Methylprednisolone inhibits inflammation mediated by activated microglia after spinal cord injury in rat models

doi:10.3969/j.issn.2095-4344.1250

Abstract

Abstract:

BACKGROUND: Microglia, an innate immunocyte of central nervous system, has been shown to be involved in the regulation of neuroinflammation following central nervous system injury. Methylprednisolone pulse therapy is a common clinical treatment for early spinal cord injury, but its effect on the neuroinflammation after spinal cord injury is unclear.
OBJECTIVE: To observe the effect of methylprednisolone on inflammation mediated by activated microglia after spinal cord injury.
METHODS: Thirty-six Sprague-Dawley rats (provided by Shanghai Slack Laboratory Animals Co., Ltd.) were randomly divided into sham (laminectomy plus 0.9% sodium chloride ), modeling (spinal cord injury model plus 0.9% sodium chloride) and methylprednisolone groups (spinal cord injury model plus injection of methylprednisolone via tail vein) (n=12/group). The spinal cord injury model was established with NYU spinal cord impactor. Three days after administration, the morphology of Nissl body in neurons was observed under light microscope. The levels of interleukin-1β and interleukin-18 in venous blood were detected by ELISA. The number of IBA-1/ED-1 positive activated microglia was tested by laser confocal scanning microscope. The expression of ED-1 protein was detected by western blot assay. The expression levels of interleukin-1β and interleukin-18 mRNA were detected by qPCR.
RESULTS AND CONCLUSION: (1) Nissl staining results revealed that methylprednisolone can improve the morphology of neuron after spinal cord injury, and promote neuron repair. (2) Compared with the modeling group, the levels of interleukin-1β and interleukin-18 in the methylprednisolone group were significantly decreased (P < 0.05). (3) The number of IBA-1/ED-1 positive activated microglia in the methylprednisolone group was significantly less than that in the modeling group (P < 0.05). (4) The expression level of ED-1 protein in the methylprednisolone group was significantly lower than that in the modeling group (P < 0.05). (5) The expression levels of interleukin-1β and interleukin-18 mRNA in the methylprednisolone group were significantly lower than those in the modeling group. (6) To conclude, the mechanism of methylprednisolone inhibiting inflammation is related to inhibiting inflammation induced by activated microglial after spinal cord injury, which is conducive to the recovery of neurons.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: spinal cord injury, methylprednisolone, microglia, inflammation, ED-1 protein, interleukin-18 mRNA, injection via tail vein

CLC Number:

R446
R318

Huang Xingqiu, Liao Xuqiang, Shi Chenglong, Zhang Xiuli . Methylprednisolone inhibits inflammation mediated by activated microglia after spinal cord injury in rat models [J]. Chinese Journal of Tissue Engineering Research, 2019, 23(19): 3050-3055.

Figures/Tables 1

2.1 实验动物数量分析实验选用大鼠36只，分为3组，实验过程无脱失，全部进入结果分析。 2.2 尼氏染色观察神经元形态假手术组神经元形态规则，结构完整，细胞内可见虎斑样尼氏体，尼氏体饱满，数量较多；模型组神经元形态不规则，部分神经元水肿明显，部分神经元固缩坏死，可见神经元溶解、液化后形成的空泡样结构，尼氏体碎裂，较小，数量较少；甲强龙组神经元形态较模型组改善，虽然可见少量神经元水肿以及空泡样结构形成，但程度较模型组轻，尼氏体较饱满，数量较多。见图1。 2.3 Elisa检测静脉血白细胞介素1β和白细胞介素18水平见图2。假手术组白细胞介素1β和白细胞介素18水平较低，模型组白细胞介素1β和白细胞介素18水平较高；与假手术组相比，模型组和甲强龙组白细胞介素1β和白细胞介素18表达水平显著增高(P < 0.05)；与模型组比较，甲强龙组白细胞介素1β和白细胞介素18表达水平显著降低(P < 0.05)。 2.4 激光共聚集检测IBA-1/ED-1双阳性细胞 IBA-1阳性细胞呈红色，为小胶质细胞；ED-1阳性细胞呈绿色，为被激活的小胶质/巨噬细胞；IBA1/ED-1双阳性细胞呈黄色，为活化的小胶质细胞。阳性反应细胞均呈树桠状，散在分布于脊髓中，见图3。与假手术组比较，模型组和甲强龙组IBA1/ED1双阳性细胞数显著增多(P < 0.05)；与模型组比较，甲强龙组IBA1/ED1双阳性细胞数显著减少(P < 0.05)，见图4。 2.5 Western blot检测ED-1蛋白表达假手术组ED-1蛋白表达量较低，模型组ED-1白表达量较高，见图5；与假手术组相比，模型组和甲强龙组ED-1蛋白表达量显著增高(P < 0.05)；与模型组比较，甲强龙组ED-1蛋白表达量显著降低(P < 0.05)。见图6。 2.6 qPCR检测白细胞介素1β mRNA和白细胞介素18 mRNA表达假手术组白细胞介素1β mRNA和白细胞介素18 mRNA表达水平较低，模型组白细胞介素1β mRNA和白细胞介素18 mRNA表达水平较高；与假手术组相比，模型组和甲强龙组白细胞介素1β mRNA和白细胞介素18 mRNA表达水平显著增高(P < 0.05)；与模型组比较，甲强龙组白细胞介素1β mRNA和白细胞介素18 mRNA表达水平显著降低(P < 0.05)，见图7。"

References

[1] 高永良,李琰.脊髓损伤的药物治疗进展[J]. 浙江创伤外科. 2013, 18(1):141-144.

