This work was carried out with the use of facilities and resources at the VA Puget Sound Health Care Center. The work is that of the author and does not necessarily reflect the position or policy of the Department of Veterans Affairs or the United States government. Dr Tang is currently funded via the VA (Merit 5 I01 BX004975-02).

Animal experiments were performed according to a protocol approved by the University of Washington Institutional Animal Care and Use Committee.
1. Scanner preparation
<ol>
	<li>Adjust the scanner height so that the distance to the scanned subject is approximately 30 cm.</li>
	<li>Turn on the imager and launch the associated software.</li>
	<li>Open the Measurement program. If the software is correctly communicating with the scanner, the infrared laser turn on warning will appear.</li>
	<li>Calibrate the machine with manufacturer provided standards (not shown in the video and will depend on the specific model of machine being used).</li>
	<li>Adjust the scanner settings to be appropriate for the background material and lighting setup in the room.
	<ol>
		<li>Set the gain levels DC FLUX and CONC, per the manufacturer&#39;s instructions (not shown in the video).</li>
		<li>Set the Background Threshold by pointing the laser beam at the black background material, and press Auto BK Set.</li>
	</ol>
	</li>
</ol>
2. Mouse pre-scanning preparation
<ol>
	<li>Set up the isoflurane induction chamber with appropriate scavenging of the waste gas. 
	NOTE: Placing the induction chamber on a warming pad will help prevent mouse temperature loss during anesthesia induction.</li>
	<li>Turn on the homeothermic blanket, which is placed in the scanning area underneath a nonreflective surface (in this case a black neoprene fabric). Set the homeothermic blanket to maintain a body temperature of 37 &#176;C.</li>
	<li>Position the temperature probe for the homeothermic blanket and lubricant so they are ready for insertion.</li>
	<li>Place the anesthesia mask and scavenging system in the scanning area.</li>
	<li>Anesthetize the mouse with an isoflurane vaporizer. Set the oxygen rate to 1 L/min of flow and adjust the isoflurane to 4% for anesthesia induction. Turn on the flow to the anesthesia induction chamber, and the mouse breathing rate will slow. Adequate anesthesia is achieved when the mouse loses its righting reflex.</li>
	<li>Transfer the mouse to an anesthetic mask/nose cone with attached waste gas scavenger and adjust the isoflurane to 1.5%. 
	NOTE: This anesthesia level is generally adequate to keep the mouse lying relatively still during scanning, but is not intended to provide surgical levels of anesthesia, so the depth of anesthesia is not checked. Changing the isoflurane level causes changes in heartbeat, respiration, and dermal perfusion, so a consistent percentage should be used throughout any time course experiment and for all experimental subjects. Alternative anesthetic techniques such as IP injection of ketamine xylazine can also be used, but the same anesthetic technique should be used throughout any time course study as different anesthetics affect dermal perfusion differently.</li>
	<li>(Optional depending on scanning area) If the planned region of interest to be scanned is covered by fur, use a small electric trimmer or depilatory cream to remove the hair from the region of interest. 
	NOTE: The depilatory cream should be completely removed, and the mouse skin dried prior to scanning.</li>
	<li>Place the mouse in the appropriate scanning position on the black nonreflective surface covering the homeothermic blanket, confirming that both hindlimbs remain on the heat source throughout equilibration and scanning (Figure 1). 
	NOTE: It is important to maintain both feet on the homeothermic blanket to prevent regional variation in temperature.</li>
	<li>Insert the lubricated rectal temperature probe associated with the homeothermic blanket.</li>
	<li>Equilibrate the mouse temperature to desired scanning temperature (37 &#176;C); approximately 5-10 minutes.</li>
	<li>Select Scanner Setup, which can be accessed from the top menu or from the scanner setup icon. Adjust the scan area by changing the X-Y coordinates to accommodate the region of interest. Scan speed will depend on the scan resolution. Higher resolution will result in longer scan times. For repeat scanning focusing on global perfusion, as opposed to higher resolution focusing on anatomic perfusion, a scan speed of 4 ms/pixel is adequate. 
