激光多普勒灌注成像在鼠标欣德利姆
Summary
在这里，我们提出了一个协议，演示了激光多普勒灌注成像的技术和必要的控制，以测量小鼠后肢的血流。
Abstract
血流恢复是实验性后肢缺血或缺血性输液后的关键结果测量。激光多普勒灌注成像 （LDPI） 是评估血流恢复的常见、非侵入性、可重复性的方法。该技术计算了当激光击中移动的红血球时产生的多普勒频率变化的样本组织的整体血流量。测量以任意灌注单元表示，因此腿部的对交不干预通常用于帮助控制测量。测量深度在0.3-1毫米范围内：对于后肢缺血，这意味着皮肤灌注被评估。皮肤灌注取决于几个因素-最重要的是皮肤温度和麻醉剂，必须仔细控制，以产生可靠的读数。此外，头发和皮肤色素着色可以改变激光到达或穿透真皮的能力。本文演示了鼠标后肢的 LDPI 技术。
Introduction
皮肤溃疡与伤口愈合不足是人类患者截肢的主要原因1。足够的伤口愈合需要更高的动脉灌注水平比需要保持完整的皮肤，这是妥协的外周动脉疾病2，3，4患者。其他几个类风湿病和糖尿病也可能导致不安和皮肤微循环不足，以愈合伤口5，6。许多糖尿病患者患有伴随的外周动脉疾病，使他们面临截肢的特别高风险。激光多普勒灌注成像 （LDPI） 用于临床情况，用于评估皮肤微循环，以及在研究情况中评估实验后肢缺血、缺血-输液和显微外科皮瓣7后的血流和血流恢复。
LDPI 系统投射低功率激光束，该光束由扫描镜偏转，以移动到感兴趣的区域。这与激光多普勒流量计不同，后者为与流测探针8直接接触的较小组织区域提供了灌注测量。当激光束与微血管中移动的血液相互作用时，它就会经历多普勒频率变化，这种移位由扫描仪照射并转换为任意灌注单元。由于LDPY是一种基于光的技术，因此其渗透深度限制在0.3-1毫米，这意味着大部分皮肤灌注被评估为7。皮肤流量可以通过皮肤温度和交感神经系统改变，这可能受到各种麻醉剂9的影响。光学激光的测量也受到环境照明条件、皮肤色素沉着的影响，并可能被过度的毛皮或头发堵塞。
LDPI 是最常用的研究技术，用于监测缺血后的灌注恢复，因为它具有非侵入性，不需要对比度管理，并且具有快速扫描时间，允许对多个动物进行数据收集。这使得它非常理想，以帮助确定治疗动脉生成或血管生成的治疗是否有效的小动物模型。根据LDBI测量的后肢缺血后的血流量恢复与辅助动脉发育密切相关，当通过其他方法评估，如微滤铸造或微CT10，11。本协议的目的是演示使用 LDPI 对后肢灌注的评估。
Protocol
动物实验是根据华盛顿大学机构动物护理和使用委员会批准的协议进行的。 1. 扫描仪准备 调整扫描仪高度，使扫描对象的距离约为 30 厘米。 打开成像器并启动相关软件。 打开 测量 程序。如果软件与扫描仪进行正确通信，将显示红外激光打开警告。 使用制造商提供的标准校准机器（视频未显示，将取决于正在使用的机器的特定型?…
Representative Results
成功的 LDPI 应导致三次扫描之间一致的重复测量扫描，注液单位变化不超过 100-150 次（相当于鼠标脚垫通常平均灌注的 10% 左右）（图 2）。如图 2所示，重复扫描有助于确定鼠标已得到适当的均衡，以便缺血/控制比最能反映基础血流，而不是温度变化导致的皮肤灌注变化。使用单次扫描数据点将增加变异性，导致需要更多的实验鼠。当用于后肢缺血时…
Discussion
一致的技术对于通过 LDPI 获得可靠的结果至关重要。在整个过程中应使用相同的麻醉剂、温度设置、鼠标位置和感兴趣的区域。不同的麻醉剂将导致更高或更低的灌注值9。异氟乙烯麻醉是方便的，因为它的快速发病和出现，以及整体安全。应使用一致百分比的异氟兰作为麻醉深度与这种血管扩张剂可能会改变皮肤灌注。如果感兴趣的区域包括毛皮，那么每次都应该使用相同的脱…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是在VA普吉特健康保健中心的设施和资源使用下进行的。这项工作是作者的作品，不一定反映退伍军人事务部或美国政府的立场或政策。唐博士目前由VA资助（功绩5 I01 BX004975-02）。
Materials
| Black nonreflective material | Fabric store, black neoprene recommended by company | ||
| F/air cannister | A.M. Bickford Inc | 80120 | |
| Homeothermic blanket with rigid metal probe | Harvard Apparatus | Also comes with flexible probe, but this is less durable | |
| Isoflurane Anesthesia machine | Drager | Multiple manufacturers | |
| Isoflurane induction chamber | VetEquip | 941444 | 2 L chamber |
| Moor laser Doppler perfusion imager | Moor Instruments | MoorLDI2-IR | Higher resolution imager (MoorLDI2-HIR) |
| Mouse Anesthesia nose cone | Multiple manufacturers | ||
| Nair | Nair | ||
| Oxygen tank | Multiple manufacturers | ||
