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Medicine

内皮素-1诱导大脑中动脉闭塞模型缺血性中风激光多普勒血流仪在大鼠指导

Published: February 16, 2013
doi:
10.3791/50014
, , , , , , ,
1Department of Neurosurgery,University of Florida , 2Department of Anatomical Sciences,Shiraz University of Medical Sciences, Shiraz, Iran , 3Department of Physiology and Functional Genomics,University of Florida , 4Department of Neurology,University of Florida

Summary

已经开发了几种脑缺血的动物模型来模拟人的条件中风。该协议描述了内皮素-1（ET-1）诱导大鼠大脑中动脉闭塞（MCAO）模型大鼠缺血性中风。此外，重要的考虑因素，此模型的优点和缺点，进行了讨论。

Abstract

2009年1，2，耗资约70亿美元在美国，中风是导致残疾和死亡的第三大原因，在世界上的头号。已经开发了几种型号的脑缺血，中风模仿人类的​​条件。曾有人建议，高达80％的所有笔划的结果由缺血性脑损伤中的大脑中动脉（MCA）的第3区。在20世纪90年代早期，内皮素-1（ET-1）4，通过将它直接相邻的表面后，在MCA开颅用于诱导缺血。后来，这个模型进行了改进，采用立体定向注射ET-1的相邻MCA局灶性脑缺血。这种模式的主要优点包括能够迅速执行过程，控制动脉收缩，通过改变剂量的ET-1交付的能力，没有需要处理的颅外血管供应血液输送到大脑，并逐渐reperfusi的利率，更接近人类再灌注损伤的5-7。另一方面，ET-1的模型具有的缺点，其中包括需要开颅，以及更高的变异性的每搏输出量8。这种变化可以减少使用激光多普勒血流仪（LDF），以验证脑缺血时ET-1次静脉滴注。变异性中风的因素影响包括输液和一批ET-1的精度，使用了6个 。另一个重要因素是，虽然再灌注损伤是一种常见的发生在人类中风，阻断ET-1诱导脑缺血的持续时间可能不会紧密地模仿人类中风病人多有局部灌注在一个小时内闭塞9天， 10。此协议中详细描述的ET-1诱导的脑缺血模型大鼠缺血性中风的。在整个过程中，它也将提请注意的特殊考虑和潜在的缺点。

Protocol

机构动物管理和使用委员会（IACUC）在佛罗里达大学和批准该协议是符合“指南的照顾和使用的实验动物”（第八版，美国国家科学院，2011年）。 物料动物：8周龄，雄性，只SD大鼠（查尔斯河农场，威​​尔明顿，MA，USA），体重250〜300克，手术时间。 麻醉吸入麻醉系统（VetEquip公司，普莱森顿，CA，USA） 异氟醚麻醉剂（巴克斯特制药，?…

Representative Results

1。术后神经学评估动物苏醒后，可用于广泛的测试，以评估神经功能障碍，包括平衡，握力​​，爪子配售，姿势不对称和楼梯攀登。葵花籽任务的运动和感觉功能，具有显着的相关性梗死第7卷，12是一个总的评价。在这个任务中，老鼠是定时开放和消费5葵花籽。五个种子放置在一个角落里，一个空的，干燥，塑料保持架和操纵的种子所花费的时间被记录。?…

Discussion

ET-1诱导脑缺血是一个既定的模式，经常使用在多个品系的实验缺血性中风。许多变量，如大鼠株，动物年龄，体温，麻醉方法和操作者的专业知识，可导致梗死体积的变异性增加时，使用此模型5中，14。然而，一些研究者已经证明，这种模式的优势，包括相对非侵入性的方法，剂量反应的脑血流ET-1，和能力，以避免完全由麻醉注射ET-1在清醒大鼠14日，15。值得注意的是，有很多…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作是支持由美国心脏协会大中华区东南亚联盟（09GRNT2060421），美国医学协会，并从大学的佛罗里达州的临床与转化科学研究所的资助。亚当麦加是一个NIH / NINDS，NRSA博士前研究员（F30 NS-060335）。高血压（T32 HL-083810）从佛罗里达州的多学科培训计划的大学，博士前奖学金罗伯特·Regenhardt收到支持。

