| Objective:l.To establish experimental cerebral ischemia/reperfusion model in rats and to explore the neuroprotective effect of basic fibroblast growth factor (bFGF) pretreatment on cerebral ischemia-reperfusion injury.2. To explore the influence of bFGF pretreatment on the proliferation and differentiation of endogenous neural stem cells in rats with cerebral ischemia/reperfusion injury, which can provide a theoretical and experimental basis for the early application of bFGF in treating ischemic cerebrovascular disease in clinical practice.Methods:90 healthy male Sprague-Dawley(SD) rats were randomly assigned into three groups:sham-operated group, ischemia group, bFGF pretreatment group. A dose of 5 ul bFGF (50 mg/ml in saline) was administered to the animals of bFGF pretreatment group by intraventricular injection and an equal volume of sterile saline was administered to the animals of sham-operated group and ischemia group by the same method.2 hours after the injection, ischemia-reperfusion injury was induced by 2 hours of unilateral middle cerebral artery occlusion (MCAO) in the rats. The loss of neurological function was assessed by the modified Neurological Severity Score (mNSS) and the infarct volume was determined by TTC staining at 1,3 and 7 days after MCAO.1 day after operation, Toluidine blue staining (TBS) was performed to study the gross histological changes and TUNEL staining was performed for the detection of cell apotosis. The number of BrdU positive cells was determined by immunofluorescent staining. And imrnunofluorescence double-labelled staining was performed for detection of the BrdU positive cells expressing DCX, NeuN and GFAP.Results:1. mNSS results indicate that the sham-operated group has no neurological damage. Horner syndrome on the ipsilateral side of injury and neurological deficit on the contralateral side of injury have been found in the animals of the ischemia group and the bFGF pretreatment group.The neurological function defects are the most serious at 1 day after MCAO, and the mNSS has a tendency to improve on days of 3 and 7 after operation. Compared with the ischemia group, the mNSS of the rats of the bFGF pretreatment group significantly decreases at different observation time points after operation.2. The results of TTC staining indicate that there was no infarction in the brain of the sham-operated animals and infarcts which appear pale in TTC staining were found in animals of both the ischemia group and the bFGF pretreatment group at all of the 3 observation time points. The infarct volume is the largest at 1 day after operation and it has a tendency to decrease with time going on. Compared with the ischemia group, the cerebral infarction volumes in rats of the bFGF pretreatment group significantly reduced at each observation time point.3. No neuron damage was found by Toluidine blue staining in the rats of the sham-operated group. In the rats of the ischemia group, extensive necrosis and apoptosis of the neurons within the cerebral cortex, striatum and hippocampus on the ipsilateral side of injuery were observed, and the number of neural cells significantly reduced in the ischemic area. Although karyopyknosis and disordered arrangement of nerve cells were found in the ischemic area, the extent of the neuronal damage and the loss of neurons significantly decreased in the brains of rats of the bFGF pretreatment group compared with that of the ischemia group.4. Few of TUNEL positive cells were seen in the brain of rats of the sham-operated group at 1 day after operation. However, a large number of TUNEL positive cells were seen in the cerebral cortex and striatum of the ipsilateral side of the injury in animals of the ischemia group and bFGF pretreatment group. The numbers of TUNEL positive cells in the animals of the bFGF pretreatment group were significantly higher than those in the animals of the ischemia group.5. Cell proliferation within the subventricular zone (SVZ) region, striatum and cortex of the hemisphere with ischemic/reperfusion injury (left) was determined by BrdU labelled immunofluorescence staining. In rats of the sham-operated group, only a small amount of BrdU positive cells were observed in the SVZ region, and no BrdU positive cells were found in the striatum and cerebral cortex. BrdU positive cells were observed in all of the three regions in the rats of the ischemia group, and the number of BrdU positive cells was significantly higher than that of the sham-operated group. In the bFGF pretreatment group, the number of BrdU positive cells significantly increased compared with the ischemia group.6. The results of immunofluorescence double-labelled staining indicate that (1) the majority of BrdU positive cells express DCX 7 days after operation, BrdU/DCX positive cells accounts for 70%-80% of the total BrdU positive cells.The number of BrdU/DCX positive cells are the least in the SVZ region of the sham-operated animals. The number of BrdU/DCX positive cells in the SVZ of rats of the ischemia group is of 1.9 times as high as that of the sham-operated group. The number of BrdU/DCX positive cells significantly increased in the SVZ regions of the bFGF pretreated rats compared with the ischemia group. No BrdU/DCX positive signals were detected in the striatum and cortex of the sham-operated animals. However, a large quantity of BrdU/DCX positive cells were seen inthe cortex and striatum of the ipsilateral side in rats of the ischemia and bFGF pretreatment groups.The numbers of BrdU/DCX positive cells in the bFGF pretreatment group were significantly higher than those in the ischemia group. (2) BrdU/GFAP positive cells account for 3%-5% of the total BrdU positive cells. BrdU/GFAP positive cells were found in the SVZ, striatum and cortex in animals of the ischemia group and bFGF pretreatment group but theywere only detected in the SVZ of the sham-operated rats. The number of the BrdU/GFAP positive cells in the bFGF pretreated rats were higher than that of the ischemia group, which is of no statiscally significant difference. (3) BrdU/NeuN positive cells account for 5%-8% of the total BrdU positive cells.No BrdU/NeuN positive signals were found in the SVZ of the rats of the three groups. A small amount of BrdU/NeuN positive cells were detected in the striatum and cortex of the rats of the ischemia and bFGF pretreatment groups. The number of BrdU/NeuN positive cells in the bFGF pretreated rats is higher than that in the rats of ischemia group but it hasno statistical significance.Conclusion:bFGF pretreatment plays an important role in neuroprotection on cerebral ischemia/reperfusion injury by reducing the cerebral infarct volume, alleviating the degeneration of nerve cells, inhibiting the apotosis of neurons within the ischemic penumbra of cerebral cortex and striatum. bFGF pretreatment also plays an important role in the recovery of neurological functions by promoting the proliferation of endogenous neural stem cells and the production of more immature neurons which can replace the dead nerve cells of ischemia region. |
PDF Full Text Request