Leading up to the World Congress of Microcirculation in Beijing later this year (Sept 20-24, 2023), we will be exploring one of our most common applications in its various perspectives: The pre-clinical use of the hind limb ischemia assessment techniques to develop novel therapies for peripheral arterial diseases and related applications.

In the run up to the congress we will be releasing a series of articles looking at various aspects of this application, to provide useful background information on current uses of the measurement, practicalities and ultimately to help you make a well-informed choice on the equipment options available to you.

The series includes Hind Limb Ischemia Techniques, Cell TreatmentsTreatment Devices & Drug Therapies and Physiological studies.

Here we briefly consider Hind Limb Ischemia techniques and whether laser Doppler Imaging (LDI) can be considered the Gold Standard for Hind Limb Ischemia studies on vascularisation following femoral artery occlusion.

As always, we invite your questions and feedback. If you’d like to be kept informed of the release of future articles in this series, please sign up to our newsletter at the bottom of the page. If you have any questions or comments on the above information or our applications,please don’t hesitate to contact us.

Hind Limb Ischemia Assessment Techniques

Image submitted by Dr Natalie Haywood, Leeds, UK – Laser Doppler images of Hind Limb Ischemia model, day 7 post op.
Image courtesy of Dr Natalie Haywood, Leeds, UK – Laser Doppler images of Hind Limb Ischemia model, day 7 post op.

The most common practice for Hind Limb Ischemia occlusion is a surgical procedure to ligate the rodent femoral artery in one limb, with the other, sham operated, as a control. Surgical ligation techniques have been explored by Goto et al, 2006, concluding that a ‘relatively severe stable ischemia model is created by stripping the femoral artery from the distal site of the bifurcation of the deep femoral artery to the saphenous artery and the mild ischemic model should be made by cutting the femoral artery just below the bifurcation of the deep femoral artery’.

More recently, Aref et al, 2019 have described electrocoagulation at various sites and the respective collaterals that can be studied, in terms of reperfusion time course, ischemia rate, and suitability for studying arteriogenesis in various muscle compartments and angiogenesis in ischemic distal tissues.

Han et al, 2019, have proposed a photochemical technique, with local thrombosis triggered by erythrosine B, citing various advantages for this compared with surgery: modifying endothelial function and occluding the vessels lumen by blood clot, thereby getting closer to the human pathology than by ligation. It is still early days for wider uptake and assessments by this technique.

Is Laser Doppler Imaging the Gold Standard for Vascular Perfusion Studies?

The Hind Limb Ischemia model is one of macrovascular disease. For this reason, changes in the macrovasculature can be considered a primary observational objective; changes in the distal microvascular perfusion is an excellent surrogate for following progress to reperfusion and decline to necrosis/auto-amputation, and for assessing the effects on distal perfusion in its own right.

Laser Doppler Imaging (LDI) has the depth of penetration to visualise and assess blood flow in collaterals when the LD signal from their flow is not masked by the LD signal from overlying skin or muscle blood flow. Deeper faster blood flow is appropriately represented by LDI because the LD signal is frequency-weighted, i.e. an integral of power spectrum amplitudes multiplied by respective Doppler frequency shifts.

Laser Speckle Contrast Analysis (LASCA) is dominated by superficial blood flow, probably to the depth of the superficial venous and arteriolar plexus. These are likely to be the dominant sources of LASCA flux signal rather than nutritional capillaries or deep dermal perfusion; collaterals are not seen with LASCA. When superficial tissue blood flow is low, the LASCA signal is likely to be dominated by tissue movement caused by respiration and cardiac pulse proximal to the ligation.

A potential advantage of LASCA is its scan speed (up to 100 frames per second in spatial mode; resolution of about 20 microns/pixel for the minimum scan area of 6mm x 8mm; 200microns/pixel at 60mm x 80mm scan area for Hind Limb Ischemia). However, the Hind Limb Ischemia is a relatively chronic condition so neither dynamic assessments nor oscillatory characteristics seem to be of current interest to take advantage of LASCA speed. In practice, to obtain high resolution, temporal mode is used and, to reduce noise, includes at-least 10 seconds per frame.

In temporal mode, LASCA has very high resolution (about 4 microns/pixel for the minimum scan area of 6mm x 8mm; about 40microns/pixel at a typical Hind Limb Ischemia study area of 60mm x 80mm) but this confers little or no advantage for Hind Limb Ischemia because the laser light is diffused by epidermis and connective tissues. To take advantage of the high resolution of LASCA, the microvasculature has to be exposed at surgery, as in mesenteric and cortical spreading depression brain studies.

For comparison, the moorLDI2-HIR has a resolution of about 240microns/pixel for a scan area of 60mm x 60mm, scan duration depending on scan speed (about 4.4min at 4ms/pixel and in proportion for 10 and 50ms/pixel; for a maximum 256 x 256 pixel scan, proportionately faster for reduced scan lines).

A disadvantage of LASCA is its susceptibility to movement. This is minimised or avoided for LDI by use of a high pass filter and differential detection. The bandwidth of LDI is about 15KHz, whereas the dynamic range of LASCA is limited by the camera exposure time (e.g. selected from 1ms up to 20ms).  The 15kHz bandwidth of LDI enables a wide dynamic range to capture small changes of low perfusion up to the fast flows seen as collaterals develop and expand, enabling accurate comparison with the contralateral control at each stage.

In conclusion, for these reasons, Laser Doppler Imaging, LDI can be considered the Gold Standard for Hind Limb Ischemia vascular studies.

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Aref Z, de Vries MR and Quax PHA
Variations in Surgical Procedures for Inducing Hind Limb Ischemia in Mice and the Impact of These Variations on Neovascularization Assessment
Int. J. Mol. Sci. 2019, 20, 3704

Goto T, Fukuyama N, Aki A, Kanabuchi K, Kimura K, Taira H, Tanaka E, Wakana N, Mori H, Inoue H.
Search for appropriate experimental methods to create stable hind-limb ischemia in mouse.
Tokai J Exp Clin Med., 2006; 31 (3); 118-122.

Han S-S, Jin Z, Lee B-S, Han J-S, Choi J-J, Park S-J, Chung H-M, Mukhtar AS, Moon S-H6, Kang S-W.
Reproducible hindlimb ischemia model based on photochemically induced thrombosis to evaluate angiogenic effects.
Microvascular Research, 2019; 126; 103912