Comparison of Different Animal Models in Hindlimb Functional Recovery after Acute Limb Ischemia-Reperfusion Injury

Abstract

Acute limb ischemia (ALI) is a sudden lack of blood flow to a limb, primarily caused by arterial embolism and thrombosis. Various experimental animal models, including non-invasive and invasive methods, have been developed and successfully used to induce limb ischemia-reperfusion injuries (L-IRI). However, there is no consensus on the methodologies used in animal models for L-IRI, particularly regarding the assessment of functional recovery. The present study aims to compare different approaches that induce L-IRI and determine the optimal animal model to study functional limb recovery. In this study, we applied a pneumatic cuff as a non-invasive method and ligated the aorta, iliac, or femoral artery as invasive methods to induce L-IRI. We have measured grip strength, motor function, creatine kinase level, inflammatory markers such as nuclear factor NF-κB, interleukin-6 (IL-6), hypoxia markers such as hypoxia-induced factor-1α (HIF-1α), and evaluated the muscle injury with hematoxylin and eosin (H&E) staining in Sprague Dawley rats after inducing L-IRI. The pneumatic pressure cuff method significantly decreased the muscle strength of the rats, causing the loss of ability to hold the grid and inducing significant limb function impairment, while artery ligations did not. We conclude from this study that the tourniquet cuff method could be ideal for studying functional recovery after L-IRI in the rat model.

Keywords: SD rat; aorta; artery; creatine kinase; femoral; grip strength; iliac; ligation; limb ischemia; motor function; muscle injury; pneumatic cuff.

Figure 1

Creatine kinase (CK) concentrations. Plasma samples were collected from rats with L-IRI induced by the cuff or clamping of the aortic, iliac, or femoral artery (n = 4 rats for each group) 24 h after surgery and compared with the sham group. Statistical analysis was performed with one-way ANOVA; * p < 0.05 was considered significant.

Figure 2

Measurement of grip strength of rats from different experimental groups. The grip strength of the left hindlimbs of the rats was measured with a grip strength meter (Ugo Basile 47,200 Grip Strength Meter) 24 h (A) for all groups and up to 7 days (B) after surgery for the cuff group. The ischemia-reperfusion injury of left hindlimbs was induced with cuff (n = 4) or clamping of the aortic (n = 5), iliac (n = 5), or femoral (n = 4) artery and compared with the sham group (n = 9). One-way ANOVA and two-way ANOVA followed by Sidak multiple comparison was performed to compare the grip strengths among different groups at 24 h after IRI and between the sham and cuff groups for up to 7 days, respectively (* p < 0.05, ** p < 0.01, ns, not significant).

Figure 3

Evaluation of motor functions with a modified Tarlov scale. Motor functions of the hindlimbs were evaluated with a modified Tarlov scoring scale. The position, motion against gravity, gait, and walking ability of the animal in a transparent 1 × 2 ft bucket was observed and graded in all groups of rats (sham, cuff, aortic, iliac, femoral-clamping-induced ischemia, n = 4 rats/group) 24 h (A) and 7 days (B) after surgery. The recovery from L-IRI was assessed in the experimental cohort, referred to as the cuff group, over seven days (C). One-way ANOVA and two-way ANOVA followed by Sidak multiple comparison was performed to compare the Tarlov scales among different groups at 24 h after IRI and between the sham and cuff groups for up to 7 days, respectively. (* p < 0.05, ** p < 0.01 and *** p < 0.001).

Figure 4

(A) Western blot analysis of the expression of inflammatory factors in the hindlimb tissue homogenates from SD rats after 3 h of limb ischemia compared with sham controls. The limb ischemia samples (n = 5) exhibited significantly higher NF-κB expression than the sham control group. The GraphPad Prism 10 software was used as a statistical analytical tool with an unpaired t-test. (B) Densitometry data: The relative expression of a target protein was calculated using the ChemiDoc MP Imager and Image Lab V 6.1 software. GAPDH was used as a loading control (* p < 0.05; ns, not significant).

Figure 5

Evaluation of muscle injury with H&E staining. (A) Representative images of hematoxylin and eosin (H&E) staining of hindlimb muscle tissue sections from control and LI-cuff-ischemia-induced SD rat; (B) histological scoring was performed using the histology scoring system described in the methods; (C) size distribution of muscle fiber diameter; (D) average muscle fiber cross-section area. Student’s 1-tailed t test was applied (n = 3 mice, n = 140 fibers per mouse) (* p < 0.05).