	&#8203;NOTE: Higher resolution and single scan should be considered if the researcher is attempting to directly study the developing collateral circulation (best imaged in the ventral thigh and calf where it is closer to the skin). Repeated scan at lower resolution/speed (e.g., 4 ms/pixel) is adequate when assessing global perfusion to the end organ of the mouse footpad. The software shown in the video automatically loads the previously used template for scanning area, speed, and resolution when restarted, or it can be retrieved from a stored file if different regions of interest are being used for various experiments.</li>
	<li>If performing repeat scans, select the Repeat and Line Scan tab. The number of scans can be changed (in this case 3 scans) as well as the repeat interval. The minimum time for the repeat interval would be the estimated scanning time, which is shown in the grayed-out area on the right of the box determined by scan area and scan resolution. Adding a few seconds allows the user to pause and potentially reposition the mouse if needed between scans.</li>
</ol>
3. Scanning
<ol>
	<li>Select the Image Scan tab and select the Mark button. The laser will move to outline the scanning area. Adjust the mouse position so that the target to be scanned is within the marked area. 
	NOTE: For footpad or footpad and calf scanning, prone positioning with the hindlimbs extended provides a more consistent region of interest than supine positioning. The femoral artery and saphenous artery and collaterals are very close to the ventral surface of the thigh and calf, so supine positioning is preferred if using these regions of interest.</li>
	<li>Start repeated measurement by selecting the Repeat Scanning icon and press the Play Button to initiate the scan.</li>
	<li>Confirm the scanning distance in the pop-up window and click OK to start scanning.</li>
	<li>Monitor the mouse during scanning for mouse movement; if the mouse moves sufficiently that the hindpaws are no longer in the scanning region in the middle of a scan, restart the scan. Small variations in mouse hindpaw position can be accommodated for in the analysis software.</li>
	<li>Monitor the mouse temperature during the scanning process as it may fluctuate even with the use of the homeothermic blanket. If there is too much variation in the mouse temperature, this may result in significant variation between scans. Generally, a temperature range of 36.8-37.2 &#176;C will result in acceptable data.</li>
	<li>Save the captured scan under the Save as window with a file name that includes mouse identifier and timepoint for easier data analysis. Enter mouse and timepoint details if desired in the subject details window.</li>
	<li>Turn off the isoflurane and remove the rectal temperature probe.</li>
	<li>Disinfect the rectal temperature probe with 70% ethanol so it is ready for use in the next mouse.</li>
	<li>Allow the mouse to recover from anesthesia to the point where it displays a righting reflex by flipping from the supine position to the prone position prior to returning it to the cage. 
	&#8203;NOTE: Recovery can be carried out either on a warming blanket for isoflurane since recovery is very quick or in a warmed recovery cage for ketamine/xylazine.</li>
</ol>
4. Capturing LDPI data (Figure 3)
<ol>
	<li>Open the imaging review software program.</li>
	<li>Go to the file menu, open, and locate the saved file.</li>
	<li>Select the ROI icon from the toolbar.</li>
	<li>Select the Add Polygon button.</li>
	<li>Trace the region of interest (ROI) for the control hindlimb using the mouse. Polygon tracing does not have to be exact as the gray background will not be included in the calculated averages.</li>
	<li>Repeat steps 4.3-4.5&#160;for the surgical hindlimb.</li>
	<li>Choose the Statistics icon to open the Image ROIs Statistics Results (PU) window.</li>
	<li>Export the results for Polygon 1 (control hindlimb) and Polygon 2 (surgical hindlimb) to a data collection worksheet via copy/paste.</li>
</ol>
5. Analysis
<ol>
	<li>Capture the data as Surgical/Control ratio for each scan.</li>
	<li>Use the averaged surgical/control for all three scans for the data point for that particular mouse at that timepoint. Because of biologic variability in the response to hindlimb ischemia, in general 8-10 mice are required per timepoint to achieve reproducible results with ~10% standard error. 
	NOTE: Before allowing the mouse to recover from anesthesia, it is worthwhile to perform a quick analysis of the repeated scans to check if the data is too variable (e.g., more than 100-150 perfusion units different between scans 1-3). High variation between repeated scans suggests the mouse was not completely equilibrated during the scan (Figure 2), and a repeat scan can be performed without losing a datapoint, which would occur if the images are not analyzed until a later date. Changing the color palette to optimize the dynamic range of displayed flux values may be necessary to better display scan variation (Figure 2).</li>
</ol>

