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Mouse Transient Global Ischemia Two-Vessel Occlusion Model
小鼠短暂性脑缺血双血管阻塞模型   

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Abstract

Transient global ischemia in rodents induces delayed death of hippocampal CA1 neurons, as well as in some hilar neurons of the dentate gyrus, medium aspiny neurons of the striatum, pyramidal neurons in neocortical layers II, V and VI, and Purkinje neurons of the cerebellum. In contrast to focal ischemia that mimics regional stroke in humans, this model of global ischemia mimics the brain injury that occurs after human cardiac arrest. Early events include caspase activation, cleavage of anti-death Bcl-2 family proteins and large mitochondrial channel activity. Genetically engineered mice provide opportunities for study such as the knock-in mouse expressing a caspase-resistant form of Bcl-xL found to exhibit markedly reduced mitochondrial channel activity and reduced vulnerability to ischemia-induced neuronal death1. It is therefore relevant to adapt and develop a simple protocol for producing transient global ischemia in mouse2. The two-vessel occlusion model has been specifically developed to provide optimal outcomes in mouse and offers several advantages over the four-vessel occlusion model traditionally used in rat including the relative ease of the procedure as well as only a single day of surgery. However it should be noted that this procedure has a higher morbidity rate compared to other ischemia models as well as a higher degree of variability. These two disadvantages necessitate the use of a larger cohort of animals, which for many healthy breeding transgenic animals is a non-deterring factor.

Keywords: Bcl-xL Bcl-2(Bcl-xL蛋白), Stroke(划), Cell death(细胞死亡), Apoptosis(细胞凋亡), Mitochondria(线粒体)

Material and Reagents

  1. Eight-week-old C57 mice or a variety of transgenic/knockout mice, body weight 20-25 g
  2. Isoflurane USP (ISOTHESIA) (Butler Schein, catalog number: 029404 )
  3. 2% lidocaine hydrochloride jelly USP (Healthhotline, catalog number: 1024140 )
  4. Analgesic: Flunixiject (Flunixin Meglumine) (Butler Schein, catalog number: 029405 )
  5. 0.9% normal saline or other vehicle for your reagents
  6. Betadine surgical scrub (7.5% povidone-Iodine) (Healthhotline, catalog number: 6900581 )
  7. Sterile ocular lubricant (puralube) (Butler Schein, catalog number: 008897 )

Equipment

  1. Anesthesia induction chamber (VetEquip, catalog number: 941444 )
  2. Vapomatic anesthetic vaporizer (Bickford)
  3. Dissecting microscope with a surgical board included
  4. Facial mask
  5. Electric shaver
  6. Timer
  7. Q- tips
  8. Gauze
  9. Scissors
  10. Heating lamp
  11. Scalpel handle (Fine Science Tools, catalog number: 10003-12 )
  12. Blade #11 (Fine Science Tools, catalog number: 10011-00 )
  13. 2x tissue forceps (Fine Science Tools, Dumont 5/45, catalog number: 11251-35 )
  14. 2x micro serrefine clamp (Fine Science Tools, catalog number: 18055-04 )
  15. Forceps style clamp Applicator (Fine Science Tools, catalog number: 18057-14 )
  16. Hot bead sterilizer (Fine Science Tools, catalog number: 18000-50 )
  17. Wound closing clip-EZ clip kit (Stoelting, catalog number: 59020 )
  18. Silk surgical suture (Healthhotline, catalog number: 100-5597 )
  19. BD 1 ml syringe 26G x 3/8 (0.45 mm x 10 mm) (Thermo Fischer Scientific, catalog number: 14-823-2E )
  20. Surgical shaver (Stoelting, catalog number: 5148/Blade , Surgical/Shaving, catalog number: 50, mice, Steel, catalog number: 5148 )

Procedure

  1. Anesthesia
    1. Set the mouse in the anesthesia chamber where a flow of Isoflurane 4% in a mixture of N2:O2 (40:30) will be delivered by a tube attached to a Vapomatic Anesthetic Vaporizer (CWE). Mouse is adequately anaesthetized when it fails to twitch upon tail or foot pinch.

  2. Animal preparation
    1. Shave the area that will undergo procedure (neck), with a surgical shaver.
    2. Set the mouse on the surgical board (supine position) with the head placed into a facial mask connected to the Vapomatic Anesthetic Vaporizer (CWE) through which a flow of Isoflurane 2% in a mixture of N2:O2 (40:30) is delivered constantly until carotid clamping.
    3. Stabilize the mouse by taping down the forelegs (surgical tape or scotch tape will suffice).

  3. Surgical procedure
    1. With a Q-Tip apply Betadine Surgical Scrub to decontaminate the skin.
    2. With a scalpel, make a ventral midline vertical 0.5-1 cm skin incision on the neck (at exactly, and parallel to, the ventral midline).
    3. Lift up fat and connective tissue with forceps and cut horizontally at bottom to enter the tissue plane underneath.
    4. Locate sternocleidomastoid muscle and muscles around the trachea. Open the space between these muscles to expose blood vessels.
    5. While observing the field in the microscope, using forceps gently isolate (CRITICAL: Avoid any direct contact with the vagal nerve-this step requires some practice) common carotid artery (CCA) from nerves (vagal nerve etc.) and veins. Nerves are white. The artery is medial to the vagal nerve. Perform this step for both CCAs.
    6. Loop the CCA using 4-0 silk suture to allow exposure of the CCA as shown in the image below.



