三种方式构建脑低灌注大鼠模型的比较研究
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中国医学科学院药用植物研究所

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国家重点研发计划课题(No. 2022YFC3501105)


A comparative study of three ways to construct a rat model of cerebral hypoperfusion
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Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences

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National Key R&D Program of China

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    摘要:

    【】 目的:通过比较三种不同大鼠脑灌注不足模型的脑血流量、脑组织中神经细胞形态改变及血清氧化与炎症因子水平的变化,为脑血管疾病及神经退行性疾病机制及治疗药物研究提供实验动物模型。方法:将88只雄性SD大鼠随机分为假手术组(n=16)、经典双侧颈总动脉结扎组(2-VO组,n=24)、改良2-VO组(n=24)和线栓法组(n=24)。经典2-VO组行双侧颈总动脉结扎术,改良2-VO组在结扎之前先从颈总动脉抽血(1mL/100g),线栓法组行大脑中动脉闭塞术。前两种模型的假手术组仅分离颈总动脉但不结扎,线栓法模型的假手术组仅结扎颈总动脉近心端和颈外动脉,但不插入栓线。术后1天、3天和7天时检测三组大鼠的脑血流量、脑梗死体积、血清炎症因子水平、海马组织HE染色和超微结构。结果:激光散斑的结果显示:三组模型大鼠手术后脑血流迅速下降,随后缓慢上升,其中改良2-VO组大鼠脑血流下降幅度较其他两组更明显。7天时,仅有改良2-VO组大鼠脑血流量相较于假手术组仍有显著性差异,且仍处于低灌注状态(与术前相比脑血流量下降30%)。TTC染色的结果显示:随着造模时间延长,三组大鼠纹状体区域的白色梗死灶逐渐变大,其中改良2-VO组大鼠有4只(约26.7%)在皮层和纹状体都出现了白色梗死灶,线栓法组有10只(约66.7%)在患侧皮层和纹状体出现了白色梗死灶。ELISA结果显示:三组模型大鼠造模后炎症因子TNF-α、IL-1β、hs-CRP水平升高,氧化因子ROS的水平升高,抗氧化因子SOD水平下降。在术后7天时,经典2-VO与线栓法组大鼠的hs-CRP与假手术组相比无明显差异,但改良2-VO组与假手术组相比上述所有指标仍有显著性差异。HE染色的结果显示,改良2-VO组相较于经典2-VO组和线栓法组能造成更严重的海马CA1和CA3区损伤。透射电镜的结果显示,改良2-VO组相较于经典2-VO组和线栓法组能造成海马区细胞更严重的线粒体和内质网损伤。结论:相较于经典2-VO法和线栓法,血清、脑组织影像学、病理水平结果表明改良2-VO法能在相同的时间内造成更完全的脑低灌注和更严重的神经损伤,更接近人类脑低灌注发病机制。

    Abstract:

    【】 Objective: By comparing the cerebral blood flow, the morphological changes of nerve cells in brain tissue and the levels of serum oxidation and inflammatory factors in three different rat models of cerebral hypoperfusion, the experimental animal models were provided for the study of mechanisms and therapeutic drugs of cerebrovascular diseases and neurodegenerative diseases. Method: A total of 88 male SD rats were randomly divided into sham operation group (n=16), classic bilateral common carotid artery occlusion group (2-VO group, n=24), modified 2-VO group (n=24) and intraluminal thread technique group (n=24). Bilateral common carotid artery ligation was performed in the classic 2-VO group, while blood was drawn from the common carotid artery before ligation in the modified 2-VO group (1mL/100g). Middle cerebral artery occlusion was performed in the intraluminal thread technique group. In the sham operation group of the first two models, the common carotid artery was separated but not ligated, while the proximal end of the common carotid artery and the external carotid artery were ligated but not insert the bolt thread in the sham group of intraluminal thread technique model. Cerebral blood flow, infarct volume, serum inflammatory factor level, HE staining and ultrastructure of hippocampal tissue were measured at 1, 3 and 7 days after operation. Results: Laser speckle showed that the cerebral blood flow of rats of the three groups decreased rapidly after surgery, and then increased slowly. The decrease of cerebral blood flow of the modified 2-VO group was more obvious than that of the other two groups. At day 7, only the modified 2-VO group still had significant differences in cerebral blood flow compared with the sham group, and remained in a state of hypoperfusion (cerebral blood flow decreased by 30% compared with the operation). The results of TTC staining showed that the infarcts in the striatum of the three groups gradually increased with the extension of the time after operation, among which 4 rats (about 26.7%) in the modified 2-VO group had infarcts in both the cortex and striatum, while 10 rats (about 66.7%) in the intraluminal thread technique group. ELISA showed that the levels of inflammatory factors like TNF-α, IL-1β and hs-CRP in the three groups were increased after operation, and the levels of pro-oxidation factors ROS were also increased. In contrast, the level of antioxidant factor SOD decreased. At postoperative day 7, there was no significant difference in hs-CRP between the classic 2-VO and the intraluminal thread technique groups compared to the sham group. However, the modified 2-VO group still exhibited significant differences in all the above indicators compared to the sham group. HE staining showed that the modified 2-VO group caused more severe damage to the hippocampal CA1 and CA3 regions compared to the classic 2-VO and the intraluminal thread technique groups. Transmission electron microscopy demonstrated that the modified 2-VO group induced more severe damage to mitochondrial and endoplasmic reticulum in the hippocampal region compared to the the classic 2-VO and the intraluminal thread technique groups. Conclusion: Compared to the classic 2-VO and the intraluminal thread technique, the modified 2-VO method can induce more complete cerebral hypoperfusion and more severe neural damage within the same time frame, resembling the pathogenesis of human cerebral hypoperfusion more closely.

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  • 收稿日期:2024-04-30
  • 最后修改日期:2024-05-22
  • 录用日期:2024-09-30
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