Materials

  1. Animals: Eight-week-old, male, Sprague Dawley rats (Charles River Farms, Wilmington, MA, USA) weighing 250-300 g at the time of surgery.
  2. Anesthesia
    1. Inhalation anesthesia system (VetEquip Inc., Pleasanton, CA, USA)
    2. Isoflurane anesthetic (Baxter Pharmaceutics, Deerfield, IL, USA)
  3. Stereotaxic system (David Kopf Instruments, Tujunga, CA, USA)
    1. Small animal stereotaxic system
    2. Non-rupture ear bars for rats
    3. Gas anesthesia head holder for rats
  4. Temperature regulation
    1. BAT-12 microprobe thermometer (World Precision Instruments, Inc., Sarasota, FL, USA)
    2. T/PUMP, TP600 Thermal blanket (Gaymar Industries, Inc., Orchard Park, NY, USA)
  5. Surgical instruments
    1. Scalpel handle and #11 blade, iris forceps, Graefe forceps, bulldog clamp retractors, screwdriver, 10 μl syringe with 26 gauge beveled needle (World Precision Instruments, Inc., Sarasota, FL, USA)
    2. Micromotor drill and stereotaxic holder, Quintessential Stereotaxic Injector (Stoelting, Wood Dale, IL, USA)
    3. 1.0 mm round drill bur, 1.0 mm inverted cone drill bur (Roboz Surgical Instrument Co., Inc., Gaithersburg, MD, USA)
  6. Surgical Supplies
    1. Mounting screws 0-80 X 3/32 with 2.4 mm shaft length, 21-gauge guide cannula [4mm long below the pedestal] and cannula dummy (Plastics one, Roanoke, VA, USA)
    2. Jet denture acrylic and liquid (Lang Dental Manufacturing Co., Inc., Wheeling, IL, USA)
    3. 3.0 nylon suture (Oasis, Mettawa, IL, USA)
    4. Cotton swabs, Puralube eye ointment (Fisher Scientific, Pittsburg, PA, USA)
    5. Electric hair clippers (Oster, Providence, RI, USA)
  7. Chemicals
    1. Endothelin-1 (American Peptide, Sunnyvale, CA, USA)
    2. Chlorhexidine 2% (Agrilabs, St. Joseph, MO, USA)
    3. Buprenorphine HCl (Hospira Inc., Lake Forest, IL, USA)
  8. Visualization Equipment
    1. Surgical microscope (Seiler Instrument and Manufacturing; St. Louis, MO, USA)
    2. Fiber Optic illuminator (TechniQuip Corp., Livermore, CA, USA)
  9. Laser Doppler flowmetry system (ADInstruments, Inc., Colorado Springs, CO, USA)
    1. Standard Pencil Probe
    2. Probe holder
    3. Blood FlowMeter
    4. Powerlab 4/30 with LabChart 7
  10. Measurement of infarct volume
    1. Rat brain matrix (Zivic-Miller Lab., Inc., Allison Park, PA, USA)
    2. 2,3,5-triphenyltetrazolium chloride (Sigma-Aldrich Co., St Louis, MO, USA) diluted to 0.05% in PBS
    3. Flatbed scanner (Epson Perfection V30, Epson America, Inc., Long Beach, CA, USA)
    4. Image J software (ImageJ 1.42q software, U.S. National Institutes of Health, Bethesda, MA, USA)
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References

