| Stroke is a rapid developmental brain injury caused by blocked brain blood supply, including ischemic damage caused by thrombus and hemorrhagic injury due to hematoma formation. Stroke, which is of high incidence and morbidity, is a common and frequently-occurring disease in older adults. With the rapid development of social economy, stroke also showed a young tendency of occurrence.Spatial cognitive deficits including spatial learning and memory damage is very common after the occurrence of ischemic stroke. About 43.5% stroke patients have cognitive impairment, among which half of them are with memory deficits. The existence of memory deficits not only influences the efficiency of recovery, but also the function recovery. Furthermore, it brings the social and family heavy economic burden and spirit stress.It is well accepted that exercise is beneficial for stroke recovery which becomes an indispensable method in therapeutic schedule. But little therapy is for memory recovery. Recently, it has confirmed by many animal experiments and clinical studies that neurogenesis in adult mammalian is critical for the reconstruction of brain function after stroke. Spatial learning and memory mainly depends on the hippocampus, thus neurogenesis in dentate gyrus (DG) is essential for learning and memory formation. A lot of literatures have confirmed focal cerebral ischemia in adult mammals can stimulate neurogenesis in lateral ventricle subventricular zone (SVZ) and DG in hippocampus. These newborn neurons after ischemic injury were shown to migrate to injured brain regions, become actively integrated into the existing circuitry, and form appropriate synapses, which contributed to ameliorate neurological deficits and form hippocampus-dependent memory. These studies raise the possibility that spatial cognitive deficits after ischemic stroke may be improved through enhancing neurogenesis in hippocampus.Aiming to solve this problem, we investigate the contribution of enriched environment on hippocampal neurogenesis with spatial learning recovery after ischemic stroke. It would provide a safe and effective method of rehabilitation for spatial cognitive deficits after ischemic stroke. We examined the effect of enriched environment on hippocampal neurogenesis and functional recovery, especially spatial cognitive function after focal cerebral ischemia. Middle cerebral artery occlusion (MCAO) model, which is stable and feasible, was used in this project.Part I: Investigate influence of enriched environment on spatial learning recovery after cerebral ischemia.On day 0, mice were subjected to middle cerebral artery occlusion (MCAO). One week after surgery, the mice with hemiparesis were randomly divided into 2 groups: MCAO model and enriched environment. The enriched group of 15 mice was placed in a large, enriched-environment cage, equipped with horizontal and vertical boards, ladders, swings, wooden tunnels, two running wheels and other objects of different sizes and materials. These objects were moved around once daily and some objects were exchanged with new ones twice a week. Sham and MCAO model animals were treated identically and housed in standard cages.Firstly, we tested mice in Morris water maze from day 45 to 49 after MCAO to examine whether postischemic exposure to different experiences could improve spatial cognitive performance. All groups of mice improved in their ability to locate the platform over the 5 days'trials. There was significant difference between sham and model groups for latency to reach the platform, suggesting that focal cerebral ischemia impaired spatial cognitive performance. Living in an enriched environment significantly reduced the latency time, compared with MCAO control mice. The differences of latency time among groups were not likely to be attributed to a deficit in their swimming ability, as the swimming velocities measured during the tasks were not significantly different. These data indicate that postischemic environmental enrichment reverses spatial cognitive impairment caused by stroke.The improved spatial memory is possibly due to reduced ischemic injury after exposure to experiences. To exclude this possibility, we carried out infarct volume measurement and neurons loss detection. A well-demarcated infarct in the striatum and cortex was detected 49 days after MCAO. No lesions were found in sham-operated animals. There were no significant differences in infarct volume among groups. Moreover, Fluoro-Jade staining showed no animal in any group with ischemic damage in the dentate gyrus, although focal cerebral ischemia caused a marked neuronal loss in the cortex. These data suggest that enriched environment does not affect ishcemic injury.The mortality of animals during the first week after MCAO, except for the mice in the sham group, was 44.4% (95/214). During physical exercises, the mortality was 0.0% (0/24) in sham, 27.5% (11/40) in model, 42.2% (19/42) in enriched