激光多普勒成像与监测在大鼠脊髓微循环分析中的应用
Summary
在这里, 我们提出了激光多普勒灌注成像 (LDPI) 和激光多普勒灌注监测 (LDPM) 来测量脊髓局部血流量和氧饱和度 (因此,2), 以及一个标准化的程序, 脊髓的介绍老鼠受伤了
Abstract
激光多普勒血流仪是一种无创的血流 (BF) 测量方法, 它更适合于测量脊髓微循环的变化。在本文中, 我们的目标是使用激光多普勒成像和监测来分析在脊髓损伤后 BF 的变化。激光多普勒图像扫描器和探头/显示器都被用来获取每个读数。LDPI 的数据提供了高炉的局部分布, 对损伤部位的灌注进行了概述, 使高炉在不同地点进行了比较分析。通过在一段时间内对探测区域进行强烈测量, 采用联合探针同时测量脊髓的 BF 和氧饱和度, 显示整体脊髓灌注和供氧量。它本身有一些局限性, 如相对通量, 运动敏感性和生物零信号。然而, 由于高炉的简易设置和快速测量, 该技术已在临床和实验研究中得到应用。
Introduction
脊髓损伤 (SCI) 的组织是高血管化的, 对缺氧非常敏感。我们以前的研究表明, 脑震荡后脊髓血流明显减少1,2, 这可能与运动功能的缺陷有关。最近的研究表明, SCI 后血管的完整性与感官运动功能的改善有很好的相关性3。据报道, 改良的血管可能会拯救白质, 间接导致改善功能4。因此, 维持损伤后脊髓灌注似乎是至关重要的维持生存能力和功能。
许多研究者在 sci5、6、7的实验模型中使用多种技术, 对 sci 后的各种治疗效果进行了研究。激光多普勒作为一种公认的技术, 无疑是定量研究几种动物和人体实验的有效方法8,9,10,11。该技术是基于测量的多普勒移位12诱导的红色血细胞到照明光。自二十世纪八十年代代初技术商业化以来, 激光技术、光纤和信号处理在激光多普勒仪器测量中的应用已经取得了很大的进展, 这使得该方法成为一种可靠的技术.
本研究采用激光多普勒测量方法对震荡大鼠脊髓血流 (BF) 进行评价。由于该技术的无创性和简单的设置, 我们的协议提供了一个敏感, 快速, 可靠的方法, 对高炉测量脊髓。更重要的是, 这种方法允许对高炉后震荡 SCI 的纵向研究没有动物牺牲在每个时间点。
由于有能力评估高炉的组织和快速灌注变化的刺激, 有可能应用此协议评估脑 BF14,15以及测量其他组织, 如肝16,17、皮肤18、19和肠道20。在大脑中动脉短暂闭塞的大鼠模型中, 采用激光多普勒读数, 以确保 BF 率适当地降低到缺血性半影14中预期的水平。在发生严重肢体缺血 (cli) 诱导的大鼠中, 应用激光多普勒扫描观察了术前和术后的后肢 BF, 并在治疗后的不同时期进行了21。此外, 一些药物的生物利用度和代谢清除依赖于肝 BF, 这是由16检测到的。因此, 可广泛应用于实验模型、药效学和药动学评价。
Protocol
涉及实验动物的动物议定书遵循了国家卫生研究院 (NIH) 制定的指导方针, 并得到了首都医科大学动物保育和使用委员会的批准。 使用以下描述的激光多普勒设备介绍 SCI 和测量脊髓 BF 的程序在发布的研究1中使用。 1. 手术准备 在生理盐水中准备戊巴比妥钠溶液 3% (w/v), 在35毫克/千克的剂量下进行管理。注意: 戊巴比妥钠是一种?…
Representative Results
LDPI 用于测量脊髓中的 BF, 这是通过提取线性轮廓 (图 4) 在脊髓的延髓-尾鳍轴上进行量化的。图 5A和图 5B分别代表了假组和 SCI 组脊髓的通量成像。图 5C和图 5D分别代表了假组和 SCI 组脊髓的延髓-尾轴的改变。图 5A和图 5B</s…
Discussion
执行此协议时应注意一些细节。首先, 应尽可能快速、优雅地进行麻醉和手术, 尽量减少对动物的压力。为了减少对结果的干扰, 保持动物处于相对平静和稳定的状态。其次, 在使用激光多普勒设备测量时, 应更加注意出血, 因为血液可能会干扰阅读。最后, 在数据记录过程中, 应将动物保存在温度控制的环境中, 避免因温度变化而产生不一致的结果。
研究人员在使用激光多普勒?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者没有确认。
Materials
| Laser Doppler Line Scanner | Moor Instruments | moorLDLS2 | |
| Laser Doppler Monitor | Moor Instruments | moorVMS-LDF | |
| Probe for Monitor | Moor Instruments | VP3 | Blunt needle end delivery probe |
| Impactor | Precision Systems and Instrumentation | IH-0400 | |
| Phenobarbital sodium | Sigma-Aldrich | P3761 | |
| Buprenorphine | Sigma-Aldrich | B-908 | |
| Syringe | Becton Dickinson Medica (s) Pte.Ltd | 300841 | |
| Surgical suture needles with thread | Shanghai Pudong Jinhuan Medical Products Co., Ltd | 18T0329 (batch number) /4-0 | |
| Scalpel | Operation instrument factory of Shanghai Medical Instrument Co., Ltd. | J11030 4# | |
| Scalpel blade | Operation instrument factory of Shanghai Medical Instrument Co., Ltd. | J12130 20# | |
