Laser Doppler Perfusion Imaging in the Mouse Hindlimb

Skin ulceration with inadequate wound healing is a leading cause of amputation in human patients. Adequate wound healing requires higher levels of arterial profusion that are needed to maintain intact skin, which is compromised in patients with peripheral arterial disease. Several other rheumatologic conditions and diabetes can also lead to disturbed and inadequate skin microcirculation to heal wounds.

Many diabetic patients have concomitant peripheral arterial disease, placing them at especially high risk for amputation. Laser Doppler perfusion imaging or 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 flaps. 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 probe. 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 to 1 millimeter, meaning that for the most part dermal perfusion is assessed.

Dermal flow can be altered by skin temperature and the sympathetic nervous system, which may be affected by various anesthetic agents. Measurements from the optical laser are also affected by ambient lighting conditions, skin pigmentation and can be blocked by overlying fur or hair. LDPI is the most commonly used research technique to monitor profusion recovery after ischemia because it is non-invasive, 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 microfill casting or micro CT.The goal of this protocol is to demonstrate the assessment of hindlimb perfusion using LDPI. Adjust the scanner height so that the distance to the scan subject is approximately 30 centimeters.

Turn on the imager and launch the associated software. Open the measurement program. If the software is correctly communicating with the scanner the infrared laser turn on warning will appear.

Adjust the scanner settings to be appropriate for the background material and lighting setup in the room. Set the background threshold by pointing the laser beam at the black background material and press Auto BK Set. Set up the isoflurane induction chamber with appropriate scavenging of waste gas.

Placing the induction chamber on a warming pad will help prevent mouse temperature loss during anesthesia induction. Turn on the homeothermic blanket, which is placed in the scanning area underneath a non-reflective surface. In this case, a black neoprene fabric.

The homeothermic blanket is set to maintain a body temperature of 37 degrees Celsius. Position the temperature probe and lubricants so they're ready for insertion. Place the anesthesia face mask and scavenging system into the scanning area as well.

Anesthetize the mouse with an isoflurane vaporizer. The oxygen rate is set to one liter per minute of flow. And the isoflurane is adjusted to 4%for anesthesia induction.

The flow is turned on to the anesthesia induction chamber and the mouse breathing rate will slow. Adequate anesthesia is achieved when the mouse loses its righting reflex. Transfer the mouse to an anesthetic mask nose cone with attached waste gas scavenger and adjust the isoflurane to 1.5%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 heart rate, respiration, and dermal profusion. 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 profusion differently. If the plan region of interest to be scanned is covered by fur, use a small electrical trimmer and/or depilatory cream to remove the hair from the region of interest. The depilatory creams should be completely removed and the mouse skin dried prior to scanning as wet skin can cause reflections from the laser Insert the lubricated rectal temperature probe associated with the homeothermic blanket into the mouse rectum.

Equilibrate the mouse temperature to desire scanning temperature, 37 degrees Celsius. This can take up to five to 10 minutes. Select Scanner Setup which can be accessed from the top menu or from the Scanner Setup icon.

The scan area should be adjusted 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 profusion, as opposed to higher resolution focusing on anatomic perfusion, the highest scan speed of four milliseconds per pixel is adequate. If performing repeat scans, select the Repeat and Line Scan tab. The number of scans can be changed.

In this case, three scans, as well as the repeat interval. The minimum time for the repeat interval would be the estimated scanning times 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.

The total scan time in this case is three minutes and 21 seconds. Select the Image Scan tab and select the Mark button. This will cause the laser to outline the scanning area.

Adjust the mouse position so that the target to be scanned is within the marked area. Start repeated measurement by selecting the Repeat Scanning icon and pressing the Play button to initiate the scan. A pop-up window confirming scanning distance will appear.

Click OK to start scanning. The initial scan of a mouse prior to induction of hindlimb ischemia is shown in real-time. The second and third repeat scans are accelerated to 16x speed.

The main window shows the results of the scan as it is generated by the software. The inset at the bottom right shows the laser scanning the mouse footpads. For footpad or footpad and dorsal calf scanning, prone positioning with the hindlimbs extended toward the tail provides a more consistent region of interest in supine positioning.

The femoral artery, 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. Monitor the mouse during scanning for mouse movement.

