| Part1PURPOSE:To determine whether hippocampal swelling occurs on 48 hours after blood reperfusion and whether hippocampal swelling relates with delayed neuronal death (DND) in CA1 region and long-term cognitive impairment in transient middle cerebral artery occlusion (tMCAO) of SD rats.METHODS:The model of middle cerebral artery occlusion (tMCAO) was established by a silicone thread in SD rats. The thread was retreated after 2 hours ischemia. Infarct volume (V) and hippocampal volume were assessed by magnetic resonance imaging (MRI) after 48 hours of blood reperfusion. Hippocampal swelling ratio (SR) was calculated in tMCAO rats. SR= (the ispilateral hippocampal volume of infarct-the contralateral hippocampal volume)/the contralateral hippocampal volume. Meanwhile, a pseudo operation group (P group, n=10) was build. During the fifth week after reperfusion, Morris water maze was performed to evaluate spatial learning and memory function of rats. Nissl and TUNEL staining and glial fibrillary acidic protein (GFAP) immunohitochemistry staining were performed to observe neuronal loss and apoptosis and astrogliosis in hippocampus CA1 at 36 days after reperfusion. Then, tMCAO rats were divided into the DND group or the nDND group according to the presence of neuronal damage assessed by Nissl staining in CA1 region.RESULTS:The volume in the ipsilateral hippocampus of infarct is larger than the contralateral hippocampal volume in SD rats on 48 hours after blood reperfusion of tMCAO. Rats in the DND group displayed a significantly longer escape latency, less time in the target quadrant, higher percentage of dementia, less pyramidal neuronal density, higher histological grade of neuronal loss, higher apoptotic cell density (ACD) and higher IOD of GFAP in CA1 region, compared to the P and nDND groups. The hippocampus swelling ratio (SR) on 48 hours after blood reperfusion is positively correlated with the ratio of dementia, the apoptotic cell density (ACD), the incidence of DND, histological grade of neuronal loss, and GFAP expression in CA1 region. On ther contrary, hippocampus swelling ratio (SR) is correlated negatively with the percentage of platform quadrant swimming time/the total time of swimming time (tP/tT), and pyramidal neuronal density (ND) in CA1 region. No correlation was found between infarct volume and the above test indexes.CONSLUSION:MRI identified that hippocampal swelling occurs on 48 hours after blood reperfusion in tMCAO models. Evaluation of the delayed neuronal death (DND) of CA1 region and long-term cognitive impairment in rats after tMCAO, the hippocampal swelling ratio based on MRI measurements at 48 hours after reperfusion may be useful.Part 2Purpose:Hypertonic saline (HS) has not only been advocated as a hyperosmolar agent for the treatment of cerebral edema after cerebral infarction, but also attenuates astrocyte hypertrophy in a model of traumatic brain injury. We tested the hypothesis that a single-dose HS administered on 6 hours after reperfusion from transient focal cerebral ischemia attenuates hippocampal swelling and delayed neuronal death (DND) in CA1 region and long-term spatial cognitive impairment.Methods:Focal ischemia in SD rats was produced by 2-h occlusion of the middle cerebral artery (tMCAO). Rats were divided into the ischemia-reperfusion group (IR group, rats without infusion of saline after tMCAO), hypertonic saline group (H group, 4ml/kg of 7.5% saline with an injection velocity of 0.2ml/min was administered on 6 hours after reperfusion from tMCAO), and the normal saline group (N group,4ml/kg of 0.9% saline with an injection velocity of 0.2ml/min was administered on 6 hours after reperfusion from tMCAO). And a pseudo operation group (P group, surgery was performed on rats but without MCAO). Serum was collected at 10 min before and after infusion, and 4 h after reperfusion. Plasma osmolarity and sodium concentrations were measured. Hippocampal edema was assessed by comparing wet-to-dry weight ratios on 48 hours after reperfusion. Morris water maze test was performed to evaluate learning and memory function of rats during the fifth week after reperfusion. Nissl and TUNEL staining and glial fibrillary acidic protein (GFAP) immunohitochemistry staining were performed to observe neuronal loss and apoptosis and astrogliosis in hippocampus CA1 at 36 days after reperfusion.Results:Infusion of 7.5% hypertonic saline resulted elevated significantly sodium and osmolarity for at least 4 h. Water content was increased in the ipsilateral hippocampus of infarction in rats of the IR and N groups compared with the P and H groups on 48 hours after reperfusion of MCAO. Water content of the ipsilateral hippocampus of infarction (or the right hippocampus) was similar between the H and P groups. Water content decreased in contralateral hippocampus of infarction in rats of the H group compared with the other groups. In the H group, the pyramidal neuronal density in CA1 region was significantly increased compared to the N and IR groups. On the contrary, GFAP in CA1 region was significantly decreased in the H group compared to N and IR group. The N and IR groups displayed significantly longer escape latency and less time in the target quadrant compared to the P group, suggesting that ischemia/reperfusion strongly induces learning dysfunction. Hypertonic saline-treated group behaved completely different compared to the N and IR groups. Furthermore, results demonstrated that the hypertonic saline-treated rats spent significantly more time in the target quadrant when the hidden platforms were removed on the last day of training. However, there was not a significant difference of spatial cognitive performance, GFAP, pyramidal neuronal density (ND) in CA1 region between rats of the H group and rats of the P group.Conclusion:Our study identified that the ipsilateral hippocampal edema of infarction occurred in rats on 48 hours after tMCAO. The present results indicated that administration of 7.5% hypertonic saline on 6 hours after reperfusion reduce hippocampal edema on 48 hours after reperfusion, and protects long-term spatial cognitive function, and alleviated DND and astrogliosis in CA1 region in rats with tMCAO. |
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