environment with MCAO, respectively, suggesting that postischemic exercises do not attenuate ischemic injury.Part II: Investigate the relationship of neurogenesis with enriched environment on spatial learning and memory improvement after cerebral ischemia, and also the molecular mechanism.To examine whether enriched environment increases neurogenesis, mice received i.p. injections of 5-bromo-2'-deoxyuridine (BrdU) 50 mg?kg-1 daily during day 14-20 after MCAO. For the cellular proliferation study, animals were sacrificed 24 hours after the last BrdU injection to examine the number of newly formed cells in the DG. To determine the survival of the newly born cells, animals were sacrificed 28 days after the last BrdU injection. The number of BrdU+ cells in the ipsilateral dentate gyrus increased substantially in model compared with sham. Significantly more BrdU+ cells were observed in the ipsilateral dentate gyrus than in the contralateral in model groups. The BrdU~+ cells number in the ipsilateral in other groups was also higher than that in the contralateral even though without significant differences. This result indicates that progenitor cells proliferation induced by ischemia is still persisting during the duration. Moreover, we did not see any significant effect of enriched environment on proliferation of cells in the dentate gyrus. These results suggest that exposure to enriched environment after focal ischemia for 2 weeks do not stimulate progenitor cells proliferation in the dentate gyrus.The number of surviving BrdU+ cells after 28 days significantly decreased in MCAO control either in ipsilateral or in contralateral dentate gyrus compared with sham, although the BrdU+ cells number of MCAO control was higher than that of sham at 1 d after the last injection. However, Living in an enriched environment significantly enhanced the number of surviving BrdU+ cells in both ipsilateral and contralateral dentate gyrus. Interestingly, there were fewer BrdU+ cells in the ipsilateral dentate gyrus of MCAO control mice than in the contralateral. This difference between ipsilateral and contralateral was more obvious in enriched group. When the number of surviving cells was expressed as a ratio of the number of BrdU+ cells at 1 d after the last injection of BrdU, it was found that environmental enrichment could significantly increase the cell survival rate in both ipsilateral and contralateral dentate gyrus, compared with model group. As shown in the number of surviving progenitor cells, survival rate of BrdU~+ cells in the ipsilateral dentate gyrus was significantly lower than that in the contralateral in enriched group. These results suggest that exposure to enriched environment for 6 weeks promotes the survival of the dividing progenitor cells. To determine whether BrdU+ cells in the dentate gyrus became neurons, we used antibodies against neuronal marker NeuN and MAP-2. We found that majority of BrdU+ cells in the GCL were co-labeled with NeuN or MAP-2 4 weeks after the last BrdU injection. These findings provided evidence that most of the newborn cells in the dentate gyrus matured into granule neurons. Therefore, living in an enriched environment after focal ischemia could improve neurogenesis in the dentate gyrus by increasing the number of new neurons.There was a negative correlation between the total number of surviving BrdU+ cells in both dentate gyri and mean latency (day 45-49) to reach the platform. These data indicate that the survival of dividing progenitor cells in the dentate gyrus is associated with the improved spatial cognitive performance and that exposure to enriched environment improves spatial memory by enhancing the survival of dividing progenitor cells in the dentate gyrus.Western blots showed a substantial increase of pCREB level in both ipsilateral and contralateral dentate gyrus in enriched group, compared with MCAO control mice. However, there was no significant difference between groups with regard to protein content of CREB. Consistent with the Western blots, staining of the hippocampal sections with anti-pCREB antibody demonstrated that the number of pCREB labeled nuclei was markedly increased in enriched group on day 28 after surgery, compared with MCAO control. The pCREB-postive cells aligned in the lowest part of GCL, adjacent to or including the SGZ, of dentate gyrus. The pCREB protein content and the number of pCREB labeled nuclei in ipsilateral dentate gyrus were fewer than that in contralateral in enriched group. These data suggest that enriched environment after focal cerebral ischemia up-regulates the level of pCREB in the dentate gyrus.In summary, our data suggest that, in the adult mice, exposure to enriched environment after ischemic stroke may promote newborn cells survival in the dentate gyrus by up-regulating CREB phosphorylation and consequently restore impaired hippocampus-dependent memory. |
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