在小鼠永久性大脑中动脉结扎术中的应用
Summary
大脑中动脉（MCA）结扎是一种技术，研究在动物模型局灶性脑缺血。在此方法中，通过开颅暴露大脑中动脉和烧灼结扎。此方法提供了高度重复性的脑梗死体积和增加手术后的生存率率比现有的其他方法。
Abstract
局灶性脑缺血之间看到中风患者最常见的类型。由于临床意义出现了长时间的努力，制定合适的动物模型研究缺血期间开展的活动。这些技术包括短暂或永久的，局灶性或全球的许多不同的动物模型，使用最常见的啮齿类动物的缺血模型。
永久性MCA结扎的方法，这也是在文献中提到pMCAo，被广泛使用作为一个在啮齿类动物中1-6局灶性脑缺血模型。这种方法最初是老鼠田村等。在1981年7。开颅手术是在这个协议用于访问由电闭塞MCA和近端地区。梗死涉及大多是皮层和纹状体有时的地区，根据闭塞的位置。现在这项技术是建立和使用在许多实验室8-13。早期使用这种技术的定义和描述的“梗塞核心”和“半影”14日至16日，它往往是用来评估潜在的神经保护作用的化合物10，12，13， 17。虽然最初的研究是在大鼠进行的，永久的马华结扎已成功地使用在18-20稍作修改的小鼠。
这种模式产生重现梗死和增加后的存活率。在人类缺血性中风的约80％发生在MCA 面积 21，因此这是一个中风的研究非常相关的模型。目前，中风患者提供有效的治疗方法缺乏，因此需要有一个很好的模式，来测试潜在的药理的化合物和评估生理成果。这种方法也可以用于研究体内细胞缺氧反应机制。
在这里，我们目前在C57/BL6鼠标MCA结扎手术。我们描述的术前准备，马华结扎手术和2,3,5 – 三苯基氯化四氮唑（TTC）染色脑梗死体积的定量。
Protocol
该协议被批准用于对动物研究的伦理（UCAR）的大学罗切斯特委员会。在协议应遵循无菌技术。无菌手套和口罩的使用是必需的。 表1描述了所有的设备，材料，化学品，并在协议使用工具。 1。手术前准备注入小鼠皮下丁丙诺啡（0.05mg/kg），术前2小时后，立即手术，然后在第一个24小时后手术期每3-5 h。 高压灭菌器消毒所有的手术器械?…
Discussion
永久性MCA结扎方法给予高度重复性的脑梗死体积和增加手术后的生存率率比现有的其他方法。程序的难易程度和持续时间短（约30分钟），使其更加实用。该方法被广泛用于在小鼠和大鼠。
这种方法需要一个立体显微镜下微创手术。因此，在显微镜下操作和完善一个成功的开颅手术的经验是必不可少的。最好是建立在实验室进行实验前，一致梗死。为了实现可重复性的结果是…
Disclosures
The authors have nothing to disclose.
Acknowledgements
手术技术的最初收购的威廉D.山博士在乔治亚医学院的实验室。作者还要感谢大卫博士A. Rempe和兰大Prifti解剖相机使用。这项研究是由美国国立卫生研究院NS041744 NS051279，F31 NS064700和果酸30815697D支持。
Materials
| NAME | COMPANY AND CATALOGUE NUMBER |
|---|---|
| A. Solutions and Chemicals | |
| Povidone-Iodine Solution, Surgical Scrub | Aplicare, 82-209 |
| 70% Ethanol | Koptec, V1001 |
| 2,3,5 Triphenyltetrazolium chloride (TTC) | Sigma, T8877 |
| 4% Paraformaldehyde | Electron Microscopy Sciences, 159SP |
| Phosphate Buffered Saline, pH:7.4 | Made and sterilized in the lab. |
| Mineral Oil | PML Microbiologicals, R6570 |
| Isofluorane | The Butler Company, 029405 |
| Buprenorphine (0.3mg/ml) | Hospira, NDC 0409-2012-32 |
| Artificial tears | The Butler Company, 007312 |
| Recovery Gel | Clear H2O, 72-01-5022 |
| B. Surgical Materials and Equipment | |
| C57/BL6 mouse, 10-12 weeks old | The colony was bred in the university vivarium |
| 100% oxygen tank | Airgas |
| Anesthetic Vaporizer and Flow meter | Surgivet, Model 100 |
| Heating Pad with Rectal Probe | Fine Science Tools, TR200 |
| Dissecting microscope | Zeiss, Stemi 2000 |
| Heating panel | Petco Services Ltd. 0307-013 |
| Small Vessel Cauterizer Set | Fine Science Tools, 18000-00 |
| Mini Peristaltic Pump | Harvard Apparatus, MPII |
| 1ml syringe | BD, 329650 |
| 18G needle | Becton Dickinson, 305196 |
| Surgical 5.0 Nylon Suture | Ethilon, 698H |
| Sectioning Block | Kent Scientific, 1mm |
| Single Edge Razor Blades | Electron Microscopy Sciences, 71962 |
| Label Tape | Scienceware, Bel-art Products F13463-2075 |
| Gauze Sponges | Kendall Curity 2252, Johnson and Johnson 6415 |
| Cotton Tip Applicators | Puritan 806-WCL |
| C. Surgery Tools | |
| Surgical Spring Scissors | Fine Science Tools 15003-08 |
| Microdissecting forceps angled | Fine Science Tools 5/45 |
| Microdissecting forceps curved | Fine Science Tools 5 Ti |
| Fine straight scissors | Fine Science Tools 14568-12 |
| Curved bone rongeur | Fine Science Tools 16021-14 |
| Hartman Hemostat Curved | Fine Science Tools 13003-10 |
Table 1. The list of materials, tools and chemicals used for the MCA ligation procedure. A. Solutions and Chemicals, B. Surgical Materials and Equipment, C. Surgery Tools.