<ol>
	<li>Varma, P., Stineman, M. G., Dillingham, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+limb+loss.">Epidemiology of limb loss.</a> Physical Medicine and Rehabilitation Clinics of North America. 25 (1), 1-8 (2014).</li><li>Farber, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+Limb-Threatening+Ischemia.">Chronic Limb-Threatening Ischemia.</a> New England Journal of Medicine. 379 (2), 171-180 (2018).</li><li>Abularrage, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+microcirculation+in+vascular+disease.">Evaluation of the microcirculation in vascular disease.</a> Journal of Vascular Surgery. 42 (3), 574-581 (2005).</li><li>Houben, A., Martens, R. J. H., Stehouwer, C. D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+Microvascular+Function+in+Humans+from+a+Chronic+Disease+Perspective.">Assessing Microvascular Function in Humans from a Chronic Disease Perspective.</a> Journal of the American Society of Nephrology. 28 (12), 3461-3472 (2017).</li><li>Mahe, G., Humeau-Heurtier, A., Durand, S., Leftheriotis, G., Abraham, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+skin+microvascular+function+and+dysfunction+with+laser+speckle+contrast+imaging.">Assessment of skin microvascular function and dysfunction with laser speckle contrast imaging.</a> Circulation: Cardiovascular Imaging. 5 (1), 155-163 (2012).</li><li>Murray, A. K., Herrick, A. L., King, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+Doppler+imaging:+a+developing+technique+for+application+in+the+rheumatic+diseases.">Laser Doppler imaging: a developing technique for application in the rheumatic diseases.</a> Rheumatology (Oxford). 43 (10), 1210-1218 (2004).</li><li>Greco, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeatability,+reproducibility+and+standardisation+of+a+laser+Doppler+imaging+technique+for+the+evaluation+of+normal+mouse+hindlimb+perfusion.">Repeatability, reproducibility and standardisation of a laser Doppler imaging technique for the evaluation of normal mouse hindlimb perfusion.</a> Sensors (Basel). 13 (1), 500-515 (2012).</li><li>Sonmez, T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+laser-Doppler+flowmetry+assisted+murine+model+of+acute+hindlimb+ischemia-reperfusion+for+free+flap+research.">A novel laser-Doppler flowmetry assisted murine model of acute hindlimb ischemia-reperfusion for free flap research.</a> PLoS One. 8 (6), 66498 (2013).</li><li>Gargiulo, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+some+anesthetic+agents+on+skin+microcirculation+evaluated+by+laser+Doppler+perfusion+imaging+in+mice.">Effects of some anesthetic agents on skin microcirculation evaluated by laser Doppler perfusion imaging in mice.</a> BMC Veterinary Research. 9, 255 (2013).</li><li>Ankri-Eliahoo, G., Weitz, K., Cox, T. C., Tang, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p27(kip1)+Knockout+enhances+collateralization+in+response+to+hindlimb+ischemia.">p27(kip1) Knockout enhances collateralization in response to hindlimb ischemia.</a> Journal of Vascular Surgery. 63 (5), 1351-1359 (2016).</li><li>McEnaney, R. M., Shukla, A., Madigan, M. C., Sachdev, U., Tzeng, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P2Y2+nucleotide+receptor+mediates+arteriogenesis+in+a+murine+model+of+hind+limb+ischemia.">P2Y2 nucleotide receptor mediates arteriogenesis in a murine model of hind limb ischemia.</a> Journal of Vascular Surgery. 63 (1), 216-225 (2016).</li><li>Padgett, M. E., McCord, T. J., McClung, J. M., Kontos, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+Acute+and+Subacute+Murine+Hindlimb+Ischemia.">Methods for Acute and Subacute Murine Hindlimb Ischemia.</a> Journal of Visualized Experiments. (112), e54166 (2016).</li><li>Niiyama, H., Huang, N. F., Rollins, M. D., Cooke, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+model+of+hindlimb+ischemia.">Murine model of hindlimb ischemia.</a> Journal of Visualized Experiments. (23), e1035 (2009).</li><li>Chalothorn, D., Faber, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-dependent+variation+in+collateral+circulatory+function+in+mouse+hindlimb.">Strain-dependent variation in collateral circulatory function in mouse hindlimb.</a> Physiological Genomics. 42 (3), 469-479 (2010).</li><li>Helisch, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+mouse+strain+differences+in+innate+hindlimb+collateral+vasculature.">Impact of mouse strain differences in innate hindlimb collateral vasculature.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 26 (3), 520-526 (2006).</li><li>Faber, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+causes+collateral+rarefaction+and+increased+severity+of+ischemic+injury+in+multiple+tissues.">Aging causes collateral rarefaction and increased severity of ischemic injury in multiple tissues.