| Objective: To detect the changes of immune cells(T cells and NK cells) in peripheral blood after hypoxic preconditioning and ischemic brain injury; to detected the changes of surface marker expression levels and cell number and morphology of central immune cells(microglia cells, astrocytes, and oligodendrocytes) in cerebral cortex after hypoxic preconditioning and ischemic brain injury; and to investigate the change of immune system in hypoxic preconditioning and ischemic brain injury and its possible mechanism involved.Methods: 70 SPF-grade Wistar male SD(Sprague Dawley) rats were randomized into 5 groups: normal control group(N, n = 12), sham operated group(S, n=15), hypoxic preconditioning group(HP, n=13), cerebral infarction group(M, n =15), and hypoxic preconditioning + cerebral infarction group(HM, n=15). Neurological function of rats were evaluated 24 hours after MCAO using Longa 5-point scoring method; volume of cerebral infarction in rats was measured and analyzed using Image J software; peripheral arterial blood T cells(CD4 +, CD8 + percentage) and NK cells(CD161+ percentage)were determined using flow cytometry; m RNA expression levels of Iba1, CNPase, and GFAP in infracted cerebral cortex were detected using real-time quantitative polymerase chain reaction(PCR); Number and morphological changes of microglia cells, astrocytes, and oligodendrocytes in brain tissues of normal and cerebral infarction rats were observed by using immunohistochemistry.Results: Rats of HM group showed milder neurological defect symptoms, lower neurological function scores(p<0.05), and decreased cerebral infarction area(p<0.05) when compared with those of M group; No difference was revealed by flow cytometry concerning T cells(CD4 + and CD8 + percentage) and NK cells(p>0.05) among all groups; PCR results from each group showed that expression level of Iba1 of the H group was decreased compared with the N group(p<0.05), while that of the HM group was lower than the M group(p<0.05); expression level of CNPase of the HP group was lower than that of the N group(p<0.05); no significant change of GFAP level was found in each group(p>0.05); Immunohistochemistry results showed that microglia cells were highly branched in the N group, while the cell morphology and cell number showed no significant variations in the HP group(p>0.05); microglial cells were activated and projection number was reduced or even disappeared in the M group, with round or amoeba-shaped forms. Microglial cell number was increased at the ischemic border zone; the decreased number of processes of microglia in the HM group, most of which were branched, was not as sharp as those of the M group. Microglial cell number was lower at the ischemic border zone versus the M group; astrocytic processes of the HP group was thickened and cell number was not significantly changed compared with the N group(p>0.05); M group was increased compared with the N group(p<0.05); astrocytes in both M and HM groups were activated, with enlarged volume of cell bodies and lengthened and thickened processes. The number of astrocytes increased at the ischemic border zone, but there was no significant difference between the M and the HM groups in astrocyte cell numbers(p>0.05); morphological changes of oligodendrocytes in both the M and HM groups were found, as the projections increased and thickened, but the numbers of oligodendrocyte between the two groups showed no significant differences(p>0.05); HP group was decreased compared with the N group(p<0.05).Conclusions:1. Hypoxia preconditioning can alleviate neurological defect symptoms and reduce the cerebral infarction area after ischemic brain damage, playing a role in brain protection;2. The number of neurons is significantly reduced in center area of infarction 24 hours after cerebral infarction, so were the microglial cells, astrocytes, and oligodendrocytes, and the morphology of glial cells is also changed, suggesting that infarction effects brain cells soon after its onset.3. The expression level of microglia in rats with cerebral infarction is decreased after hypoxic preconditioning, with a reduction in activated cell number, indicating that hypoxia preconditioning is likely to inhibit the further activation of microglia cells and thereby to inhibit their neurotoxicity and exert the neural protect function; oligodendrocytes and astrocytes may be involved in the cerebral protect in case of hypoxia preconditioning.4.The percentages of peripheral blood T lymphocytes(CD3 + / CD4 +, and CD3) and NK cells(CD161+) were no significantly increased 24 hours after cerebral infarction in rats, suggesting that the changes of peripheral immune cells may varies at different periods after the onset of stroke, and the inhibition or reduction of the functions of immune cells may be involved in the immune suppression syndrome induced by stroke. |
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