探索基于基因修饰猪血红细胞灌注液亚低温有氧机械灌注对食蟹猴缺血缺氧脑损伤的保护作用

Exploring the protective effects of subnormothermic normoxic mechanical perfusion of genetically modified porcine erythrocyte perfusate on ischemic and hypoxic brain injury in cynomolgus monkeys

  • 摘要:
    目的  探讨基因修饰猪血红细胞作为亚低温有氧机械灌注液对创伤性失血引起的食蟹猴脑组织缺氧缺血脑损伤的保护作用。
    方法  按随机数字表法将食蟹猴分为阳性对照组与阴性对照组(共3只,其中3只取左脑半球作为阳性对照组,3只取右脑半球作为阴性对照组)和亚低温灌注组(3只)。阳性对照组为自循环停止1 h直接取材,阴性对照组为自循环停止1 h亚低温放置6 h,亚低温灌注组为自循环停止1 h后利用基于基因修饰猪血红细胞灌注液对食蟹猴脑缺氧缺血损伤模型双侧颈总动脉进行6 h亚低温有氧机械灌注。灌注前进行六基因修饰猪与食蟹猴交叉配血实验,灌注开始后检测0 h、1 h、2 h、3 h、4 h、5 h和6 h各时间点灌注液血常规指标水平变化,记录血氧饱和度,检测灌注液Na+、K+、Ca2+、葡萄糖水平和血液酸碱度(pH值),以及灌注液IgG和IgM水平。灌注6 h后,检测食蟹猴脑组织含水量。取额叶皮质区和海马区进行尼氏染色,免疫荧光染色检测胶质纤维酸性蛋白(GFAP)、离子钙结合适配器分子1(Iba1)和神经元核抗原(NEUN)的表达情况。
    结果  六基因修饰猪与食蟹猴交叉配血结果呈阴性。在灌注3 h时灌注液红细胞数量下降较为明显,血红蛋白水平在1、3、5、6 h均呈下降趋势,白细胞和血小板数量在各时间点均呈下降趋势。亚低温灌注组血氧饱和度始终保持在95%~98%,亚低温灌注组血氧饱和度、Na+、Ca2+、葡萄糖水平以及pH值均保持稳定,K+水平先升高后降低。灌注前后IgG和IgM水平差异无统计学意义。亚低温灌注组灌注终末脑组织含水量显著高于阳性对照组(P<0.001)。尼氏染色结果显示,与阳性对照组比较,亚低温灌注组前额叶皮质区锥体神经元保持较好的形态结构完整性,肿大变型细胞无明显增多;海马CA1区肿大变型细胞稍增多,少数结构未被破坏细胞出现胞体缩小;海马齿状回区有较少颗粒神经元细胞结构完整性被破坏,细胞水肿增加。NEUN免疫荧光染色显示,与阳性对照组比较,亚低温灌注组前额叶皮质区和海马CA1区锥体神经元细胞形态结构状态较好,轴突清晰可见,海马齿状回区颗粒细胞保存完整,但细胞核保护欠佳。GFAP免疫荧光染色显示,与阳性对照组比较,亚低温灌注组突起片段较为稀疏,与神经元结合较为紧密。Iba1免疫荧光染色显示,与阳性对照组比较,亚低温灌注组突起片段较粗壮,数量较少。
    结论  与阳性对照组相比,基因修饰猪血红细胞灌注液亚低温有氧机械灌注后食蟹猴脑组织水肿增加,神经元细胞和胶质细胞结构形态保存相对完整,保护作用可能与该技术持续供氧供能、平衡离子稳态和灌注液pH值、降低排斥反应及维持全脑低代谢相关。

     

    Abstract:
    Objective  To explore the protective effects of genetically modified porcine erythrocyte suspension as a subnormothermic normoxic mechanical perfusate on hypoxic-ischemic brain injury in cynomolgus monkeys caused by traumatic hemorrhage.
    Methods  Cynomolgus monkeys were randomly divided into positive and negative control groups (a total of 3 monkeys, with 3 left cerebral hemispheres as the positive control group and 3 right cerebral hemispheres as the negative control group) and the subnormothermic perfusion group (n=3). The positive control group was directly sampled 1 hour after circulatory arrest, while the negative control group was placed at subnormothermic conditions for 6 hours after circulatory arrest. The subnormothermic perfusion group underwent 6 hours of subnormothermic normoxic mechanical perfusion of the bilateral common carotid arteries of the cynomolgus monkey hypoxic-ischemic brain injury model using genetically modified porcine erythrocyte suspension 1 hour after circulatory arrest. Before perfusion, cross-matching experiments were conducted between the six genetically modified pig and the cynomolgus monkeys. After the start of perfusion, the levels of routine blood indicators in the perfusate were detected at 0, 1, 2, 3, 4, 5 and 6 hours. Blood oxygen saturation was recorded, and the levels of Na+, K+, Ca2+, glucose and blood pH in the perfusate were measured, as well as the levels of IgG and IgM in the perfusate. After 6 hours of perfusion, the water content of the brain tissue was measured. Nissl staining was performed on the frontal cortex and hippocampal regions, and immunofluorescence staining was used to detect the expression of glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1 (Iba1), and neuronal nuclear antigen (NEUN).
    Results  The cross-matching results between the six genetically modified pigs and the cynomolgus monkeys were negative. The number of red blood cells in the perfusate decreased significantly at 3 hours of perfusion, and the hemoglobin level showed a downward trend at 1, 3, 5 and 6 hours. The number of white blood cells and platelets decreased at all time points. The blood oxygen saturation in the subnormothermic perfusion group remained stable at 95%–98%, and the levels of blood oxygen saturation, Na+, Ca2+, glucose and pH were stable, while the K+ level first increased and then decreased. There was no significant difference in the levels of IgG and IgM before and after perfusion. The water content of brain tissue at the end of perfusion in the subnormothermic perfusion group was significantly higher than that in the positive control group (P<0.001). Nissl staining results showed that compared with the positive control group, the pyramidal neurons in the prefrontal cortex of the subnormothermic perfusion group maintained better morphological integrity, with no significant increase in enlarged and deformed cells. In the hippocampal CA1 region, there was a slight increase in enlarged and deformed cells, and a few cells with undamaged structures showed reduced cell size. In the hippocampal dentate gyrus, fewer granule neurons had compromised structural integrity, with increased cell edema. NEUN immunofluorescence staining showed that compared with the positive control group, the pyramidal neurons in the prefrontal cortex and hippocampal CA1 region of the subnormothermic perfusion group had better morphological states, with clear axons. The granule cells in the hippocampal dentate gyrus were well preserved, but the nuclei were less well protected. GFAP immunofluorescence staining showed that compared with the positive control group, the subnormothermic perfusion group had sparser protrusions that were more tightly associated with neurons. Iba1 immunofluorescence staining showed that compared with the positive control group, the subnormothermic perfusion group had thicker and fewer protrusions.
    Conclusions  Compared with the positive control group, subnormothermic normoxic mechanical perfusion with genetically modified porcine erythrocyte perfusate increased brain tissue edema in cynomolgus monkeys, but better preserved the morphological integrity of neurons and glial cells. The protective effects may be related to the continuous oxygen and energy supply, maintenance of ion homeostasis and perfusate pH, reduced rejection, and low metabolic state of the whole brain.

     

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