    7. Expose the CCA by pulling it up through the silk suture (left) and clip both CCAs using micro serrefine clamps (right).
    8. Set timer to 30 min (clamping time may vary depending on laboratory conditions i.e. clamp position and strength; investigators should perform pilot experiments and test times between 20-45* min to obtain optimal ischemia with minimal mortality).
    9. Reduce the isoflurane to 1% (depending on how the animals respond after clamping). The mouse should have no response to tail pinch. Respirations will be variable.
    10. *At 30 min remove clips and silk suture underneath the CCA.
    11. Close the skin incision with autoclips.

  4. Check point during clipping:
    1. Diaphragm Contraction (~hiccups); if this happens the vagal nerve has been scratched. If there is vagal damage the animals rarely survive the procedure.

  5. Post-operative care:
    1. Give (analgesic) Flunixin Subcutaneously, 2.2 mg/kg.
    2. Apply Lidocaine directly to the incision.
    3. Apply the ocular lubricant to prevent drying of the eyes.
    4. Set the mouse in a recovery cage and keep it warm by using a heating lamp until recovered.
    5. Daily monitoring of the animal’s condition is necessary to determine if there is pain or abnormal behavior.
    6. At desired time points, mouse will be anesthetized and perfused transcardially with saline 0.9%, followed by buffered 4% paraformaldehyde for immunohistochemistry.
      Note: At 4-5 day post ischemia FluoroJade staining can be used to determine the efficacy of ischemia. It is critical to check the CA1 region of the hippocampus bilaterally as mouse ischemia is not always bilateral. Because of the non-stereotypical vasculature of mice approximately 50% of mice will show histological signs of ischemia with this method.

Acknowledgments

This work was supported by the NIH (grant number NS045876).

References

  1. Ofengeim, D., Chen, Y. B., Miyawaki, T., Li, H., Sacchetti, S., Flannery, R. J., Alavian, K. N., Pontarelli, F., Roelofs, B. A., Hickman, J. A., Hardwick, J. M., Zukin, R. S. and Jonas, E. A. (2012). N-terminally cleaved Bcl-xL mediates ischemia-induced neuronal death. Nat Neurosci 15(4): 574-580.
  2. Zhen, G. and Dore, S. (2007). Optimized protocol to reduce variable outcomes for the bilateral common carotid artery occlusion model in mice. J Neurosci Methods 166(1): 73-80.

简介

啮齿类动物的瞬时全局性缺血诱导海马CA1神经元的延迟死亡,以及在齿状回的一些肝门神经元,纹状体的中度aspiny神经元,新皮质层II,V和VI中的锥体神经元和小脑的浦肯野神经元。与模拟人类的局部中风的局部缺血相反,这种全局缺血模型模拟了在人类心脏骤停后发生的脑损伤。早期事件包括胱天蛋白酶活化,抗死亡Bcl-2家族蛋白的切割和大线粒体通道活性。基因工程小鼠提供了研究的机会,如敲入小鼠表达的caspase抗性形式的Bcl-xL发现表现出显着减少的线粒体通道活动和减少缺血诱导的神经元死亡的脆弱性1。因此,适应和开发一个简单的协议,以在小鼠中产生瞬时全局缺血是相关的。双血管闭塞模型已被专门开发以在小鼠中提供最佳结果,并且相对于传统上用于大鼠的四脉管闭塞模型提供了若干优点,包括相对容易的程序以及仅一天的手术。然而,应当注意,与其他缺血模型相比,该程序具有更高的发病率以及更高程度的变异性。这两个缺点需要使用更大的动物群,对于许多健康的育种转基因动物是非威慑因子。

关键字:Bcl-xL蛋白, 划, 细胞死亡, 细胞凋亡, 线粒体

材料和试剂

  1. 8周龄的C57小鼠或各种转基因/基因敲除小鼠,体重为20-25g
  2. 异氟烷USP(ISOTHESIA)(Butler Schein,目录号:029404)
  3. 2%利多卡因盐酸盐冻胶USP(Healthhotline,目录号:1024140)
  4. 止痛剂:Flunixiject(Flunixin Meglumine)(Butler Schein,目录号:029405)
  5. 0.9%生理盐水或其他用于您的试剂的载体
  6. Betadine手术擦洗(7.5%聚维酮碘)(Healthhotline,目录号:6900581)
  7. 无菌眼润滑剂(puralube)(Butler Schein,目录号:008897)