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 31 (8), 1748-1756 (2011).</li><li>Forrester, K. R., Stewart, C., Tulip, J., Leonard, C., Bray, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+laser+speckle+and+laser+Doppler+perfusion+imaging:+measurement+in+human+skin+and+rabbit+articular+tissue.">Comparison of laser speckle and laser Doppler perfusion imaging: measurement in human skin and rabbit articular tissue.</a> Medical &amp; Biological Engineering &amp; Computing. 40 (6), 687-697 (2002).</li><li>Briers, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+Doppler,+speckle+and+related+techniques+for+blood+perfusion+mapping+and+imaging.">Laser Doppler, speckle and related techniques for blood perfusion mapping and imaging.</a> Physiological Measurement. 22 (4), 35-66 (2001).</li><li>Heeman, W., Steenbergen, W., van Dam, G., Boerma, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+applications+of+laser+speckle+contrast+imaging:+a+review.">Clinical applications of laser speckle contrast imaging: a review.</a> Journal of Biomedical Optics. 24 (8), 1-11 (2019).</li><li>Nguyen, T., Davidson, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrast+Enhanced+Ultrasound+Perfusion+Imaging+in+Skeletal+Muscle.">Contrast Enhanced Ultrasound Perfusion Imaging in Skeletal Muscle.</a> Journal of Cardiovascular Imaging. 27 (3), 163-177 (2019).</li><li>Zaccagnini, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+Resonance+Imaging+Allows+the+Evaluation+of+Tissue+Damage+and+Regeneration+in+a+Mouse+Model+of+Critical+Limb+Ischemia.">Magnetic Resonance Imaging Allows the Evaluation of Tissue Damage and Regeneration in a Mouse Model of Critical Limb Ischemia.</a> PLoS One. 10 (11), 0142111 (2015).</li><li>Penuelas, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PET+as+a+measurement+of+hindlimb+perfusion+in+a+mouse+model+of+peripheral+artery+occlusive+disease.">PET as a measurement of hindlimb perfusion in a mouse model of peripheral artery occlusive disease.</a> Journal of Nuclear Medicine. 48 (13), 1216-1223 (2007).</li><li>Jia, Y., Qin, J., Zhi, Z., Wang, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrahigh+sensitive+optical+microangiography+reveals+depth-resolved+microcirculation+and+its+longitudinal+response+to+prolonged+ischemic+event+within+skeletal+muscles+in+mice.">Ultrahigh sensitive optical microangiography reveals depth-resolved microcirculation and its longitudinal response to prolonged ischemic event within skeletal muscles in mice.</a> Journal of Biomedical Optics. 16 (8), 086004 (2011).</li><li>Turaihi, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+Intravital+Microscopy+and+Contrast-enhanced+Ultrasonography+of+the+Mouse+Hindlimb+to+Study+Insulin-induced+Vasodilation+and+Muscle+Perfusion.">Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion.</a> Journal of Visualized Experiments. (121), e54912 (2017).</li><li>Liu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+autophagy+alleviates+injury+during+hindlimb+ischemia/reperfusion+in+mice.">Enhanced autophagy alleviates injury during hindlimb ischemia/reperfusion in mice.</a> Experimental and Therapeutic Medicine. 18 (3), 1669-1676 (2019).</li><li>Liu, D. L., Svanberg, K., Wang, I., Andersson-Engels, S., Svanberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+Doppler+perfusion+imaging:+new+technique+for+determination+of+perfusion+and+reperfusion+of+splanchnic+organs+and+tumor+tissue.">Laser Doppler perfusion imaging: new technique for determination of perfusion and reperfusion of splanchnic organs and tumor tissue.</a> Lasers in Surgery and Medicine. 20 (4), 473-479 (1997).</li><li>Jing, Y., Bai, F., Chen, H., Dong, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Laser+Doppler+Imaging+and+Monitoring+to+Analyze+Spinal+Cord+Microcirculation+in+Rat.">Using Laser Doppler Imaging and Monitoring to Analyze Spinal Cord Microcirculation in Rat.</a> Journal of Visualized Experiments. (135), e56243 (2018).</li><li>Zhang, D., Li, S., Wang, S., Ma, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+the+effect+of+a+gastric+ischemia-reperfusion+model+with+laser+Doppler+blood+perfusion+imaging.">An evaluation of the effect of a gastric ischemia-reperfusion model with laser Doppler blood perfusion imaging.</a> Lasers in Medical Science. 21 (4), 224-228 (2006).</li><li>Fitzal, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulatory+changes+after+prolonged+ischemia+in+the+epigastric+flap.">Circulatory changes after prolonged ischemia in the epigastric flap.</a> Journal of Reconstructive Microsurgery. 17 (7), 535-543 (2001).</li></ol>

