each of the groups.Results:①The rNEI at centre and margin of the cerebral infarction both enhanced in group A at 3h after stroke compared with untreated group in part 2, with no recovery of rMSI at centre. The rNEI at centre and margin of the infarction in group A at 6h grew to the degree of the contralateral area, with rTTM at centre prolonged and no recovery of rMSI at centre. There were over reperfusion at both centre and margin in group A at 9h, with rTTM increased to 111% and no enhancement of rMSI in according degree. Although there were no difference of rNEI when compared B group and C group with A gourp at 3h and 6h, the rMSI increased significantly, which indicated BBB was improved. There was no over reperfusion in C group at 9h, with rMSI increased to 107%. rNEI in B group at 9h was still high, but rTTM decreased accordingly and rMSI improved obviously, which indicated the speed of expurgation of contrast drug increased.②The electron microscope showed a nomal morphology of neuron, mild swelling of mitochondrion, moderate vacuolation of end-foot process of astrocyte, and no press of the lumen of the vascular in group C at 6h and 9h compared with group A at 6h after stroke.③LSCM showed a slight, local exudation of fluorescent materials with a mild dilation of the microvascular in group C at 6h and 9h after stroke, and the fluorescent materials was remarkably reduced compared with group A at 6h after stroke.④Immuno-histochemistry confirmed that GFAP in group C at 6h and 9h after stroke increased and activity at the margin of the cerebral infarction with a clear border between the central and margin of the cerebral infarction compared with group A at 6h after stroke.Conclusion: These results demonstrate that magnesium sulfate improves neurological score; albumin reduces the leakage of BBB at ischemic area when combining with thrombolysis. So, albumin, magnesium sulfate combining with thrombolysis could extend thrombolytic time window. |
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