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SD 大鼠 MCAO 线栓模型

SD 大鼠线栓法 MCAO 模型。用于缺血性脑卒中与再灌注损伤研究。大脑中动脉及其分支是人类缺血性卒中最常受累的脑血管(约占梗死的 70%),短暂性 MCAo 是最接近模拟人类缺血性卒中的模型之一,具有与人类卒中相似的缺血半暗带,并可产生可逆的缺血-再灌注。

Outcome measures

记录每次定位试验的到台潜伏期和游泳轨迹,比较相继试验中的空间定位表现。到达平台与达到时限未到台的情况按对应条件路径分别处理。

存活脑组织染为红色、梗死区呈白色;以 2 mm 连续冠状切片逐孔染色成像后,测量患侧与对侧半球的梗死面积及总面积,计算患侧相对对侧的梗死面积百分比。

Experimental animals

大鼠 Morris 水迷宫空间定位与再学习测定
Age
成年(来源未报告具体周龄)
Tissue
脑/海马(空间学习与记忆)
脑切片 TTC 染色与梗死面积测定
Age
成年(来源未报告具体周龄)
Tissue
脑组织(2 mm 冠状切片;梗死区与存活区)
脑切片 TTC 染色与梗死面积测定
Age
成年(来源未报告具体周龄)
Tissue
脑组织(2 mm 冠状切片;梗死区与存活区)
大鼠 Morris 水迷宫空间定位与再学习测定
Age
成年(来源未报告具体周龄)
Tissue
脑/海马(空间学习与记忆)

Experimental procedures

检查摄像机设置 检查摄像机设置。 将 Atlantis 自动升降平台置于水池,确认升降运动正常,避免平台上下移动时产生水波 将 Atlantis 自动升降平台置于水池,确认升降运动正常,避免平台上下移动时产生水波。 将平台放置在与前一天不同的位置 将平台放置在与前一天不同的位置。 实验第一天不执行与前一天位置比较的要求 实验第一天不执行与前一天位置比较的要求。 启动 Bonsai 工作流,确认以 30 fps 记录视频 启动 Bonsai 工作流,确认以 30 fps 记录视频。 降下平台,使动物暂时无法使用平台 降下平台,使动物暂时无法使用平台。 将动物放在所选起点,面向水池中心 将动物放在所选起点,面向水池中心。 轻放动物入水,避免抛掷和溅水 轻放动物入水,避免抛掷和溅水。 释放动物且操作者离开实验区域时,启动计时器或计算机跟踪程序 释放动物且操作者离开实验区域时,启动计时器或计算机跟踪程序。 经过 1 分钟 后,按下按钮升起平台 经过 1 分钟 后,按下按钮升起平台。 动物到达并接触平台时停止计时;本次试验时限为 2 分钟 Choose the matching condition: Platform — Rats will find the platform. They will do this in two minutes or less; Not platform — If the rat does not find the platform within 2 minutes. 动物到达并接触平台时停止计时;本次试验时限为 2 分钟。 返回取出动物,放入笼内擦干 Condition: Platform — Rats will find the platform. They will do this in two minutes or less 返回取出动物,放入笼内擦干。 间隔 30 分钟 后开始第 2 次试验 Condition: Platform — Rats will find the platform. They will do this in two minutes or less 间隔 30 分钟 后开始第 2 次试验。 升起平台,使动物能够登台,但平台仍不可直接看见 Condition: Platform — Rats will find the platform. They will do this in two minutes or less 升起平台,使动物能够登台,但平台仍不可直接看见。 将动物放在所选起点,面向水池中心;每次试验随机选择不同的起点 Condition: Platform — Rats will find the platform. They will do this in two minutes or less 将动物放在所选起点,面向水池中心;每次试验随机选择不同的起点。 轻放动物入水,避免抛掷和溅水 Condition: Platform — Rats will find the platform. They will do this in two minutes or less 轻放动物入水,避免抛掷和溅水。 释放动物且操作者离开实验区域时,启动计时器或计算机跟踪程序 Condition: Platform — Rats will find the platform. They will do this in two minutes or less 释放动物且操作者离开实验区域时,启动计时器或计算机跟踪程序。 动物到达并接触平台,或经过 1 分钟 时停止计时 Condition: Platform — Rats will find the platform. They will do this in two minutes or less Choose the matching condition: Platform — Rats will find the platform. They will do this in 1 minute or less; Not platform — If the rat does not find the platform within 1 minute 动物到达并接触平台,或经过 1 分钟 时停止计时。 返回取出动物,放入笼内擦干 Condition: Platform — Rats will find the platform. They will do this in two minutes or less Condition: Platform — Rats will find the platform. They will do this in 1 minute or less 返回取出动物,放入笼内擦干。 间隔 5 分钟 后进行下一次试验 Condition: Platform — Rats will find the platform. They will do this in two minutes or less Condition: Platform — Rats will find the platform. They will do this in 1 minute or less 间隔 5 分钟 后进行下一次试验。 将大鼠放在平台上 Condition: Platform — Rats will find the platform. They will do this in two minutes or less Condition: Not platform — If the rat does not find the platform within 1 minute 将大鼠放在平台上。 离开实验区域 Condition: Platform — Rats will find the platform. They will do this in two minutes or less Condition: Not platform — If the rat does not find the platform within 1 minute 离开实验区域。 等待 10 秒 Condition: Platform — Rats will find the platform. They will do this in two minutes or less Condition: Not platform — If the rat does not find the platform within 1 minute 等待 10 秒。 返回取出动物,放入笼内擦干并等待 Condition: Platform — Rats will find the platform. They will do this in two minutes or less Condition: Not platform — If the rat does not find the platform within 1 minute 返回取出动物,放入笼内擦干并等待。 间隔 5 分钟 后进行下一次试验 Condition: Platform — Rats will find the platform. They will do this in two minutes or less Condition: Not platform — If the rat does not find the platform within 1 minute 间隔 5 分钟 后进行下一次试验。 将大鼠放在平台上 Condition: Not platform — If the rat does not find the platform within 2 minutes. 将大鼠放在平台上。 离开实验区域 Condition: Not platform — If the rat does not find the platform within 2 minutes. 离开实验区域。 等待 10 秒 Condition: Not platform — If the rat does not find the platform within 2 minutes. 等待 10 秒。 返回取出动物,放入笼内擦干并等待 Condition: Not platform — If the rat does not find the platform within 2 minutes. 返回取出动物,放入笼内擦干并等待。 间隔 30 分钟 后开始第 2 次试验 Condition: Not platform — If the rat does not find the platform within 2 minutes. 间隔 30 分钟 后开始第 2 次试验。 升起平台,使动物能够登台,但平台仍不可直接看见 Condition: Not platform — If the rat does not find the platform within 2 minutes. 升起平台,使动物能够登台,但平台仍不可直接看见。 将动物放在所选起点,面向水池中心;每次试验使用不同的起点 Condition: Not platform — If the rat does not find the platform within 2 minutes. 将动物放在所选起点,面向水池中心;每次试验使用不同的起点。 轻放动物入水,避免抛掷和溅水 Condition: Not platform — If the rat does not find the platform within 2 minutes. 轻放动物入水,避免抛掷和溅水。 释放动物且操作者离开实验区域时,启动计时器或计算机跟踪程序 Condition: Not platform — If the rat does not find the platform within 2 minutes. 释放动物且操作者离开实验区域时,启动计时器或计算机跟踪程序。 动物到达并接触平台时停止计时;本次试验时限为 1 分钟 Condition: Not platform — If the rat does not find the platform within 2 minutes. 动物到达并接触平台时停止计时;本次试验时限为 1 分钟。

