中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2020, Vol. 15 ›› Issue (4): 667-675.doi: 10.4103/1673-5374.266914

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

抑制内源性组织型纤溶酶原激活物可增强创伤性脑损伤后神经细胞凋亡和轴索损伤

  

  • 出版日期:2020-04-15 发布日期:2020-05-28

Inhibiting endogenous tissue plasminogen activator enhanced neuronal apoptosis and axonal injury after traumatic brain injury

Jun-Jie Zhao1, Zun-Wei Liu2, 3, Bo Wang1, Ting-Qin Huang4, Dan Guo5, Yong-Lin Zhao6, Jin-Ning Song1   

  1. 1 Department of Neurosurgery, the First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi Province, China
    2 Institute of Organ Transplantation, the First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi Province, China
    3 Department of Renal Transplantation, Nephropathy Hospital, the First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi Province, China
    4 Department of Neurosurgery, the Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi Province, China
    5 Department of Science and Technology, Xi’an Medical University, Xi’an, Shaanxi Province, China
    6 Department of Oncology, the Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi Province, China
  • Online:2020-04-15 Published:2020-05-28
  • Contact: Jin-Ning Song, MD,jinningsong@126.com.

PDF

158

摘要：

组织型纤溶酶原激活物常用于治疗急性缺血性卒中，但是内源性组织型纤溶酶原激活物对创伤性脑损伤的作用尚不明确。为此，实验设计以重物打击法建立创伤性脑损伤大鼠模型。(1)实验的干预方法：然后以立体定位注射侧脑室5μL组织型纤溶酶原激活物抑制剂neuroserpin（0.25mg/mL）；（2）实验的评估手段：以神经严重性评分评估神经功能，以苏木精-伊红染色和Bielschowsky银染法评估损伤脑组织中神经元和轴突损伤情况，以Western blot分析损伤脑组织中组织型纤溶酶原激活物的表达水平，以免疫组化染色分析损伤脑组织中凋亡标记物c-caspase3、神经元标记物神经丝蛋白轻链、星形胶质细胞标记物胶质纤维酸性蛋白和小胶质细胞标记物Iba-1阳性反应，以免疫荧光双标记检测细胞凋亡的细胞类型，以TUNEL染色检测损伤皮质中细胞的凋亡情况，以Fluoro-Jade B染色检测损伤皮质中损伤的神经元；（3）结果显示：6h时组织型纤溶酶原激活物的表达增加，且在3d时达到峰值；创伤性脑损伤后出现大量的神经细胞凋亡和轴突损伤，且neuroserpin可进一步增加神经细胞凋亡、神经元损伤以及轴索损伤，同时还能激活小胶质细胞和星形胶质细胞。(3)neuroserpin进一步恶化创伤性脑损伤大鼠的神经行为表现；(4)结果表明：抑制内源性组织纤溶酶原激活剂可加重创伤性脑损伤后神经细胞凋亡和轴突损伤，活化小胶质细胞和星形胶质细胞。实验于2015年6月经陕西省动物实验生物医学伦理委员会批准，批准号SYXK[Shaan] 2015-002）。

orcid: 0000-0002-0620-8983 (Jin-Ning Song)

关键词: 组织型纤溶酶原激活物, neuroserpin, 细胞凋亡, 轴索损伤, 神经元损伤, 创伤性脑损伤, 神经元, 星形胶质细胞, 小胶质细胞, 炎症, 神经再生

Abstract:

Tissue plasminogen activator is usually used for the treatment of acute ischemic stroke, but the role of endogenous tissue plasminogen activator in traumatic brain injury has been rarely reported. A rat model of traumatic brain injury was established by weight-drop method. The tissue plasminogen activator inhibitor neuroserpin (5 μL, 0.25 mg/mL) was injected into the lateral ventricle. Neurological function was assessed by neurological severity score. Neuronal and axonal injuries were assessed by hematoxylin-eosin staining and Bielschowsky silver staining. Protein level of endogenous tissue plasminogen activator was analyzed by western blot assay. Apoptotic marker cleaved caspase-3, neuronal marker neurofilament light chain, astrocyte marker glial fibrillary acidic protein and microglial marker Iba-1 were analyzed by immunohistochemical staining. Apoptotic cell types were detected by immunofluorescence double labeling. Apoptotic cells in the damaged cortex were detected by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP-biotin nick-end labeling staining. Degenerating neurons in the damaged cortex were detected by Fluoro-Jade B staining. Expression of tissue plasminogen activator was increased at 6 hours, and peaked at 3 days after traumatic brain injury. Neuronal apoptosis and axonal injury were detected after traumatic brain injury. Moreover, neuroserpin enhanced neuronal apoptosis, neuronal injury and axonal injury, and activated microglia and astrocytes. Neuroserpin further deteriorated neurobehavioral function in rats with traumatic brain injury. Our findings confirm that inhibition of endogenous tissue plasminogen activator aggravates neuronal apoptosis and axonal injury after traumatic brain injury, and activates microglia and astrocytes. This study was approved by the Biomedical Ethics Committee of Animal Experiments of Shaanxi Province in China in June 2015 (approval No. SYXK [Shaan] 2015-002).

Key words: apoptosis, astrocytes, axonal injury, inflammation, microglia, nerve regeneration, neural regeneration, neuronal injury, neurons, neuroserpin, tissue plasminogen activator, traumatic brain injury