Dr Tang has no conflicts of interest to disclose.

Consistent technique is critical for obtaining reliable results with LDPI. The same anesthetic, temperature settings, mouse position, and region of interest should be used throughout the entire time course. Different anesthetic agents will result in higher or lower perfusion values9. Isoflurane anesthesia is convenient because of its rapid onset and emergence as well as overall safety. A consistent percentage of isoflurane should be used as depth of anesthesia with this vasodilatory agent may alte...

Skin ulceration with inadequate wound healing is a leading cause of amputations in human patients1. Adequate wound healing requires higher levels of arterial perfusion than are needed to maintain intact skin, which is compromised in patients with peripheral arterial disease2,3,4. Several other rheumatologic conditions and diabetes can also lead to disturbed and inadequate skin microcirculation to heal wounds5,6. Many diabetic patients have concomitant peripheral arterial disease, placing them at especially high risk for amputation. Laser Doppler perfusion imaging (LDPI) is used in clinical situations to evaluate the skin microcirculation, as well as in research situations to evaluate blood flow and blood flow recovery after experimental hindlimb ischemia, ischemia-reperfusion, and microsurgical flaps7.
The LDPI system projects a low power laser beam that is deflected by a scanning mirror to move over a region of interest. This differs from Laser Doppler flowmetry, which provides a perfusion measurement for the small area of tissue in direct contact with the flowmetry probe8. When the laser beam interacts with moving blood in the microvasculature, it undergoes a Doppler frequency shift, which is photodetected by the scanner and converted to arbitrary perfusion units. Because LDPI is a light-based technique, it is limited in terms of depth of penetration to 0.3-1 mm, meaning that for the most part dermal perfusion is assessed7. Dermal flow can be altered by skin temperature and the sympathetic nervous system, which may be affected by various anesthetic agents9. Measurements from the optical laser are also affected by ambient lighting conditions, skin pigmentation, and can be blocked by overlying fur or hair7.
LDPI is the most commonly used research technique to monitor perfusion recovery after ischemia because it is noninvasive, does not require contrast administration, and has quick scan times allowing data collection on multiple animals. This makes it ideal to help determine whether treatments aimed at therapeutic arteriogenesis or angiogenesis are effective in small animal models. Blood flow recovery after hindlimb ischemia as measured by LDPI correlates well with collateral artery development when assessed by other means such as Microfil casting or micro-CT10,11. The goal of this protocol is to demonstrate the assessment of hindlimb perfusion using LDPI.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Black nonreflective material</td>
			<td></td>
			<td></td>
			<td>Fabric store, black neoprene recommended by company</td>
		</tr>
		<tr>
			<td>F/air cannister</td>
			<td>A.M. Bickford Inc</td>
			<td>80120</td>
			<td></td>
		</tr>
		<tr>
			<td>Homeothermic blanket with rigid metal probe</td>
			<td>Harvard Apparatus</td>
			<td></td>
			<td>Also comes with flexible probe, but this is less durable</td>
		</tr>
		<tr>
			<td>Isoflurane Anesthesia machine</td>
			<td>Drager</td>
			<td></td>
			<td>Multiple manufacturers</td>
		</tr>
		<tr>
			<td>Isoflurane induction chamber</td>
			<td>VetEquip</td>
			<td>941444</td>
			<td>2 L chamber</td>
		</tr>
		<tr>
			<td>Moor laser Doppler perfusion imager</td>
			<td>Moor Instruments</td>
			<td>MoorLDI2-IR</td>
			<td>Higher resolution imager (MoorLDI2-HIR)</td>
		</tr>
		<tr>
			<td>Mouse Anesthesia nose cone</td>
			<td></td>
			<td></td>
			<td>Multiple manufacturers</td>
		</tr>
		<tr>
			<td>Nair</td>
			<td>Nair</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen tank</td>
			<td></td>
			<td></td>
			<td>Multiple manufacturers</td>
		</tr>
		<tr>
			<td>Surgilube</td>
			<td>Multiple distributors</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

