An optimized low-pressure tourniquet murine hind limb ischemia reperfusion model: Inducing acute ischemia reperfusion injury in C57BL/6 wild type mice

Abstract

Acute ischemia reperfusion injury in skeletal muscle remains an important issue in several fields of regenerative medicine. Thus, a valid model is essential to gain deeper insights into pathophysiological relations and evaluate possible treatment options. While the vascular anatomy of mice regularly prevents sufficient vessel occlusion by invasive methods, there is a multitude of existing models to induce ischemia reperfusion injury without surgical procedures. Since there is no consensus on which model to prefer, this study aims to develop and evaluate a novel, optimized low-pressure tourniquet model. C57BL/6 mice underwent an ischemic procedure by either tourniquet or invasive artery clamping. A sham group served as control. With exception of the sham group, mice underwent 2 hours of ischemia followed by 4 hours of reperfusion. Groups were compared using microcirculatory and spectroscopic measurements, distinctions in tissue edema, histological and immunohistochemical analyses. Both procedures led to a significant decrease in tissue blood flow (- 97% vs. - 86%) and oxygenation (- 87% vs. - 75%) with a superiority of the low-pressure tourniquet. Tissue edema in the tourniquet cohort was significantly increased (+ 59%), while the increase in the clamping cohort was non-significant (+ 7%). Haematoxylin Eosin staining showed significantly more impaired muscle fibers in the tourniquet group (+ 77 p.p. vs. + 11 p.p.) and increased neutrophil infiltration/ROI (+ 51 vs. + 8). Immunofluorescence demonstrated an equal increase of p38 in both groups (7-fold vs. 8-fold), while the increase in apoptotic markers (Caspase-3, 3-Nitrotyrosine, 4-Hydroxynonenal) was significantly higher in the tourniquet group. The low-pressure tourniquet has been proven to produce reproducible and thus reliable ischemia reperfusion injury. In addition, significantly less force was needed than previously stated. It is therefore an important instrument for studying the pathophysiology of ischemia reperfusion injury and for the development of prophylactic as well as therapeutic interventions.

Fig 1. Low-pressure tourniquet.

Fig 2. Microcirculatory measurement of oxygenation, flow and relative hemoglobin amount in the tourniquet and operative clamping model.

(A) Using the O2C device, relative oxygenation of the murine hind limb was significantly reduced at a force of 0.4 N with no further reduction by increasing pressure. The operative clamping model (bar) showed a significant reduction but not as high as the low-pressure tourniquet. (B) Microvascular flow was significantly decreased at a force of 0.2 N and upwards compared to the control as well as in the operative technique (bar). (C) The relative hemoglobin amount was not significantly changed by either the operative clamping technique or increasing tourniquet pressure. Results are shown as means ± SEM. P-value: # < 0.05; § < 0.001; (two sample t-test), n = 4.

Fig 3. Measurement of oxygenation, perfusion and tissue hemoglobin in the tourniquet and operative clamping model with a hyperspectral camera system.

(A) Using the TIVITA Tissue system, acquisition of the tissue Oxygenation (StO2) showed a significant reduction of oxygenation in the low-pressure tourniquet group at a force of 0.4 N with no further reduction of oxygenation by rising pressure. The low-pressure tourniquet group showed a higher reduction of oxygenation than the operative clamping method. (B) Live images of the oxygenation visualization exemplary at a force of 0 N, 2 N, after reperfusion and with the open clamping technique. The yellow arrow marks the compromised hind limb. (C) Measurement of the near-infrared (NIR) perfusion index showed a significant steady reduction of the hind limb perfusion starting with a force of 0.4 N. In the tourniquet group this force showed a significantly higher reduction of perfusion than the operative clamping technique. (D) Live images of the NIR exemplary at a force of 0 N, 2 N, after reperfusion and with the open clamping technique. The yellow arrow marks the compromised hind limb. (E) Measurement of Tissue-Hemoglobin-Index showed no significant change either in the tourniquet group or in the operative clamping group compared to control (0 N). (F) Live images of the Tissue-Hemoglobin-Index exemplary at a force of 0 N, 2 N, after reperfusion and with the open clamping technique. The yellow arrow marks the compromised hind limb. Results are shown as means ± SEM. P-value: # < 0.05; § < 0.001 (two sample t-test), n = 4.

Fig 4. Measurement of wet to dry ratio in the low-pressure tourniquet group and operative clamping cohort.

After 2 hours of ischemia and 4 hours of reperfusion the low-pressure tourniquet group showed a significant increase in edema compared to both sham and operative clamping. Results are shown as means ± SEM. P-value: *** < 0.001; (ANOVA), n = 4.

Fig 5. Low-pressure tourniquet leads to destruction of muscle fibers and neutrophil infiltration.

After ischemia of 2 hours and reperfusion of 4 hours (A) Neutrophils per 2000×2000 Pixels were measured. The low-pressure tourniquet led to a significant increase in neutrophil infiltration compared to control (sham) animals. The operative clamping of the femoral artery did not show the same significant increase in neutrophil infiltration. (B) Fraction of normal myofibers was significantly lower in the tourniquet cohort compared to control and operative clamping. (C) Histological images at a magnification of 100× showed a higher neutrophil infiltration (neutrophils are marked with a green arrow) in the tourniquet group compared to control and operative clamping. Results are shown as means ± SEM. P-value: * < 0.05; *** < 0.001; (ANOVA), n = 4.

Fig 6. Immunofluorescence of gastrocnemius muscle in tourniquet cohort and operative clamping group.

Stainings of Caspase-3, 3-Nitrotyrosine and 4-Hydroxynonenal showed an enhanced signal (red) in mice which underwent the low-pressure tourniquet method compared to control and operative clamping. Levels of p38 were elevated in both the tourniquet and the operative clamping group. Illustrations show regions of 1000×1000 pixels each. Results are shown as means ± SEM. Scale bar: 40 μm. P-value: * < 0.05; *** < 0.001; (ANOVA), n = 4.