| Surgilube | Multiple distributors |
References
- Varma, P., Stineman, M. G., Dillingham, T. R. Epidemiology of limb loss. Physical Medicine and Rehabilitation Clinics of North America. 25 (1), 1-8 (2014).
- Farber, A. Chronic Limb-Threatening Ischemia. New England Journal of Medicine. 379 (2), 171-180 (2018).
- Abularrage, C. J., et al. Evaluation of the microcirculation in vascular disease. Journal of Vascular Surgery. 42 (3), 574-581 (2005).
- Houben, A., Martens, R. J. H., Stehouwer, C. D. A. Assessing Microvascular Function in Humans from a Chronic Disease Perspective. Journal of the American Society of Nephrology. 28 (12), 3461-3472 (2017).
- Mahe, G., Humeau-Heurtier, A., Durand, S., Leftheriotis, G., Abraham, P. Assessment of skin microvascular function and dysfunction with laser speckle contrast imaging. Circulation: Cardiovascular Imaging. 5 (1), 155-163 (2012).
- Murray, A. K., Herrick, A. L., King, T. A. Laser Doppler imaging: a developing technique for application in the rheumatic diseases. Rheumatology (Oxford). 43 (10), 1210-1218 (2004).
- Greco, A., et al. Repeatability, reproducibility and standardisation of a laser Doppler imaging technique for the evaluation of normal mouse hindlimb perfusion. Sensors (Basel). 13 (1), 500-515 (2012).
- Sonmez, T. T., et al. A novel laser-Doppler flowmetry assisted murine model of acute hindlimb ischemia-reperfusion for free flap research. PLoS One. 8 (6), 66498 (2013).
- Gargiulo, S., et al. Effects of some anesthetic agents on skin microcirculation evaluated by laser Doppler perfusion imaging in mice. BMC Veterinary Research. 9, 255 (2013).
- Ankri-Eliahoo, G., Weitz, K., Cox, T. C., Tang, G. L. p27(kip1) Knockout enhances collateralization in response to hindlimb ischemia. Journal of Vascular Surgery. 63 (5), 1351-1359 (2016).
- McEnaney, R. M., Shukla, A., Madigan, M. C., Sachdev, U., Tzeng, E. P2Y2 nucleotide receptor mediates arteriogenesis in a murine model of hind limb ischemia. Journal of Vascular Surgery. 63 (1), 216-225 (2016).
- Padgett, M. E., McCord, T. J., McClung, J. M., Kontos, C. D. Methods for Acute and Subacute Murine Hindlimb Ischemia. Journal of Visualized Experiments. (112), e54166 (2016).
- Niiyama, H., Huang, N. F., Rollins, M. D., Cooke, J. P. Murine model of hindlimb ischemia. Journal of Visualized Experiments. (23), e1035 (2009).
