Gadd45b alleviates white matter damage in chronic ischemic rats by modulating astrocyte phenotype

doi:10.12307/2025.549

Abstract

Abstract: BACKGROUND: Previous studies have found that growth arrest and DNA damage-inducible protein 45β (Gadd45b) is beneficial to the repair of acute cerebral ischemia, but the action mechanism is still unclear.
OBJECTIVE: To investigate the effect and mechanism of Gadd45b on white matter demyelinating lesions in rats with chronic cerebral ischemia.
METHODS: SD rats were randomly divided into four groups: sham operation group, model group, empty vector group, and Gadd45b overexpression group, with 15 rats in each group. Gadd45b-overexpressing lentivirus and no-load lentivirus were injected into the bilateral hippocampus and bilateral ventricles of rats. One week after lentivirus transfection, the rat model of chronic hypoperfusion cerebral ischemia was established by bilateral common carotid artery ligation. Three weeks after the bilateral common carotid artery ligation, the learning and cognitive functions of rats were evaluated by novel object recognition test. Luxol fast blue staining was used to observe the changes of myelin structure in the corpus callosum of rats. Hematoxylin-eosin staining and Nissl staining were used to observe the damage of the rat corpus callosum. Immunofluorescence staining was used to detect the expression of myelin basic protein and neurofilament protein 200 in the corpus callosum of rats. Immunofluorescence double staining was used to detect the expression of astrocyte markers GFAP/C3d and GFAP/S100A10 in rat brain tissue. ELISA was used to detect the levels of tumor necrosis factor-α and interleukin-6 in the supernatant of brain tissue.
RESULTS AND CONCLUSION: (1) Gadd45b overexpression could significantly improve the learning and cognitive function of rats with chronic cerebral ischemia, and improve demyelination and pathological damage in the rat corpus callosum. (2) The results of immunofluorescence showed that Gadd45b overexpression significantly increased the expression levels of myelin basic protein and neurofilament protein 200 in the brain tissue of rats with chronic cerebral ischemia. (3) Gadd45b overexpression reduced GFAP/C3d double positive cells and increased GFAP/S100A10 double positive cells in the brain tissue of rats with chronic cerebral ischemia. (4) Gadd45b overexpression reduced the levels of tumor necrosis factor-α and interleukin-6 in the brain tissue of rats with chronic cerebral ischemia. It is concluded that Gadd45b overexpression improves cognitive dysfunction by promoting the A2 phenotype transformation of astrocytes, alleviating white matter myelin structure damage and neuroinflammation in rats with chronic cerebral ischemia.

Key words: ">chronic cerebral ischemia, growth arrest and DNA damage inducible protein 45β, white matter, demyelination, astrocyte, nerve inflammation, engineered cell

CLC Number:

Yu Hui, Yang Yang, Wei Ting, Li Wenli, Luo Wenqian, Liu Bin. Gadd45b alleviates white matter damage in chronic ischemic rats by modulating astrocyte phenotype[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(36): 7797-7803.

Figures/Tables 2

2.1 实验动物数量分析计划参与实验的 SD大鼠 60只，假手术组、模型组、空载组、Gadd45b过表达组每组15只。由于创伤或术后感染共死亡7只。大鼠死亡后不计入数据统计，重新造模补充相应的大鼠。 2.2 Gadd45b过表达改善慢性脑缺血大鼠学习记忆功能以及脱髓鞘病变如图1A所示，与假手术组相比，模型组分辨指数下降；与模型组和空载体组相比，Gadd45b过表达组分辨指数显著升高。表明Gadd45b可以改善大鼠慢性脑缺血后的认知障碍。如图1B，C所示，大鼠胼胝体固蓝髓鞘染色结果显示，与假手术组相比，模型组大鼠胼胝体部位着色程度显著减轻，髓鞘结构紊乱明显。与模型组和空载体组相比，Gadd45b过表达组胼胝体区着色部分加深，髓鞘紊乱得到改善。表明Gadd45b可以逆转慢性脑缺血后脱髓鞘损伤。 2.3 Gadd45b过表达改善慢性脑缺血大鼠胼胝体区脑组织病理损伤苏木精-伊红染色结果显示(图2A，C)，假手术组大鼠胼胝体区神经元排列整齐致密、形态规则，细胞核饱满圆润，核仁清晰；模型组大鼠胼胝体区神经元排列稀少杂乱，细胞间隙大，胞核深染，核膜不清晰，可见大量凋亡坏死细胞；与空载体组相比，Gadd45b过表达组大鼠胼胝体区神经元结构则明显改善，细胞排列紧密，组织完整，凋亡坏死细胞减少。尼氏染色显示(图2B，D)，假手术组大鼠神经元结构正常，神经元中等大小，排列紧密；模型组大鼠脑组织损伤，表现为松散排列的神经元、神经元结构恶化和神经元收缩；与空载体组相比，Gadd45b过表达逆转了形态学变化，增加了神经元的存活率。表明Gadd45b可以改善慢性脑缺血大鼠脑组织病理损伤。 2.4 Gadd45b过表达减轻慢性脑缺血大鼠脑白质脱髓鞘损伤髓鞘碱性蛋白和轴突神经丝蛋白200免疫荧光结果显示(图3)，与假手术组相比，模型组大鼠胼胝体区髓鞘碱性蛋白和轴突神经丝蛋白200表达明显减少；与空载体组相比，Gadd45b过表达组大鼠胼胝体区两者表达均有所升高。表明Gadd45b可以改善慢性脑缺血大鼠脑白质脱髓鞘，促进髓鞘再生。 "

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