| Ojective: The aim of this study was to evaluate the neuroprotective effects of glatiramer acetate(GA)and the underlying mechanisms in vascular dementia,we established the bilateral permanent occlusion of the common carotid arteries(2VO)in rats.And examined morris water maze(MWM)behavioral tests,long-term potentiation(LTP)in hippocampus and hematoxylin and eosin(H&E)stain.The expression of BDNF、GFAP 、Iba-1、cholinergic function and proinflammatory cytokines were measured.Methods: Male,healthy Sprague-Dawley rats were used and subjected to modified 2VO.After 2 weeks of 2VO,animals were injected(s.c.)four times with a total of 100 μg GA dissolved in 200 μL of PBS.During the first week,each rat received two injections separated by a 24 h interval and once per week for the following two weeks.They were randomly assigned three groups: sham group,2VO group and the GA treatment group.We assessed cognitive function by rats’ performance in a Morris water maze(MWM),electrophysiological recordings and by pathologic changes.The protein content of BDNF in the hippocampus was measured Western blotting.Real-time PCR was used to detect m RNA expressions of BDNF.Protein levels cytokines of were detected by enzyme-linked immunosorbent assay(ELISA)in ischemic brain tissue.The GFAP,Iba-1,Ch AT,VACh T and ACh E expressions in hippocampus were also observed by immunohistochemisty.Results:1.After sham surgery or the induction of injury to the CNS by neurosurgery(2VO)followed by GA or PBS cerebral hypoperfusion,the spatial learning memory of 2VO rats with and without GA-treatment was compared by using MWM testing.As shown in Fig.2,we observed that the two 2VO groups achieved longer latency times than the sham-operated group on day 2(P<0.05),day 3(P<0.05),day 4(P<0.05)and day 5(P<0.01).Notably,2VO rats treated with GA had significantly shorter latency swim times than did rats treated with PBS on day 3(P<0.05),day 4(P<0.05)and day 5(P<0.01)of training.As shown in Fig.2,the 2VO group spent less time in the target quadrant than the sham group(P<0.01),and the percentage of time spent in the target quadrant was significantly higher for GA-treated rats than for 2VO-PBS controls(P<0.05).These results demonstrate that GA treatment can ameliorate the behavior impairment induced by chronic cerebral hypoperfusion.2.The LTP at CA3-CA1 synapses in hippocampus was recorded,which was considered as one of the functional indexes of cognition.Fig.3 showed a representative electrophysiological profile,the field excitatory postsynaptic potential(f EPSP)slope was 139.33±1.76%,114.33±1.67%,133.33±3.28% in sham group,2VO group and GA group respectively.In 2VO group,the normalized slope of the f EPSP was significantly reduced compared with that in sham group while GA treatment could attenuate this change compared with that in 2VO group(P < 0.01 2VO group vs sham group;P < 0.01 GA group vs 2VO group).3.H&E strain is one of important measure to evaluate neuronal loss morphology in rats.Fig.4 contains representative micrographs of H&E staining in the hippocampus five weeks after 2VO.Extensive neuronal changes were visible in the CA1 area of the hippocampus in 2VO rats;i.e.,neuronal cell loss,nuclei shrinkage and dark staining of neurons.However,GA markedly reduced these pathological changes and attenuated cell loss induced by chronic cerebral hypoperfusion in the hippocampal CA1 area of treated rats.4.Representative immunohistochemical micrographs and statistical analysis determined that GA treatment increased the level of BDNF in the hippocampus.In addition,activated microglia cells and astrocytes were markedly increased in the hippocampus of the 2VO group.Administration of GA reduced the number of Iba-1-and GFAP-positive cells in the hippocampus of 2VO rats below that in the 2VO group(P<0.01 and P<0.05,respectively),which indicated that GA inhibited astrocytosis and microgliosis.Furthermore,GA significantly hindered the decrease of Ch AT and VACh T activity induced by cerebral hypoperfusion compared with those levels in the 2VO group(P<0.01 and P<0.01,respectively),whereas the content of the acetylcholinesterase(ACh E)had no observable change.These results indicated that GA can increase cholinergic activity(Fig.5,9,11).5.As shown in Fig.6,western blots denoted increases of BDNF expression in the hippocampuses of GA-treated rats compared with that in 2VO rats(P<0.05),but approximated that in sham rats.6.We detected Tunnel-positive material localized in the neurons’ nuclei.Compared with 2VO group,GA-treated rats showed a significant reduction of Tunnel-positive neurons in the hippocampal CA1 area(P<0.01).Overall these results suggested that GA treatment might decrease apoptosis(Fig.11)7.Fig.7 presented further analyses of m RNA expression in the hippocampus as carried out with real-time PCR.The GA-treated group had increased BDNF m RNA levels compared with that in the 2VO group(P<0.01).Additionally,RT-PCR analysis of BDNF secreted by GA-reactive T cells compared to unstimulated T cells(Fig.8)revealed that secretion of BDNF by GA-reactive T cells was significantly increased after stimulation of the T cells with GA(P<0.01).8.The effects of GA treatment on the cytokine profiles produced in supernatants of hippocampus tissue were analyzed semi-quantitatively using multi-cytokine ELISA kits.As shown in Fig.10,the production of interferon-g(IFN-g),interleukin 6(IL-6)and tumor necrosis factor a(TNF-a)was significantly reduced in the GA-treated groups compared to those levels in the 2VO group(P<0.01),whereas amounts of IL-4 and IL-10 were upregulated(P<0.05 and P<0.01,respectively).Conclusion: Our present findings indicate that GA is a CNS-related peptide with the ability to activate weakly self-reactive T cells.This agent ameliorated the cognitive deficits induced by chronic cerebral hypoperfusion and CNS cholinergic dysfunction.GA reestablished an optimal microenvironment with increased BDNF expression and modulation of the Th1/Th2 balance in the hippocampus stromal environment.In this context,GA is a likely candidate as an adjunct therapy for VaD. |
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