laser doppler perfusion imaging in the mouse hindlimb

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is a common, noninvasive, repeatable method for assessing blood flow recovery. The technique calculates overall blood flow in the sampled tissue from the Doppler shift in frequency caused when a laser hits moving red blood cells. Measurements are expressed in arbitrary perfusion units, so the contralateral non-intervened upon leg is usually used to help control measurements. Measurement depth is in the range of 0.3-1 mm; for hindlimb ischemia, this means that dermal perfusion is assessed. Dermal perfusion is dependent on several factors&#8212;most importantly skin temperature and anesthetic agent, which must be carefully controlled to result in reliable readings. Furthermore, hair and skin pigmentation can alter the ability of the laser to either reach or penetrate to the dermis. This article demonstrates the technique of LDPI in the mouse hindlimb.

Successful LDPI should result in consistent repeated measures scans, with no more than 100-150 perfusion unit variation (corresponding to about 10% of the usual mean perfusion for the mouse footpad) between the three scans (Figure 2). As demonstrated in Figure 2, repeat scans help determine that the mouse has been appropriately equilibrated so that the ischemic/control ratio best reflects the underlying blood flow as opposed to variation in dermal perfusion caus...

Watch this Scientific Journal Video about Laser Doppler Perfusion Imaging in the Mouse Hindlimb at JoVE.com

Laser Doppler Perfusion Imaging in the Mouse Hindlimb

Here, we present a protocol that demonstrates the technique and necessary controls for Laser Doppler perfusion imaging to measure blood flow in the mouse hindlimb.

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

5.7K Views.  University of Washington. Here, we present a protocol that demonstrates the technique and necessary controls for Laser Doppler perfusion imaging to measure blood flow in the mouse hindlimb.

Video: Laser Doppler Perfusion Imaging in the Mouse Hindlimb

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for imaging vasculature, few are able to visualize the vascular network as a whole while extending to a resolution that includes the smaller vessels1,2. Additionally, many vascular casting techniques destroy the surrounding tissue, preventing further analysis of the sample3-5. One method which circumvents these issues is micro-Computed Tomography (&mu;CT). &mu;CT imaging can scan at resolutions &lt;10 microns, is capable of producing 3D reconstructions of the vascular network, and leaves the tissue intact for subsequent analysis (e.g., histology and morphometry)6-11. However, imaging vessels by ex vivo &mu;CT methods requires that the vessels be filled with a radiopaque compound. As such, the accurate representation of vasculature produced by &mu;CT imaging is contingent upon reliable and complete filling of the vessels. In this protocol, we describe a technique for filling mouse coronary vessels in preparation for &mu;CT imaging.
Two predominate techniques exist for filling the coronary vasculature: in vivo via cannulation and retrograde perfusion of the aorta (or a branch off the aortic arch) 12-14, or ex vivo via a Langendorff perfusion system 15-17. Here we describe an in vivo aortic cannulation method which has been specifically designed to ensure filling of all vessels. We use a low viscosity radiopaque compound called Microfil which can perfuse through the smallest vessels to fill all the capillaries, as well as both the arterial and venous sides of the vascular network. Vessels are perfused with buffer using a pressurized perfusion system, and then filled with Microfil. To ensure that Microfil fills the small higher resistance vessels, we ligate the large branches emanating from the aorta, which diverts the Microfil into the coronaries. Once filling is complete, to prevent the elastic nature of cardiac tissue from squeezing Microfil out of some vessels, we ligate accessible major vascular exit points immediately after filling. Therefore, our technique is optimized for complete filling and maximum retention of the filling agent, enabling visualization of the complete coronary vascular network &#x2013; arteries, capillaries, and veins alike.

