出版简讯｜脊髓内注射内皮素-1构建具有长期功能障碍的脊髓梗死大鼠新模型
文摘
科学
2024-06-23 10:11
河北
New rat model of spinal cord infarction with long-lasting functional disabilities generated by intraspinal injection of endothelin-1Masayuki Otani, Yoshihiro Kushida, Yasumasa Kuroda, Shohei Wakao, Yo Oguma, Keisuke Sasaki, Shintaro Katahira, Ryohei Terai, Rie Ryoke, Hiroi Nonaka, Ryuta Kawashima, Yoshikatsu Saiki, Mari Dezawadoi: 10.1136/svn-2023-002962
Stroke & Vascular Neurology（SVN）最新上线文章“New rat model of spinal cord infarction with long-lasting functional disabilities generated by intraspinal injection of endothelin-1”，来自日本东北大学（Tohoku University）医学院心血管外科学系、干细胞生物学和组织学系Masayuki Otani等。目前构建脊髓（spinal cord, SC）梗死动物模型的方法具有高度侵入性，且只能进行短期观察，通常时限为28天。作者团队旨在通过诱导选择性缺血性SC损伤，建立一种以长期生存和持久SC功能障碍为特征的大鼠模型。选取8周龄雄性Wistar大鼠，采用对流增强输送技术选择性地将内皮素-1（endothelin-1, ET-1）输送至Th13水平的SC前角，致SC梗死。连续56天评估Basso、Beattie和Bresnahan（BBB）运动评分。通过激光组织血流仪、MRI、免疫组化、三苯基四氮唑氯化物（TTC）染色、Western印迹和TUNEL染色检查SC。Figure 1. (A, B) The length of the silica tubing was adjusted to 1.5 mm using a digital calliper. (C) Schematic diagram of the intraoperative view. (D) Intraoperative picture under the microscope (see online supplemental movie 1). (E) Schematic diagram of the Evans blue injection centred on the anterior horn of the spinal cord (SC). (F) Representative example of Evans blue administration (0.7 µL) at angle X. (G) Overall image of the ventral side of the SC injected with 0.7 µL of Evans blue at each angle. (H) Diffusion of the dye in the SC, with 1.3 and 0.7 µL Evans blue. (I) Diffusion of the dye in the ventral SC was observed for two different volumes: 1.3 µL and 0.7 µL.
研究结果显示，采用穿刺法从双侧SC向靠近后根起点的前角（40°角度，1.5 mm深度）注射0.7 µL ET-1（2.5 mg/mL）。动物存活至56天，BBB评分保持稳定（第14天5.5±1.0，第56天6.2±1.0）。第1天BBB评分≤1的大鼠，从第14天起至第56天，BBB评分稳定在5～6之间，而第1天BBB评分>1的大鼠仅表现出轻微功能障碍，第14天后BBB评分>12。TTC染色、免疫染色和TUNEL染色显示前角选择性缺血和神经元细胞死亡。T2加权MR图像显示SC梗死部位的信号强度随时间的推移而增加。Western印迹表明，ET-1给药后SC组织中出现细胞凋亡和随后的炎症。![]()
Figure 2. Changes in the BBB locomotor score and body weight ratio. (A) The volume of ET-1 was fixed at 0.7 µL/side, and the concentration was varied. Each rat was observed for 14 days, and mortality rates are shown below: all five rats/group administered 1.0 mg/mL and 3.0 mg/mL ET-1, respectively, survived for 14 days; 1 of the 5 rats administered 2.0 mg/mL ET-1 died on day 4, and the other rats survived for 14 days. Data for the rat that died during the observation period were excluded from the graph. (B) ET-1 (0.7 µL/side, 2.5 mg/mL) was injected. (C) The BBB score on postoperative day 1 predicted the BBB score in the chronic phase. At 2 hours postoperatively, both the severe and mild groups had a BBB score of 0. Already on day 1 after the injection, however, the BBB score differed significantly, with the severe group having a mean BBB score of 0.26±0.10 and the Mild group having a BBB score of 2.33±0.36 (p<0.01). (D) The body weight changes in each group up to day 56. The average preoperative weight was defined as 1.0. BBB, Basso, Beattie and Bresnahan; PBS, phosphate-buffered saline.
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Figure 3. (A) TTC staining. Left: intact spinal cord (SC) at the Th13 level. Right: punctured cross-section on day 7n in the ET-1 group. (B) Vasoconstriction process after ET-1 injection. Blood flow at the level of Th13 was 61.6±7.4 mL/min/100 g before ET-1 administration, 15.9±4.0 mL/min/100 g immediately after ET-1 administration, 15.3±4.3 mL/min/100 g at 15 min, 31.9±1.4 mL/min/100 g at 30 min and 55.3±9.0 mL/min/100 g at 60 min. The blood flow was significantly decreased immediately, 15 min and 30 min after ET-1 administration compared with that before ET-1 administration (before vs just after; p<0.001, before vs 15 min; p<0.001, before vs 30 min; p<0.05). After 60 min, blood flow improved to the same level as before ET-1 administration (before vs 60 min; not significant). (C) Immunostaining with neurological markers, NeuN, Iba-1, GFAP and GST-pi. (D) Number of NeuN positive cells 3 days after ET-1 injection compared with intact SC. TTC, triphenyl tetrazolium chloride.
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Figure 4. (A) TUNEL-positive cells per unit area (mm2) 3 days after ET-1 injection. (B) Double staining with TUNEL and NeuN. (C) Immunostaining for RECA-1. (D) RECA-1 positive vessels per unit area (mm2). The number of RECA-1 positive vessels (/mm2) in the anterior horn was 428.9±26.9 and 457.8±4.6 (p=0.38) and the number in the posterior horn was 505.6±17.6 and 503.3±23.1 (p=0.93) in the ET-1- (day 3) and intact groups, respectively. In the white matter, the number of RECA-1-positive vessels (/mm2) was 192.2±9.2 and 172.2±8.2 (p=0.11) on the ventral side and 188.9±10.0 and 163.3±6.6 (p=0.04) on the dorsal side in the ET-1- (day 3) and intact groups, respectively.
研究结论：选择性地将ET-1输送至SC可以更精确地定位目标部位的梗死区域，并构建具有稳定神经功能障碍（持续56天）的大鼠SC梗死模型。更多内容详见文章原文：https://svn.bmj.com/content/early/2024/06/20/svn-2023-002962，或点击文末“阅读原文”。投稿 / 转载 / 商务合作：csa.svn@chinastroke.netEmail：csa.svn@chinastroke.net地址：北京市丰台区南四环西路119号B区1号楼508室本公众号对其刊载的所有内容，包括文字、图片、音视频资料以及版式设计等享有版权，未经本公众号授权同意，其他任何机构不得以任何形式侵犯本公众号作品著作权，包括但不限于：擅自复制、链接、非法使用或转载，或以任何方式建立作品镜像。转载请联系后台或邮箱。如获转载授权，发布请注明出处，不得擅自修改文章内容
