| Chapter 1 Establishment of an acute focal cerebral ischemia model of middle cerebral artery occlusion with thrombusObjective: To evaluate the feasibility of an acute focal ischemia model of clot embolism with Sprague-Dawley (SD) rats, 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=65) were randomly assigned before surgery into two group: control group (n=5) and experimental group (n=60). The experimental group was divided in to three groups: small-size embolic group (0.5-0.6 mm, n=15), medium-sized (0.8-1.0 mm, n=30) and big-size embolic group (1.2-1.5 mm, n=15). After preparation of the carotid artery, a catheter was introduced into the external carotid artery. During injection of autologous, fibrin-rich emboli into the internal carotid artery the common carotid artery was temporarily occluded. Regional cerebral blood volumn (rCBV) and lesion size were determined.Results: Cerebral infarct lesion was found in all of the rats in medium-size and big-size embolic groups, while only nine of fifteen rats (60%) exhibited cerebral ischemia in small-size group, and the occurrence of cerebral ischemia in medium-size and big-size embolic groups was higher than that of the small-sized group (P=0.000). The position of the cerebral infarction in medium-size group was all located at the ipsilateral hemisphere, and the lesions situated in the left cortex, subcortex and basal nucleus accounted for 93.33% (28/30). The mean infarction size at 24 h or death in small-sized, medium-size and big-size group was 14.41±8.72%, 48.29±18.57% and 73.68±18.29% respectively. There was significant difference in infarct volume among the three groups (F=33.171, P=0.000). The mean survival in small-sized, medium-size and big-size group was 301.1±23.02 h, 277.43±20.27 h and 59.93± 25.03 h. The mean survival in the three groups showed a significant difference (F=24.676, P=0.000), while no significant difference was confirmed in the mean survival between the small-sized and the medium-size group (P=0.384). rCBV in infarct lesion in medium-sized group demonstrated no significant difference at 3 h to 18 h after stroke, this suggested that no spontaneous recanalization was confirmed in the medium-sized group.Conclusion: The MCAO model established with the use of medium-sized clots proved to have an appropriate infarct size, higher survival, constant infarct position and persisted low rCBV within 18 hours. These data confirmed that the MCAO model established with the use of medium-sized clots had a good repeatability and controllability.Chapter 2 Evolution of DWI and PWI after acute cerebral ischemiaObjective: To investigate the evolution of DWI and PWI after acute stroke at the central and margin of the cerebral infarction, and determined the specific criterion of MRI for assessment of penumbra in acute cerebra ischemia model.Materials and Methods: Male SD rats (n=30), weighting 300-450g, were randomly divided into two groups: group A (n=20) and group B (n=10). Continuous MRI was performed in rats in group A at different time with the aim to explore the evolution of ADC and rCBV at early stage after onset of stroke, and discontinuous MRI was done with the rats in group B to investigate the evolution of ADC and rCBV at later stage, while explore the changes of cerebral infarct size within two weeks. HE, immuno-hstochemistry, electron microscope and laser scanning confocal microscope (LSCM) were performed after finishing MRI scan.Results: The evolution of ADC over time in the central and margin of the cerebral infarction showed a significantly difference (P=0.000), and shared the same curve diagram. ADC ascended slightly after onset of stroke, peaking at 1.5 h, and then descended in a slow fashion and reached minimum at 10 h or 11 h. ADC subsequently started to increase gradually and reached the initial peak at 24 h, and after that ADC grew quickly and arrived at the maximum at 14d. The scope of ADC at the central and the margin of the cerebral infarction within 24 h was 58.60%~69.20% and 74.00 %~91.24%respectively. The evolution of rCBV over time in the central and margin of the cerebral infarction showed a significantly difference (P=0.000), and shared the same curve diagram also. rCBV grew in a slow fashion over time, started to increase quickly at 24 h, peaked at 4d, and decreased gradually later. The scope of rCBV at the central and the margin of the cerebral infarction within 24 h was 17.83%~68.36% and 50.45%~90.30% respectively. The infarct volume increased over time, peaked at 2d and reduced gradually in the following days. 106% increase of the infarct size was showed from 6 h to 24 h after onset of the stroke, and 53.56% reduction of the infarct size