TTC Staining TTC Staining The brain is removed from the skull and placed into a brain slicer mold placed in ice. Razor blades are positioned in the mold to obtain 2mm thick sections. Sections are placed in individual wells of a 24 well tissue culture dish with 500 μl of 2% TTC in 0.1M PBS. The plate is wrapped in foil and placed on a 37°C slide warmer for 30 minutes. TTC is removed and 500ul of fixative is added to each well for storage. Images of the sections are captured by Olympus S2H10 research stereo- microscope and Adobe Photoshop software. The infarct area and total area of the contralateral and ipsilateral hemispheres are measured with Scion image software (Scion Corporation). The percent infarct area is calculated as a percentage of the ipsilateral hemisphere relative to the contralateral hemisphere as previously described. [F2.JPG]]()

Model identity

Model name
SD 大鼠 MCAO 线栓模型
Species
rat
Taxonomy ID
10116
Strain
Sprague-Dawley
Genetic background
SD 大鼠为远交(封闭群)大鼠群体,非近交品系;两大商业来源 Charles River 与 Harlan 的 SD 群体高度遗传分化(FST>0.4),同一来源不同繁育设施乃至房间之间也存在明显群体结构。

Biological characteristics

Research questions
局灶性脑缺血与缺血-再灌注损伤的病理生理机制;以梗死体积与神经功能评分为终点的神经保护干预临床前评价(如四环素类药物经抑制白细胞功能能否减轻脑缺血损伤并改善功能结局)。
Limitations
任何单一动物模型都不能复现人类卒中的所有方面,该类研究多在无共病条件的青年动物中进行,而人类卒中尤其影响伴有多种脑血管危险因素的老年人群;SD 大鼠 MCAO 的梗死体积偏小且个体变异大,变异程度甚至与供应商选择有关;缝线硅胶涂层长度、动物年龄与体重(动脉直径随年龄与体重变化,缝线规格需按体重选择)直接影响梗死大小;Meta 分析显示 Wistar 品系的大鼠梗死体积变异最小,品系选择本身即为跨研究比较的影响条件。

Experimental methods

Experimental protocols

  • 大鼠 Morris 水迷宫空间定位与再学习测定

    A rat water-maze procedure using an automated Atlantis platform and distal cues, with a new platform location each day. Preserve the separate platform-found and platform-not-found paths, and record escape latency and swim trajectories across the successive trials.