[2] 孙祥耀,海涌.脊髓损伤非手术治疗的研究进展[J]. 中国骨与关节杂志, 2016, 5(6):437-443.

[3] Alvismiranda HR,Alcalacerra G, Moscotesalazar LR.Microglia: roles and rules in brain traumatic injury. Romanian Neurosurgery. 2013;20(1):34-45.

[4] Wolf SA, Boddeke HW, Kettenmann H.Microglia in Physiology and Disease.Annu Rev Physiol. 2017;79:619-643.

[5] 张立志,张志成,李放,等.甲基强的松龙在围手术期“大剂量冲击疗法”以外的应用[J].中国脊柱脊髓杂志, 2018(2):188-192.

[6] Chen WF, Chen CH, Chen NF, et al. Neuroprotective Effects of Direct Intrathecal Administration of Granulocyte Colony‐Stimulating Factor in Rats with Spinal Cord Injury. CNS Neurosci Ther. 2015;21(9):698-707.

[7] 孟珂, 蒲玉洁,张晓明.小胶质细胞与脊髓损伤关系的研究进展[J].中国临床解剖学杂志,2017;35(4):478-9.

[8] 万峪岑,孙师,赵利娜,等.小剂量超短波治疗对大鼠脊髓损伤后炎症反应及水肿的影响[J]. 中国康复理论与实践, 2016, 22(2): 150-155.

[9] Zhou X, He X, Ren Y. Function of microglia and macrophages in secondary damage after spinal cord injury. Neural Regen Res. 2014;9(20):1787-1795.

[10] Greenhalgh AD , David S. Differences in the Phagocytic Response of Microglia and Peripheral Macrophages after Spinal Cord Injury and Its Effects on Cell Death. J Neurosci. 2014;34(18):6316-6322.

[11] Xu J, Xq E, Liu H Y, et al. Angelica Sinensis attenuates inflammatory reaction in experimental rat models having spinal cord injury. Int J Clin Exp Pathol. 2015; 8(6):6779-6785.

[12] Peng M , Wang Y L , Wang C Y , et al. Dexmedetomidine attenuates lipopolysaccharide-induced proinflammatory response in primary microglia. J Surg Res. 2013;179(1): e219-25.

[13] Guglielmetti C, Le Blon D, Santermans E, et al. Interleukin-13 immune gene therapy prevents CNS inflammation and demyelination via alternative activation of microglia and macrophages. Glia. 2016;64(12):2181-2200.

[14] Swaroop S, Sengupta N, Suryawanshi AR , et al. HSP60 plays a regulatory role in IL-1β-induced microglial inflammation via TLR4-p38 MAPK axis. J Neuroinflammation. 2016 Feb 2;13:27.

[15] Tang Y,Le W.Differential Roles of M1 and M2 Microglia in Neurodegenerative Diseases. Mol Neurobiol. 2016;53(2): 1181-1194.

[16] Liddelow SA, Guttenplan KA, Clarke L E, et al. Neurotoxic reactive astrocytes are induced by activated microglia. Nature.2017; 541(7638):481-487.

[17] Abdanipour A,Tiraihi T,Taheri T, et al. Microglial activation in rat experimental spinal cord injury model. Iran Biomed J. 2013;17(4):214-220.

[18] Mcgeer PL,Mcgeer EG.Targeting microglia for the treatment of Alzheimer's disease.Glia.2016; 64(10):1710-1732.

[19] Tewari M, Monika, Varghese RK, et al. Astrocytes mediate HIV-1 Tat‐induced neuronal damage via ligand‐gated ion channel P2X7R. J Neurochem. 2015;132(4):464-476.

[20] Bracken MB.Steroids for acute spinal cord injury. Cochrane Database Syst Rev. 2002;(3):CD001046.

[21] 徐静磊.甲强龙与葛根素联用在预防急性脊髓缺血再灌注损伤中的疗效探讨[D]. 郑州:郑州大学,2013.

[22] 俞勇,陈明,杜利娜.甲强龙对环扎法所致大鼠脊髓损伤后神经细胞凋亡的相关性研究[J].临床和实验医学杂志, 2016, 15(16): 1559-1561.

[23] 杨新明,成垚昱,张振梁,等.甲强龙在骨髓间充质干细胞治疗大鼠脊髓损伤中的作用及对肿瘤坏死因子-α和白细胞介素-1β表达的影响[J].中国医学科学院学报,2017,39(05):615-622.

[24] Liu JT, Zhang S, Gu B, et al.Methotrexate combined with methylprednisolone for the recovery of motor function and differential gene expression in rats with spinal cord injury. Neural Regen Res. 2017;12(9):1507-1518.

[25] 成垚昱,杨新明,张振梁,等.大剂量甲强龙在BMSC移植治疗大鼠脊髓损伤中的作用[J].山东医药,2017, 57(21):13-16.