was exhibited from 24 h to 14 d in this study. Electron microscope at the central of the cerebral infarction at 3h after stroke displayed a severe swelling of the nuclear of neuron, severe vacuolation of the end-foot process of astrocyte, and a narrowed lumen of the vascular, which indicated the cerebral tissue was in a state of irreversible alteration; while electron microscope at the margin of the cerebral infarction at each time after stroke showed that a mild to moderate swelling of the nuclear of neuron, moderate swelling of mitochondrion, mild to moderate vacuolation of the end-foot process of astrocyte, and a narrowed lumen of the vascular, which indicated that the cerebral tissue was reversible. Laser scanning confocal microscope (LSCM) at 14d after stroke showed a diffused exudation of fluorescent materials with a moderate dilation of the microvascular, and immuno-hstochemistry confirmed that there was no expression of GFAP in the central of the cerebral infarction and a slight expression of GFAP in the margin of the cerebral infarction with a clear border between the central and margin of the cerebral infarction. This also demonstrated that the cerebral tissue was in a state of mild to moderate degeneration. In combination of the evolution of ADC and rCBV with the changes of pathology, we could see that the changes of ADC could reflect the degree of the injured neuron and astrocyte within 24h after stroke, and ADC less than 70% of the contralateral indicated the cerebral tissue was irreversible.Conclusion: With the combination of semi-quantitative parameters of DWI and PWI with pathology in acute cerebral ischemia can not only distinguish the penumbra from ischemic necrosis but provided a strong theoretical basis for evaluation of the therapeutic efficacy after pharmaceutical intervention or therapy. Chapter 3 Strategies to extend the thrombolytic window with combination of human albumin and magnesium sulfateObjective: To evaluate the neuroprotective effects of thrombosis with recombinant tissue plasminogen activator (rt-PA) combined human albumin and magnesium sulfate in a SD rat model of embolic stroke, 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) 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 onset of MACO; an infusion of rt-PA (10 mg/kg) and 20% human albumin (2.5 g/kg) was given i.v. alternatively in animals in B group over a period of two hours at 3h, 6h and 9h after MACO; 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 (10 mg/kg) and 20% human albumin (2.5 g/kg) was administered i.v. alternatively over a period of two hours at 3h, 6h and 9h after MACO. 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 preischemia, 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 infarct volume in group B and C reduced 4.82% and 32.03% respectively compared with group A at 3 h after stroke; the infarct volume in group B and C reduced 4.52% and 33.12% respectively compared with group A at 6 h after stroke; while the infarct volume in group B and C reduced 7.11% and 34.21% respectively compared with group A at 9 h after stroke.②An increase of rCBV was confirmed in both central and marginal of the cerebral infarction from the onset of stroke to 14 d, and peaked on day 7 in each group. rCBV in group C at 6h and 9h after stroke was significantly increased in both central and marginal of the cerebral infarction from 1d to 7d (P<0.05), while no significantly difference was showed in group A and B at 6h and 9h after stroke at the same time.③No significantly difference was demonstrated in survival among the nine groups (F=0.763, P=0.636), but the survival in group C at 9h after stroke was remarkably longer than the survival in group A (P=0.043).④LSCM 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.⑤Immuno-histochemistry confirmed that GFAP in group C at 6h and 9h after stroke was increase 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.⑥The 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.Conclusion: Thrombolysis with rt-PA combined with human albumin and magnesium sulfate reduced total infarct volume, increased the survival rate and the rCBV, improved the microcirculation and reduced the intracerebral hemorrhage rate. These data demonstrated that thrombolysis with rt-PA combined with human albumin and magnesium sulfate exerted a neuroprotective effect, and the thrombolytic window up can be extended effectively when administered 6h or 9 h after stroke in the focal cerebral ischemia model in this study. The long window of opportunity may be explained with the proposed multiple mechanisms of actions for human albumin and magnesium sulfate.
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