- Chalothorn, D., Faber, J. E. Strain-dependent variation in collateral circulatory function in mouse hindlimb. Physiological Genomics. 42 (3), 469-479 (2010).
- Helisch, A., et al. Impact of mouse strain differences in innate hindlimb collateral vasculature. Arteriosclerosis, Thrombosis, and Vascular Biology. 26 (3), 520-526 (2006).
- Faber, J. E., et al. Aging causes collateral rarefaction and increased severity of ischemic injury in multiple tissues. Arteriosclerosis, Thrombosis, and Vascular Biology. 31 (8), 1748-1756 (2011).
- Forrester, K. R., Stewart, C., Tulip, J., Leonard, C., Bray, R. C. Comparison of laser speckle and laser Doppler perfusion imaging: measurement in human skin and rabbit articular tissue. Medical & Biological Engineering & Computing. 40 (6), 687-697 (2002).
- Briers, J. D. Laser Doppler, speckle and related techniques for blood perfusion mapping and imaging. Physiological Measurement. 22 (4), 35-66 (2001).
- Heeman, W., Steenbergen, W., van Dam, G., Boerma, E. C. Clinical applications of laser speckle contrast imaging: a review. Journal of Biomedical Optics. 24 (8), 1-11 (2019).
- Nguyen, T., Davidson, B. P. Contrast Enhanced Ultrasound Perfusion Imaging in Skeletal Muscle. Journal of Cardiovascular Imaging. 27 (3), 163-177 (2019).
- Zaccagnini, G., et al. Magnetic Resonance Imaging Allows the Evaluation of Tissue Damage and Regeneration in a Mouse Model of Critical Limb Ischemia. PLoS One. 10 (11), 0142111 (2015).
- Penuelas, I., et al. PET as a measurement of hindlimb perfusion in a mouse model of peripheral artery occlusive disease. Journal of Nuclear Medicine. 48 (13), 1216-1223 (2007).
- Jia, Y., Qin, J., Zhi, Z., Wang, R. K. Ultrahigh sensitive optical microangiography reveals depth-resolved microcirculation and its longitudinal response to prolonged ischemic event within skeletal muscles in mice. Journal of Biomedical Optics. 16 (8), 086004 (2011).
- Turaihi, A. H., et al. Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion. Journal of Visualized Experiments. (121), e54912 (2017).
- Liu, C., et al. Enhanced autophagy alleviates injury during hindlimb ischemia/reperfusion in mice. Experimental and Therapeutic Medicine. 18 (3), 1669-1676 (2019).
- Liu, D. L., Svanberg, K., Wang, I., Andersson-Engels, S., Svanberg, S. Laser Doppler perfusion imaging: new technique for determination of perfusion and reperfusion of splanchnic organs and tumor tissue. Lasers in Surgery and Medicine. 20 (4), 473-479 (1997).
- Jing, Y., Bai, F., Chen, H., Dong, H. Using Laser Doppler Imaging and Monitoring to Analyze Spinal Cord Microcirculation in Rat. Journal of Visualized Experiments. (135), e56243 (2018).
- Zhang, D., Li, S., Wang, S., Ma, H. An evaluation of the effect of a gastric ischemia-reperfusion model with laser Doppler blood perfusion imaging. Lasers in Medical Science. 21 (4), 224-228 (2006).
- Fitzal, F., et al. Circulatory changes after prolonged ischemia in the epigastric flap. Journal of Reconstructive Microsurgery. 17 (7), 535-543 (2001).
Tags
Laser Doppler Perfusion ImagingLDPIMouse HindlimbSkin UlcerationWound HealingPeripheral Arterial DiseaseRheumatologic ConditionsDiabetesBlood FlowIschemiaIschemia ReperfusionMicrosurgical FlapsAnesthetic AgentsSkin PigmentationArteriogenesisAngiogenesisCollateral Artery DevelopmentMicrofill CastingMicro CT
Cite This Article
Tang, G. L., Kim, K. J. Laser Doppler Perfusion Imaging in the Mouse Hindlimb. J. Vis. Exp. (170), e62012, doi:10.3791/62012 (2021).