If the mouse moves sufficiently that the footpads are no longer in the scanning region in the middle of a scan, restart the scan. Small variations in mouse footpad position can be accommodated for in analysis software. Also, monitor the mouse temperature during the scanning process as it may fluctuate even with the use of the homeothermic blanket.

If there's too much variation in the mouse temperature, this may result in significant variation between scans. Generally, a temperature range of 36.8 to 37.2 will result in acceptable data. Save the captured scan under the Save As window with a file name that includes mouse identifier and time point for easier data analysis.

Enter mouse and time point details if desired in the Subject Details window. Ventral hindlimb scanning with the mouse in the supine position is shown here after adjusting the scan area. Note that the proposition with the mouse supine results in a variable portion of the side of the foot being scanned.

Remove the rectal temperature probe and disinfect it with 70%ethanol so it is ready for the next mouse. Decrease isoflurane to 0%The mouse will recover faster if it continues to receive oxygen, but it can also be recovered without supplemental oxygen. 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.

Recovery can be carried out either on a warming blanket for isoflurane, since recovery is very quick or in a warm recovery cage for ketamine xylazine. Open the imaging review software program. A new window to review scans will open.

Go to the File menu, Open and locate the saved file. Select the ROI icon from the toolbar. Select the Add Polygon icon.

Trace an area around the region of interest using the mouse. When finished, go back to the toolbar and select the Statistics icon. This will bring up a window showing statistics from the region of interest.

On data review, the mean profusion unit, or PU, values do not vary more than 100 to 150 PUs over the repeated scans for each footpad. This is an acceptable scan and indicates the mouse was well equilibrated during the scan. Mean PU values with variation greater than 100 to 150 PUs, which corresponds to greater than 10%of the mean perfusion for a non-operated mouse footpad over the repeated scans would indicate that the mouse was not well equilibrated and a repeat scan is strongly suggested.

Performing a quick analysis immediately after the scan is completed allows a repeat scan to be performed while the mouse is still anesthetized and avoid losing a data point which could occur if the scans are analyzed at a later date and a scan is deemed unacceptable due to significant variation between the repeated scans. Results are expressed as a ratio of surgical hindlimb profusion over control hindlimb perfusion as mice initially vasodilate and then develop their intrinsic collaterals, blood flow recovery by LDPI should be seen over a post-operative time course. The degree of recovery is dependent on the mouse strain and severity of the hindlimb ischemia model.

This figure shows a completed time course experiment over 28 days where LDPI was used to measure footpad blood flow recovery over time after femoral artery ligation and P27 knockout and wild type C57Bl/6 mice. Both P27 knockout in wild type mice had impaired blood flow recovery when treated with oral doxycycline, a generalized matrix metalloproteinases inhibitor, suggesting that MMP activity facilitates arteriogenesis. The gain setup and background threshold should be kept constant for the entire time course experiment.

If the background threshold is set too low, scanning the background material will result in low profusion values. This will result in the background profusion values being averaged into the region of interest during polygon selection. To eliminate this problem, the background threshold can be adjusted in the Scanner Setup window under the General tab.

In this case, increasing the background threshold to 118 profusion units results in the gray background that will not be included in any analysis. The palette or color scale for the flux image can be adjusted both in the measurement software and the image review software. All profusion values greater than the maximum value on the scale will be displayed as the highest profusion color.

In this case, red. Increasing the dynamic range, for instance, changing the scale to range from zero to 1500, will make repeat scan variation more obvious to the eye as areas measuring between 1000 to 1500 profusion units will now be displayed using the color gradient rather than all being shown as red. The scan shown here was obtained in a mouse whose core body temperature was initially below 36.8 at the start of the repeated scan and then increased over the repeated scans.

Note that the control footpad average profusion was more effected by the colder core body temperature than the ischemic footpad. A single scan of the cooler body temperature would have resulted in a higher profusion ratio being recorded for this data point. Variation in mouse skin pigmentation, such as may be seen with C57Bl/6 mice, may result in the scan area being below the background threshold value which will make analysis of regions of interest containing pigmented areas difficult.

The mouse footpads in general do not have hyperpigmentation, making this the most convenient area to scan for this mouse strain. If the researcher wishes to study the ventral hindlimb, then a white mouse strain should be selected. LDPI is an effective, easily performed, and repeatable method for measuring hindlimb dermal profusion as a reflection of overall arterial perfusion.

Consistent technique is required when using LDPI to obtain reliable data.