Visualization of the coronary vessels is critical to advancing our understanding of cardiovascular diseases. Here we describe a method for perfusing murine coronary vasculature with a radiopaque silicone rubber (Microfil), in preparation for micro-Computed Tomography (&mu;CT) imaging.

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate postprandial delivery of nutrients and hormones to insulin-sensitive tissues. We here describe a technique for combining intravital microscopy (IVM) and contrast-enhanced ultrasonography (CEUS) of the adductor compartment of the mouse hindlimb to simultaneously visualize muscle resistance arteries and perfusion of the microcirculation in vivo. Simultaneously assessing insulin's effect at multiple levels of the vascular tree is important to study relationships between insulin's multiple vasoactive effects and muscle perfusion. Experiments in this study were performed in mice. First, the tail vein cannula is inserted for the infusion of anesthesia, vasoactive compounds and ultrasound contrast agent (lipid-encapsulated microbubbles). Second, a small incision is made in the groin area to expose the arterial tree of the adductor muscle compartment. The ultrasound probe is then positioned at the contralateral upper hindlimb to view the muscles in cross-section. To assess baseline parameters, the arterial diameter is assessed and microbubbles are subsequently infused at a constant rate to estimate muscle blood flow and microvascular blood volume (MBV). When applied before and during a hyperinsulinemic-euglycemic clamp, combined IVM and CEUS allow assessment of insulin-induced changes of arterial diameter, microvascular muscle perfusion and whole-body insulin sensitivity. Moreover, the temporal relationship between responses of the microcirculation and the resistance arteries to insulin can be quantified. It is also possible to follow-up the mice longitudinally in time, making it a valuable tool to study changes in vascular and whole-body insulin sensitivity.

Insulin-induced vasodilation regulates muscle perfusion and increases the microvascular surface area (microvascular recruitment) available for solute exchange between blood and tissue interstitium. Combined intravital microscopy and contrast-enhanced ultrasonography is presented to simultaneously assess insulin&#39;s action at the larger vessels and the microcirculation in vivo.

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate ...

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS)

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a specific anatomical region. Within the brain, metals have a highly compartmentalized distribution, depending on the primary function they play within the central nervous system. Imaging the spatial distribution of metals has provided unique insight into the biochemical architecture of the brain, allowing direct correlation between neuroanatomical regions and their known function with regard to metal-dependent processes. In addition, several age-related neurological disorders feature disrupted metal homeostasis, which is often confined to small regions of the brain that are otherwise difficult to analyze. Here, we describe a comprehensive method for quantitatively imaging metals in the mouse brain, using laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) and specially designed image processing software. Focusing on iron, copper and zinc, which are three of the most abundant and disease-relevant metals within the brain, we describe the essential steps in sample preparation, analysis, quantitative measurements and image processing to produce maps of metal distribution within the low micrometer resolution range. This technique, applicable to any cut tissue section, is capable of demonstrating the highly variable distribution of metals within an organ or system, and can be used to identify changes in metal homeostasis and absolute levels within fine anatomical structures.

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry LA-ICP-MS

Quantitatively mapping metals in tissue by laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) is a sensitive analytical technique that can provide new insight into how metals participate in normal function and disease processes. Here, we describe a protocol for quantitatively imaging metals in thin sections of mouse neurological tissue.

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a ...

