Immunosuppressed rat model of cerebral hemorrhage: construction and assessment

doi:10.3969/j.issn.2095-4344.2016.40.002

Abstract

Abstract:

BACKGROUND: Treatment after intracerebral hemorrhage can effectively suppress immune function. The immune suppression after ischemic stroke has been studied in detail.

OBJECTIVE: To construct an immunosuppressed rat model after cerebral hemorrhage, and assess its stability.

METHODS: Sixty Wistar rats were randomly divided into control group, sham group, and cerebral hemorrhage group, with 20 rats in each group. Rat models of acute cerebral hemorrhage were established by 50 μL arterial blood injection in the rat basal ganglia. Rats in the sham group were injected with 50 μL of saline, and the operation was identical to cerebral hemorrhage model. Rats in the control group received no treatment. At 24, 48, 72 and 96 hours after model establishment, leukocytes, lymphocytes, and lymphocyte percentage were analyzed by blood analyzer. Enzyme linked immunosorbent assay was used to determine the levels of serum proinflammatory cytokine interleukin-6 and anti-inflammatory cytokine transforming growth factor β in rats. Dissected rat spleen tissue was subjected to histological and histopathological detection. RT-PCR and western blotting were utilized to measure changes in transforming growth factor β, interleukin-6 gene and protein expression in the spleen.

RESULTS AND CONCLUSION: (1) Compared with the sham group and control group, leukocyte number was significantly higher, but lymphocyte percentage gradually reduced in the cerebral hemorrhage group at 24, 48, 72 and 96 hours (P < 0.05). (2) Compared with the sham group and control group, interleukin-6 levels in the blood and spleen were higher at 24 hours, peaked at 72 hours, and decreased at 96 hours in the cerebral hemorrhage group (P < 0.05). (3) Compared with the sham group and control group, transforming growth factor β expression was lower at 24 hours, gradually increased at 72 hours, and higher at 96 hours in the rat blood and spleen of the cerebral hemorrhage group (P < 0.05). (4) These findings indicate that immune function excitement first appeared after cerebral hemorrhage, and immune suppression appeared at 96 hours, indicating successful model establishment and good stability.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

Key words: Models, Animal, Cerebral Hemorrhage, Leukocytes, Lymphocytes, Tissue Engineering

CLC Number:

R318

Zhang Jian-jun, Shi Huan-chang. Immunosuppressed rat model of cerebral hemorrhage: construction and assessment[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(40): 5939-5945.

Figures/Tables 2

2.1 实验动物数量分析纳入60只Wistar大鼠，随机分为3组，即对照组、假手术组、脑出血组，每组20只，全部进入结果分析，无脱失。 2.2 模型创新性实验重点为脑出血后周围免疫抑制的研究，故采用了目前国内外认证的经典方法，具有稳定性好，可重复性强，行为学和形态学和剂量的相关性都很一致，该模型更接近人类疾病模式，具有转化医学意义。 2.3 白细胞、淋巴细胞、淋巴细胞百分数表达与假手术组及对照组相比，脑出血组大鼠于造模后24 h血液中白细胞数明显升高，48，72，96 h血液中白细胞数较前降低，差异有显著性意义(P < 0.05)；血液中淋巴细胞及淋巴细胞百分数24，48，72，96 h均逐渐降低，差异有显著性意义(P < 0.005)，见表1。 2.4 组织学及病理学检查结果与假手术组及对照组相比，脑出血组大鼠于造模后72 h淋巴结及脾脏组织可见炎性细胞浸润，但未见明显淋巴类细胞存在，说明淋巴结及脾脏组织脑出血后处于免疫抑制状态，见图1。 2.5 转化生长因子β、白细胞介素6的表达采用双抗体夹心ELISA法测定大鼠血清促炎细胞因子白细胞介素6和抗炎细胞因子转化生长因子β的表达结果表明，与对照组及假手术组比较，脑出血组大鼠血液中的白细胞介素6于24 h时高表达，96 h时表达降低，差异有显著性意义(P < 0.05)；血液中的转化生长因子β 24 h时低表达，96 h时高表达，差异有显著性意义(P < 0.05)，见表2及图2。 2.6 脾组织转化生长因子β、白细胞介素6 mRNA的表达与假手术组及对照组相比，脑出血大鼠于造模后 24 h脾组织的白细胞介素6 mRNA高表达，96 h时表达降低，差异有显著性意义(P < 0.05)；脾组织的转化生长因子β mRNA 24 h 时低表达，96 h时高表达，差异有显著性意义(P < 0.05)，结果见图3及表3。 2.7 脾组织转化生长因子β、白细胞介素6蛋白的表达与假手术组及对照组相比，脑出血大鼠于造模后24 h脾的白细胞介素6高表达，96 h时表达降低，差异有显著性意义(P < 0.05)；脾的转化生长因子β 24 h时低表达，96 h时高表达，差异有显著性意义(P < 0.05)，结果见图4及表4。"

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