| Ophthalmic forceps | Operation instrument factory of Shanghai Medical Instrument Co., Ltd. | JD1040 | |
| Hemostatic forceps | Operation instrument factory of Shanghai Medical Instrument Co., Ltd. | J31050 | |
| Benzyl penicillin sodium | North China Pharmaceutical Co., Ltd | F6072116 (batch number) | |
| 75% alcohol | Dezhou Anjie Gaoke disinfection products Co., Ltd | 150421R (batch number) | |
| Iodine | Shandong Lierkang Medical Technology Co., Ltd | 20170102 (batch number) | |
| Rat | Laboratory Animal Center, The Academy of Millitery Medical Sciences | Sprague-Dawly (rat strain) |
References
- Jing, Y. L., Bai, F., Chen, H., Dong, H. Meliorating microcirculatory with melatonin in rat model of spinal cord injury using laser Doppler flowmetry. Neuroreport. 27 (17), 1248-1255 (2016).
- Jing, Y. L., Bai, F., Chen, H., Dong, H. Melatonin prevents blood vessel loss and neurological impairment induced by spinal cord injury in rats. J Spinal Cord Med. , 1-8 (2016).
- Han, S., et al. Rescuing vasculature with intravenous angiopoietin-1 and alpha v beta 3 integrin peptide is protective after spinal cord injury. Brain. 133 (Pt 4), 1026-1042 (2010).
- Gerzanich, V., et al. De novo expression of Trpm4 initiates secondary hemorrhage in spinal cord injury. Nat Med. 15 (2), 185-191 (2009).
- Phillips, J. P., Cibert-Goton, V., Langford, R. M., Shortland, P. J. Perfusion assessment in rat spinal cord tissue using photoplethysmography and laser Doppler flux measurements. Journal of Biomedical Optics. 18 (3), 037005 (2013).
- Garcia-Lopez, P., Martinez-Cruz, A., Guizar-Sahagun, G., Castaneda-Hernandez, G. Acute spinal cord injury changes the disposition of some, but not all drugs given intravenously. Spinal Cord. 45 (9), 603-608 (2007).
- Brown, A., Nabel, A., Oh, W., Etlinger, J. D., Zeman, R. J. Perfusion imaging of spinal cord contusion: injury-induced blockade and partial reversal by β2-agonist treatment in rats. Journal of Neurosurgery-Spine. 20 (2), 164-171 (2014).
- Olive, J. L., McCully, K. K., Dudley, G. A. Blood flow response in individuals with incomplete spinal cord injuries. Spinal Cord. 40 (12), 639-645 (2002).
- Yamada, T., et al. Spinal cord blood flow and pathophysiological changes after transient spinal cord ischemia in cats. Neurosurgery. 42 (3), 626-634 (1998).
- Gordeeva, A. E., et al. Vascular Pathology of Ischemia/Reperfusion Injury of Rat Small Intestine. Cells Tissues Organs. , (2017).
- Liu, M., et al. Insulin treatment restores islet microvascular vasomotion function in diabetic mice. J Diabetes. , (2016).
- Drain, L. . The laser Doppler technique. , (1980).