  1. Stroke–1989. Recommendations on stroke prevention, diagnosis, and therapy. Report of the WHO Task Force on Stroke and other Cerebrovascular Disorders. Stroke. 20, 1407-1431 (1989).
  2. Lloyd-Jones, D., et al. Heart disease and stroke statistics–2009 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation. 119, 480-486 (2009).
  3. Mohr, J. P., Gautier, J. C., Hier, D., Stein, R. W., Barnett, H. J. M., Stein, B. M., Mohr, J. P., Yatsu, F. M. . Stroke: pathophysiology, diagnosis, and management. , 377-450 (1986).
  4. Robinson, M. J., Macrae, I. M., Todd, M., Reid, J. L., McCulloch, J. Reduction of local cerebral blood flow to pathological levels by endothelin-1 applied to the middle cerebral artery in the rat. Neurosci. Lett. 118, 269-272 (1990).
  5. Sharkey, J., Ritchie, I. M., Kelly, P. A. Perivascular microapplication of endothelin-1: a new model of focal cerebral ischaemia in the rat. J. Cereb. Blood Flow Metab. 13, 865-871 (1993).
  6. O’Neill, M. J., Clemens, J. A. Rodent models of focal cerebral ischemia. Curr. Protoc. Neurosci. Chapter 9 (Unit 9), (2001).
  7. Mecca, A. P., O’Connor, T. E., Katovich, M. J., Sumners, C. Candesartan pretreatment is cerebroprotective in a rat model of endothelin-1-induced middle cerebral artery occlusion. Exp. Physiol. 94, 937-946 (2009).
  8. Braeuninger, S., Kleinschnitz, C. Rodent models of focal cerebral ischemia: procedural pitfalls and translational problems. Exp. Transl. Stroke Med. 1, 8 (2009).
  9. Tomsick, T. A. Intravenous thrombolysis for acute ischemic stroke. J. Vasc. Interv. Radiol. 15, 67-76 (2004).
  10. Olsen, T. S., Lassen, N. A. A dynamic concept of middle cerebral artery occlusion and cerebral infarction in the acute state based on interpreting severe hyperemia as a sign of embolic migration. Stroke. 15, 458-468 (1984).
  11. Pritchett-Corning, K. R., Luo, Y., Mulder, G. B., White, W. J. Principles of rodent surgery for the new surgeon. J. Vis. Exp. (47), e2586 (2011).
  12. Gonzalez, C. L., Kolb, B. A comparison of different models of stroke on behaviour and brain morphology. Eur. J. Neurosci. 18, 1950-1962 (2003).
  13. Ansari, S., Azari, H., McConnell, D. J., Afzal, A., Mocco, J. Intraluminal middle cerebral artery occlusion (MCAO) model for ischemic stroke with laser doppler flowmetry guidance in mice. J. Vis. Exp. (51), e2879 (2011).
  14. Sharkey, J., Butcher, S. P. Characterisation of an experimental model of stroke produced by intracerebral microinjection of endothelin-1 adjacent to the rat middle cerebral artery. J. Neurosci. Methods. 60, 125-131 (1995).
  15. Macrae, I. M., Robinson, M. J., Graham, D. I., Reid, J. L., McCulloch, J. Endothelin-1-induced reductions in cerebral blood flow: dose dependency, time course, and neuropathological consequences. J. Cereb. Blood Flow Metab. 13, 276-284 (1993).
  16. Mecca, A. P., et al. Cerebroprotection by angiotensin-(1-7) in endothelin-1-induced ischaemic stroke. Exp. Physiol. 96 (1-7), 1084-1096 (2011).
  17. Fisher, M., et al. Update of the stroke therapy academic industry roundtable preclinical recommendations. Stroke. 40, 2244-2250 (2009).

Tags

Endothelin-1Middle Cerebral Artery OcclusionIschemic StrokeLaser Doppler FlowmetryRat Model
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Ansari, S., Azari, H., Caldwell, K. J., Regenhardt, R. W., Hedna, V. S., Waters, M. F., Hoh, B. L., Mecca, A. P. Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat. J. Vis. Exp. (72), e50014, doi:10.3791/50014 (2013).
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