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon dioxide and waste products. The impairment of microvascular vasomotion might be a crucial step in the development of microcirculation-related diseases. In addition, the highly vascularized pancreatic islet is adapted to support endocrine function. In this respect, it seems possible to infer that the functional status of pancreatic islet microvascular vasomotion might affect pancreatic islet function. Analyzing the pathological changes of the functional status of pancreatic islet microvascular vasomotion may be a feasible strategy to determine contributions that pancreatic islet microcirculation makes to related diseases, such as diabetes mellitus, pancreatitis, etc. Therefore, this protocol describes using a laser Doppler blood flow monitor to determine the functional status of pancreatic islet microvascular vasomotion, and to establish parameters (including average blood perfusion, amplitude, frequency, and relative velocity of pancreatic islet microvascular vasomotion) for evaluation of the microcirculatory functional status. In a streptozotocin-induced diabetic mouse model, we observed an impaired functional status of pancreatic islet microvascular vasomotion. In conclusion, this approach for assessing pancreatic islet microvascular vasomotion in vivo may reveal mechanisms relating to pancreatic islet diseases.

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

Pancreatic islet microvascular vasomotion regulates islet blood distribution and maintains the physiological function of islet &#946; cells. This protocol describes using a laser Doppler monitor to determine the functional status of pancreatic islet microvascular vasomotion in vivo and to assess the contributions of pancreatic islet microcirculation to pancreatic-related diseases.

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...

Evaluation of Cerebral Blood Flow Autoregulation in the Rat Using Laser Doppler Flowmetry

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained using laser Doppler flowmetry (LDF). This paper demonstrates a closed skull preparation that allows cerebral blood flow to be assessed without penetrating the skull or installing a chamber or cerebral window. To evaluate autoregulatory mechanisms, a model of controlled blood pressure reduction via graded hemorrhage can be utilized while simultaneously employing LDF. This enables the real time tracking of the relative changes in the blood flow in response to reductions in arterial blood pressure produced by the withdrawal of circulating blood volume. This paradigm is a valuable approach to study cerebral blood flow autoregulation during reductions in arterial blood pressure and, with minor modifications in the protocol, is also valuable as an experimental model of hemorrhagic shock. In addition to evaluating autoregulatory responses, LDF can be used to monitor the cortical blood flow when investigating metabolic, myogenic, endothelial, humoral, or neural mechanisms that regulate cerebral blood flow and the impact of various experimental interventions and pathological conditions on cerebral blood flow.

This article demonstrates the use of laser Doppler flowmetry to evaluate the ability of the cerebral circulation to autoregulate its blood flow during reductions in arterial blood pressure.

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained ...

A Revised Method for Inducing Secondary Lymphedema in the Hindlimb of Mice

Animal models are of paramount importance in the research of lymphedema in order to understand the pathophysiology of the disease but also to explore potential treatment options. This mouse model allows researchers to induce significant lymphedema lasting at least 8 weeks. Lymphedema is induced using a combination of fractioned radiotherapy and surgical ablation of lymphatics. This model requires that the mice get a dose of 10 Gray (Gy) radiation before and after surgery. The surgical part of the model involves ligation of three lymph vessels and extraction of two lymph nodes from the mouse hindlimb. Having access to microsurgical tools and a microscope is essential, due to the small anatomical structures of mice. The advantage of this model is that it results in statistically significant lymphedema, which provides a good basis for evaluating different treatment options. It is also a great and easily available option for microsurgical training. The limitation of this model is that the procedure can be time consuming, especially if not practiced in advance. The model results in objectively quantifiable lymphedema in mice, without causing severe morbidity and has been tested in three separate projects.

This animal model enables researchers to induce statistically significant secondary lymphedema in the hindlimb of mice, lasting at least 8 weeks. The model can be used to study the pathophysiology of lymphedema and to investigate novel treatment options.

Animal models are of paramount importance in the research of lymphedema in order to understand the pathophysiology of the disease but also to explore ...

Laser Doppler Perfusion Imaging in the Mouse Hindlimb

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Chapters in this video

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS)

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Evaluation of Cerebral Blood Flow Autoregulation in the Rat Using Laser Doppler Flowmetry

A Revised Method for Inducing Secondary Lymphedema in the Hindlimb of Mice