- Rajan, V., Varghese, B., van Leeuwen, T. G., Steenbergen, W. Review of methodological developments in laser Doppler flowmetry. Lasers Med Sci. 24 (2), 269-283 (2009).
- Dohare, P., et al. The neuroprotective properties of the superoxide dismutase mimetic tempol correlate with its ability to reduce pathological glutamate release in a rodent model of stroke. Free Radic Biol Med. 77, 168-182 (2014).
- Bai, H. Y., et al. Pre-treatment with LCZ696, an orally active angiotensin receptor neprilysin inhibitor, prevents ischemic brain damage. Eur J Pharmacol. 762, 293-298 (2015).
- Vertiz-Hernandez, A., et al. L-arginine reverses alterations in drug disposition induced by spinal cord injury by increasing hepatic blood flow. J Neurotrauma. 24 (12), 1855-1862 (2007).
- Garcia-Lopez, P., Martinez-Cruz, A., Guizar-Sahagun, G., Castaneda-Hernandez, G. Acute spinal cord injury changes the disposition of some, but not all drugs given intravenously. Spinal Cord. 45 (9), 603-608 (2007).
- Li, Z., et al. Post pressure response of skin blood flowmotions in anesthetized rats with spinal cord injury. Microvasc Res. 78 (1), 20-24 (2009).
- Boyle, N. H., et al. Scanning laser Doppler is a useful technique to assess foot cutaneous perfusion during femoral artery cannulation. Critical Care. 3 (4), 95-100 (1999).
- Emmanuel, A. V., Chung, E. A. L., Kamm, M. A., Middleton, F. Relationship between gut-specific autonomic testing and bowel dysfunction in spinal cord injury patients. Spinal Cord. 47 (8), 623-627 (2009).
- Sheu, J. J., et al. Combination of cilostazol and clopidogrel attenuates rat critical limb ischemia. J Transl Med. 10, 164 (2012).
- Basso, D. M., Beattie, M. S., Bresnahan, J. C. Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transection. Experimental Neurology. 139 (2), 244-256 (1996).
- Basso, D. M., Beattie, M. S., Bresnahan, J. C. A Sensitive and Reliable Locomotor Rating-Scale for Open-Field Testing in Rats. Journal of Neurotrauma. 12 (1), 1-21 (1995).
- Oberg, P. A. Tissue motion–a disturbance in the laser-Doppler blood flow signal?. Technol Health Care. 7 (2-3), 185-192 (1999).
- Tenland, T., Salerud, E. G., Nilsson, G. E., Oberg, P. A. Spatial and temporal variations in human skin blood flow. Int J Microcirc Clin Exp. 2 (2), 81-90 (1983).
- Kernick, D. P., Tooke, J. E., Shore, A. C. The biological zero signal in laser Doppler fluximetry – origins and practical implications. Pflugers Arch. 437 (4), 624-631 (1999).
- Rudolph, A. M., Heymann, M. A. The circulation of the fetus in utero. Methods for studying distribution of blood flow, cardiac output and organ blood flow. Circ Res. 21 (2), 163-184 (1967).
- Dubory, A., et al. Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury. Jove-Journal of Visualized Experiments. (99), e52536 (2015).
- Kuliga, K. Z., et al. Dynamics of Microvascular Blood Flow and Oxygenation Measured Simultaneously in Human Skin. Microcirculation. 21 (6), 562-573 (2014).
- Li, Z. Y., et al. Post pressure response of skin blood flowmotions in anesthetized rats with spinal cord injury. Microvascular Research. 78 (1), 20-24 (2009).
- Muck-Weymann, M. E., et al. Respiratory-dependent laser-Doppler flux motion in different skin areas and its meaning to autonomic nervous control of the vessels of the skin. Microvasc Res. 52 (1), 69-78 (1996).
- Stefanovska, A., Bracic, M., Kvernmo, H. D. Wavelet analysis of oscillations in the peripheral blood circulation measured by laser Doppler technique. Ieee Transactions on Biomedical Engineering. 46 (10), 1230-1239 (1999).
Cite This Article
Jing, Y., Bai, F., Chen, H., Dong, H. Using Laser Doppler Imaging and Monitoring to Analyze Spinal Cord Microcirculation in Rat. J. Vis. Exp. (135), e56243, doi:10.3791/56243 (2018).