设备

  1. 麻醉诱导室(VetEquip,目录号:941444)
  2. 蒸气麻醉蒸发器(Bickford)
  3. 带有手术板的解剖显微镜
  4. 面部面具
  5. 电动剃须刀
  6. 计时器
  7. Q提示
  8. 纱布
  9. 剪刀
  10. 加热灯
  11. 手术刀柄(Fine Science Tools,目录号:10003-12)
  12. 刀片#11(Fine Science Tools,目录号:10011-00)
  13. 2x组织钳(Fine Science Tools,Dumont 5/45,目录号:11251-35)
  14. 2x micro serrefine clamp(Fine Science Tools,目录号:18055-04)
  15. 镊子式夹钳施用器(Fine Science Tools,目录号:18057-14)
  16. 热珠灭菌器(Fine Science Tools,目录号:18000-50)
  17. 伤口闭合夹-EZ夹套件(Stoelting,目录号:59020)
  18. 丝绸手术缝合线(Healthhotline,目录号:100-5597)
  19. BD 1ml注射器26G×3/8(0.45mm×10mm)(Thermo Fischer Scientific,目录号:14-823-2E)
  20. 外科剃须刀(Stoelting,目录号:5148 /刀片,手术/剃刮,目录号:50,小鼠,钢,目录号:5148)

程序

  1. 麻醉
    1. 将小鼠置于麻醉室中,其中在N 2 :O 2(40:30)的混合物中的异氟醚流4%将通过附接到 Vapomatic麻醉蒸发器(CWE)。 当它不能在尾部或足部收缩时,对鼠进行充分麻醉。

  2. 动物准备
    1. 使用外科剃须刀剃刮将接受手术(颈部)的区域。
    2. 将小鼠放置在手术板(仰卧位置)上,将头部放置到与Vapomatic麻醉蒸发器(CWE)连接的面罩中,通过该面罩,在N 2/N 2混合物中的异氟烷流动2% O 2 (40:30)不断递送,直到颈动脉夹紧
    3. 通过向前绑扎前肢来稳定鼠标(外科胶带或透明胶带就足够了)。

  3. 外科手术
    1. 使用Q-Tip应用Betadine外科擦洗以净化皮肤。
    2. 用手术刀,在颈部(正中,平行于腹侧中线)处制作腹侧中线垂直的0.5-1厘米皮肤切口。
    3. 用镊子提起脂肪和结缔组织,在底部水平切开,进入下面的组织平面
    4. 找到胸锁乳突肌肉和气管周围的肌肉。打开这些肌肉之间的空间以暴露血管
    5. 在观察显微镜中的视野时,使用镊子轻轻地分离(关键:避免与迷走神经的任何直接接触,这一步需要一些实践)来自神经(迷走神经等)和静脉的颈总动脉(CCA)。神经是白色的。动脉在迷走神经的内侧。对两个CCA执行此步骤。
    6. 使用4-0丝缝线循环CCA,以允许CCA暴露,如下图所示


    7. 通过丝线缝合(左)拉出CCA,并使用微型serrefine钳夹(右)夹住两个CCA。
    8. 将定时器设置为30分钟(夹紧时间可以根据实验室条件而变化,即夹具位置和强度;研究者应当在20-45 * min之间进行中试实验和测试时间以获得最佳缺血,并且具有最小的死亡率)。
    9. 减少异氟烷至1%(取决于动物在夹紧后如何反应)。 鼠标应该没有响应尾巴捏。 呼吸是可变的。
    10. * 30分钟后,取出CCA下方的夹子和丝线
    11. 用高压灭菌器关闭皮肤切口。

  4. 剪裁期间的检查点:
    1. 膜片收缩(〜hiccups); 如果发生这种情况,迷走神经已经被刮伤。 如果有迷走神经损伤,动物很少生存的程序。

  5. 术后护理:
    1. 给予(镇痛剂)氟尼辛皮下,2.2mg/kg
    2. 将利多卡因直接应用于切口。
    3. 涂抹眼润滑剂以防止眼睛干燥。
    4. 将鼠标放在恢复笼中,使用加热灯保温,直到恢复。
    5. 每天监测动物的状况是必要的,以确定是否有疼痛或异常行为
    6. 在所需的时间点,将小鼠麻醉并用0.9%盐水经心脏灌注,然后用缓冲的4%多聚甲醛进行免疫组织化学。 注意:在缺血后4-5天,可以使用FluoroJade染色来确定缺血的功效。 因为小鼠缺血不总是双侧的,所以检查海马的CA1区域是至关重要的。 由于小鼠的非定型脉管系统,大约50%的小鼠用该方法显示缺血的组织学体征。

致谢

这项工作由NIH(授权号NS045876)支持。

参考文献

  1. 参考文献:[1] in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in ,RS和Jonas,EA(2012)。 N末端切割的Bcl-xL介导缺血诱导的神经元死亡。 Nat Neurosci 15(4):574-580。
  2. Zhen,G。和Dore,S。(2007)。 优化方案以减少小鼠双侧颈总动脉闭塞模型的可变结果。 J Neurosci Methods 166(1):73-80。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Pontarelli, F., Ofengeim, D., Zukin, R. S. and Jonas, E. A. (2012). Mouse Transient Global Ischemia Two-Vessel Occlusion Model. Bio-protocol 2(18): e262. DOI: 10.21769/BioProtoc.262.
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