References
- Britton, M., Rafols, J., Alousi, S., Dunbar, J. C. The effects of middle cerebral artery occlusion on central nervous system apoptotic events in normal and diabetic rats. Int J Exp Diabesity Res. 4, 13-20 (2003).
- Ciceri, P., Rabuffetti, M., Monopoli, A., Nicosia, S. Production of leukotrienes in a model of focal cerebral ischaemia in the rat. Br J Pharmacol. 133, 1323-1329 (2001).
- Jin, K. Delayed transplantation of human neural precursor cells improves outcome from focal cerebral ischemia in aged rats. Aging Cell. 9, 1076-1083 (2010).
- McCaig, D. Evolution of GADD34 expression after focal cerebral ischaemia. Brain Res. 1034, 51-61 (2005).
- Shirotani, T., Shima, K., Chigasaki, H. In vivo studies of extracellular metabolites in the striatum after distal middle cerebral artery occlusion in stroke-prone spontaneously hypertensive rats. Stroke. 26, 878-884 (1995).
- Wu, Y. P., Tan, C. K., Ling, E. A. Expression of Fos-like immunoreactivity in the brain and spinal cord of rats following middle cerebral artery occlusion. Exp Brain Res. 115, 129-136 (1997).
- Tamura, A., Graham, D. I., McCulloch, J., Teasdale, G. M. Focal cerebral ischaemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion. J Cereb Blood Flow Metab. 1, 53-60 (1981).
- Bederson, J. B. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke. 17, 472-476 (1986).
- Carswell, H. V. Genetic and gender influences on sensitivity to focal cerebral ischemia in the stroke-prone spontaneously hypertensive rat. Hypertension. 33, 681-685 (1999).
- Mary, V., Wahl, F., Uzan, A., Stutzmann, J. M. Enoxaparin in experimental stroke: neuroprotection and therapeutic window of opportunity. Stroke. 32, 993-999 (2001).
- Menzies, S. A., Hoff, J. T., Betz, A. L. Middle cerebral artery occlusion in rats: a neurological and pathological evaluation of a reproducible model. Neurosurgery. 31, 100-107 (1992).
- Iaci, J. F. Glial growth factor 2 promotes functional recovery with treatment initiated up to 7 days after permanent focal ischemic stroke. Neuropharmacology. 59, 640-649 (2010).
- Richard, M. J. P., Khan, B. V. C. B. J., Saleh, T. M. Cellular mechanisms by which lipoic acid confers protection during the early stages of cerebral ischemia: A possible role for calcium. Neuroscience Research. , (2011).
- Heiss, W. D. Progressive derangement of periinfarct viable tissue in ischemic stroke. J Cereb Blood Flow Metab. 12, 193-203 (1992).
- Nedergaard, M., Gjedde, A., Diemer, N. H. Focal ischemia of the rat brain: autoradiographic determination of cerebral glucose utilization, glucose content, and blood flow. J Cereb Blood Flow Metab. 6, 414-424 (1986).
- Nowicki, J. P., Assumel-Lurdin, C., Duverger, D., MacKenzie, E. T. Temporal evolution of regional energy metabolism following focal cerebral ischemia in the rat. J Cereb Blood Flow Metab. 8, 462-473 (1988).
- Butcher, S. P., Bullock, R., Graham, D. I., McCulloch, J. Correlation between amino acid release and neuropathologic outcome in rat brain following middle cerebral artery occlusion. Stroke. 21, 1727-1733 (1990).
- Arlicot, N. Detection and quantification of remote microglial activation in rodent models of focal ischaemia using the TSPO radioligand CLINDE. Eur J Nucl Med Mol Imaging. 37, 2371-2380 (2010).
- Moyanova, S. G. Protective role for type 4 metabotropic glutamate receptors against ischemic brain damage. J Cereb Blood Flow Metab. , (2010).
- Ortolano, F. Advances in imaging of new targets for pharmacological intervention in stroke: real-time tracking of T-cells in the ischaemic brain. Br J Pharmacol. 159, 808-811 (2010).
- O’Neill, M. J., A, C. J. Rodent models of focal cerebral ischemia. Current Protocols in Neuroscience. , 9.6.1-9.6.32 (2000).
- Lin, T. N., He, Y. Y., Wu, G., Khan, M., Hsu, C. Y. Effect of brain edema on infarct volume in a focal cerebral ischemia model in rats. Stroke. 24, 117-121 (1993).
- Filiano, A. J., Tucholski, J., Dolan, P. J., Colak, G., Johnson, G. V. Transglutaminase 2 protects against ischemic stroke. Neurobiol Dis. 39, 334-343 (2010).
Cite This Article
Colak, G., Filiano, A. J., Johnson, G. V. The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse. J. Vis. Exp. (53), e3039, doi:10.3791/3039 (2011).