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[Preprint]. 2024 Mar 27:2024.03.21.586197.
doi: 10.1101/2024.03.21.586197.

A 6-minute Limb Function Assessment for Therapeutic Testing in Experimental Peripheral Artery Disease Models

Victoria R Palzkill  1 , Jianna Tan  1 , Abigail L Tice  1   2   3   4 , Leonardo F Ferriera  1   2   3   4 , Terence E Ryan  1   2   3
Affiliations

A 6-minute Limb Function Assessment for Therapeutic Testing in Experimental Peripheral Artery Disease Models

Victoria R Palzkill et al. bioRxiv. .

Abstract

Background: The translation of promising therapies from pre-clinical models of hindlimb ischemia (HLI) to patients with peripheral artery disease (PAD) has been inadequate. While this failure is multifactorial, primary outcome measures in preclinical HLI models and clinical trials involving patients with PAD are not aligned well. For example, laser Doppler perfusion recovery measured under resting conditions is the most used outcome in HLI studies, whereas clinical trials involving patients with PAD primarily assess walking performance. Here, we sought to develop a 6-min limb function test for preclinical HLI models that assess muscular performance and hemodynamics congruently.

Methods: We developed an in situ 6-min limb function test that involves repeated isotonic (shortening) contractions performed against a submaximal load. Continuous measurement of muscle blood flow was performed using laser Doppler flowmetry. Quantification of muscle power, work, and perfusion are obtained across the test. To assess the efficacy of this test, we performed HLI via femoral artery ligation on several mouse strains: C57BL6J, BALBc/J, and MCK-PGC1α (muscle-specific overexpression of PGC1α). Additional experiments were performed using an exercise intervention (voluntary wheel running) following HLI.

Results: The 6-min limb function test was successful at detecting differences in limb function of C57BL6/J and BALBc/J mice subjected to HLI with effect sizes superior to laser Doppler perfusion recovery. C57BL6/J mice randomized to exercise therapy following HLI had smaller decline in muscle power, greater hyperemia, and performed more work across the 6-min limb function test compared to non-exercise controls with HLI. Mice with muscle-specific overexpression of PGC1α had no differences in perfusion recovery in resting conditions, but exhibited greater capillary density, increased muscle mass and absolute force levels, and performed more work across the 6-min limb function test compared to their wildtype littermates without the transgene.

Conclusion: These results demonstrate the efficacy of the 6-min limb function test to detect differences in the response to HLI across several interventions including where traditional perfusion recovery, capillary density, and muscle strength measures were unable to detect therapeutic differences.

Keywords: femoral artery ligation; hindlimb ischemia; peripheral artery disease.

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Conflict of interest statement

Conflict of Interest Statement: The authors have declared that no conflict of interest exists.

Figures

Figure 1.
Figure 1.. Developing a 6-minute limb function test to evaluate isotonic muscle function and perfusion.
(A) Force-frequency curve of the gastrocnemius muscle in healthy mice (n=8) showing selection of the 80Hz contraction as reference force. (B) Force-time and position-time tracings showing levels of force production and length change (shortening) at 30% and 45% of the reference force. Calculation of the Power-Time tracing allows for quantification of work. (C) Quantification of shortening velocity, power, and work (n=4/group). (D) Graphic of the experiment setup. (E) Graphical representation of the isotonic contraction protocol used in the 6-min test. (F) Force and shortening velocity across the 6-min test (n=4/group). (G) Power expressed both in absolute units (watts) and a percentage of the initial power across the 6-min test and quantification of the percent power loss at the end of the test (n=4/group). (H) Work performed across the 6-min test and the total work summed (n=4/group). (I) Laser Dopper perfusion flux across the 6-min test and the mean flux quantified during the last one-minute (n=4/group) Panel C was analyzed using unpaired, two-tailed Student’s t-test. Data were analyzed using an unpaired t-test (two-tailed). Error bars represent the standard error. The graphic and panel D was generated using BioRender.
Figure 2.
Figure 2.. 6-min Limb Function Testing Distinguishes Strains with Differing Sensitivity to HLI.
(A) Laser Doppler flowmetry quantification of perfusion recovery in the paw, gastrocnemius, and tibialis anterior muscles expressed as percentage of control limb (n=7–10/group). Perfusion recovery was analyzed using two-way ANOVA. (B) Representative immunofluorescence images and quantification of total and perfused capillaries. (C) Representative immunofluorescence images and quantification of arterioles. (D) Gastrocnemius muscle mass. (E) Representative images and quantification of the myofiber cross-sectional area (CSA). (F) Representative force traces of 80Hz contractions between strains as well as quantification of absolute and specific forces at different stimulation frequencies. (G) A representative position-time tracing and quantification of muscle power. (H) Muscle power across the 6-min test. (I) Work performed across the 6-min test and quantification of the total work completed in the test. (J) Perfusion flux during the 6-min test. Statistical analyses in panels B-J were performed using an unpaired t-test (two-tailed). Error bars represent the standard error.
Figure 3.
Figure 3.. Exercise Training Improves Limb Performance in Mice with Experimental Peripheral Artery Disease.
(A) Graphic of the experimental design and timeline (generated using BioRender). (B) Daily running distance across the intervention. (C) Laser Doppler flowmetry quantification of perfusion recovery in the paw, gastrocnemius, and tibialis anterior muscles expressed as percentage of control limb (n=10–12/group). Perfusion recovery was analyzed using two-way ANOVA. (D) Representative immunofluorescence images and quantification of total and perfused capillaries. (E) Representative immunofluorescence images and quantification of arterioles. (F) Representative images and quantification of the myofiber cross-sectional area (CSA). (G) Quantification of absolute forces at different stimulation frequencies. (H) Gastrocnemius muscle mass. (I) Quantification of specific forces at different stimulation frequencies. (J) A representative position-time tracing and quantification of muscle power. (K) Muscle power across the 6-min test and quantification of power loss at the end of the test. (L) Work performed across the 6-min test and quantification of the total work completed in the test. (M) Perfusion flux during the 6-min test. Panels D-M were analyzed using an unpaired t-test (two-tailed). Error bars represent the standard error. All panels contain n=10–12/group.
Figure 4.
Figure 4.. Muscle-specific Overexpression of PGC1α Improves Limb Function Following HLI.
(A) Graphic of the experimental design and timeline (generated using BioRender). (B) Laser Doppler flowmetry quantification of perfusion recovery in the paw, gastrocnemius, and tibialis anterior muscles expressed as percentage of control limb (n=10–12/group). Perfusion recovery was analyzed using two-way ANOVA. (C) Representative immunofluorescence images and quantification of total and perfused capillaries. (D) Representative immunofluorescence images and quantification of arterioles. (E) Quantification of absolute forces at different stimulation frequencies. (F) Gastrocnemius muscle mass. (G) Quantification of specific forces at different stimulation frequencies. (H) Representative images and quantification of the myofiber cross-sectional area (CSA). (I) Muscle power across the 6-min test. (J) A representative position-time tracing and work performed across the 6-min test as well as quantification of the total work completed. (K) Perfusion flux during the 6-min test. Panels C-K were analyzed using an unpaired t-test (two-tailed). Error bars represent the standard error. All panels contain n=7–8/group.
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