  • 脑切片 TTC 染色与梗死面积测定

    Fresh brains are cut to 2-mm coronal sections, stained per well with 2% TTC in 0.1M PBS at 37°C for 30 min protected from light, stored in fixative, imaged on a stereomicroscope, and infarct percentage computed ipsilateral-versus-contralateral.

  • 脑切片 TTC 染色与梗死面积测定

    Fresh brains are cut to 2-mm coronal sections, stained per well with 2% TTC in 0.1M PBS at 37°C for 30 min protected from light, stored in fixative, imaged on a stereomicroscope, and infarct percentage computed ipsilateral-versus-contralateral.

  • 大鼠 Morris 水迷宫空间定位与再学习测定

    A rat water-maze procedure using an automated Atlantis platform and distal cues, with a new platform location each day. Preserve the separate platform-found and platform-not-found paths, and record escape latency and swim trajectories across the successive trials.

Reagents and materials

References

  1. Clark WM, Lessov N, Lauten JD, Hazel K. Doxycycline treatment reduces ischemic brain damage in transient middle cerebral artery occlusion in the rat. J Mol Neurosci. 1997;9(2):103-108. DOI:10.1007/BF02736854; PMID:9407391. 原文在成年 Sprague-Dawley 大鼠以线栓行 2 小时可逆 MCAO,48 小时以梗死体积为终点(对照 158±44 mm3),支持 SD 大鼠 MCAO 梗死体积检测背景。
  2. Fluri F, Schuhmann MK, Kleinschnitz C. Animal models of ischemic stroke and their application in clinical research. Drug Des Devel Ther. 2015;9:3445-3454. DOI:10.2147/DDDT.S56071; PMID:26170628; PMCID:PMC4494187. 综述原文:MCA 及其分支为人类缺血性卒中最常受累血管(约占 70% 梗死);transient MCAo 最接近模拟人类缺血性卒中并伴类似人类的缺血半暗带;Mimicking all aspects of human stroke in one animal model is not feasible;研究多在无共病的青年动物中进行;SD 大鼠梗死体积小且变异大并依赖供应商(Sprague-Dawley rats exhibit small infarct volumes with considerable variability, which is even dependent on the choice of vendor);约 60% 神经保护数据来自 SD;Meta 分析显示 Wistar 变异最小;缝线涂层长度与梗死大小强相关、动脉直径随年龄变化。
  3. Gileta AF, Fitzpatrick CJ, Chitre AS, et al. Genetic characterization of outbred Sprague Dawley rats and utility for genome-wide association studies. PLoS Genet. 2022;18(5):e1010234. DOI:10.1371/journal.pgen.1010234; PMID:35639796; PMCID:PMC9187121. 摘要逐句支持:'Sprague Dawley (SD) rats are among the most widely used outbred laboratory rat populations';Charles River 与 Harlan 两群体高度分化(FST>0.4),同供应商内跨繁育设施与房间存在强群体结构。支持 genetic_background 的远交封闭群与供应商间遗传差异表述。
  4. NCBI Taxonomy record 10116: Rattus norvegicus (brown rat), rank species.
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  7. Multimodal Imaging in Rat Model Recapitulates Alzheimer's Disease Biomarkers Abnormalities. PMID:29097597; DOI:10.1523/JNEUROSCI.1346-17.2017. Model/assay applicability; not exact replication of the separate protocols.io procedure.
  8. Anxiety-like behavior as an early endophenotype in the TgF344-AD rat model of Alzheimer’s disease.. PMID:29107184; DOI:10.1016/j.neurobiolaging.2017.09.024. Model/assay applicability; not exact replication of the separate protocols.io procedure.
  9. Schisandrin ameliorates cognitive deficits, endoplasmic reticulum stress and neuroinflammation in streptozotocin (STZ)-induced Alzheimer’s disease rats. PMID:32336744; DOI:10.1538/expanim.19-0146. Model/assay applicability; not exact replication of the separate protocols.io procedure.
  10. Ian Simpson. Induce Stroke via Hypoxia-Ischemia. protocols.io, v2, 2019. DOI: 10.17504/protocols.io.8e5htg6
  11. Hu Y, Yang Z-H, Yan F, et al. CCA repair or ECA ligation - Which middle cerebral artery occlusion is better in the reperfusion mouse model? Ibrain. 2023;9(3):258-269. DOI:10.1002/ibra.12128; PMID:37786756.
  12. Bederson JB, Pitts LH, Germano SM, Nishimura MC, Davis RL, Bartkowski HM. Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke. 1986;17(6):1304-1308. DOI:10.1161/01.STR.17.6.1304; PMID:2433817. 原文以成年大鼠 MCA 闭塞后 TTC 与 H&E 对比,24 小时 TTC 梗死面积 21±2% 的冠状切片面积,r=0.91,支持 TTC 用于大鼠脑梗死检测与定量。
  13. ARRIVE 2.0 guidelines