Laser Doppler Perfusion Imaging in the Mouse Hindlimb

Summary

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.

Abstract

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—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.

Introduction

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 disease^2,3,4. Several other rheumatologic conditions and diabetes can also lead to disturbed and inadequate skin microcirculation to heal wounds^5,6. Many diabetic patients have concomitant peripheral arterial disease, placing them a....

Protocol

Animal experiments were performed according to a protocol approved by the University of Washington Institutional Animal Care and Use Committee.

1. Scanner preparation

1. Adjust the scanner height so that the distance to the scanned subject is approximately 30 cm.
2. Turn on the imager and launch the associated software.
3. Open the Measurement program. If the software is correctly communicating with the scanner, the infrared laser turn on warning will appear.
4. Calibrate the machine with manufacturer provided standards (not shown in the video and will depend on the specific model of machine be....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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