A Novel Laser-Doppler Flowmetry Assisted Murine Model of Acute Hindlimb Ischemia-Reperfusion for Free Flap Research

Tolga Taha Sönmez,René Tolba,Elisa Anamaria Liehn,Nancy Tuchscheerer,Frank Hölzle,Mersedeh Tohidnezhad,David Mitchell,Isabella Kanzler,Thomas Pufe,Gerald Brandacher,Anastasios Kanatas,Christoph Jan Wruck,Matthias Knobe,Athanassios Fragoulis,Othman Al-Sawaf,Utpal Sen

doi:10.1371/journal.pone.0066498

Abstract

Suitable and reproducible experimental models of translational research in reconstructive surgery that allow in-vivo investigation of diverse molecular and cellular mechanisms are still limited. To this end we created a novel murine model of acute hindlimb ischemia-reperfusion to mimic a microsurgical free flap procedure. Thirty-six C57BL6 mice (n = 6/group) were assigned to one control and five experimental groups (subject to 6, 12, 96, 120 hours and 14 days of reperfusion, respectively) following 4 hours of complete hindlimb ischemia. Ischemia and reperfusion were monitored using Laser-Doppler Flowmetry. Hindlimb tissue components (skin and muscle) were investigated using histopathology, quantitative immunohistochemistry and immunofluorescence. Despite massive initial tissue damage induced by ischemia-reperfusion injury, the structure of the skin component was restored after 96 hours. During the same time, muscle cells were replaced by young myotubes. In addition, initial neuromuscular dysfunction, edema and swelling resolved by day 4. After two weeks, no functional or neuromuscular deficits were detectable. Furthermore, upregulation of VEGF and tissue infiltration with CD34-positive stem cells led to new capillary formation, which peaked with significantly higher values after two weeks. These data indicate that our model is suitable to investigate cellular and molecular tissue alterations from ischemia-reperfusion such as occur during free flap procedures.

Highlights

Microsurgical composite tissue transfer has become the gold standard in reconstructive surgery [1,2]
ische miareperfusion injury (IRI) and post-ischemic reperfusion lead to acute vascular leakage and to nerve and skeletal muscle damage [3,5]
Post-ischemic reperfusion causes a burst in reactive-oxygen-species (ROS), alterations in mitochondrial function, inflammation, and tissue necrosis [6,7,8]

Summary

Introduction

Microsurgical composite tissue transfer has become the gold standard in reconstructive surgery [1,2]. IRI and post-ischemic reperfusion lead to acute vascular leakage and to nerve and skeletal muscle damage [3,5]. Post-ischemic reperfusion causes a burst in reactive-oxygen-species (ROS), alterations in mitochondrial function, inflammation, and tissue necrosis [6,7,8]. It still remains a challenge to replicate the best research conditions in murine hindlimb models, which can mimic the perioperative situation of microsurgical composite tissue transfer. Sufficient occlusion of all branches of the femoral and iliac vessels, including the rich collateral blood supply from iliac and tail branches of the hindlimb, is essential in order to induce complete, acute ischemia. Even as sufficient occlusion of hindlimb vessels is assured, care must be taken to avoid the irreversible tissue damage and neuromuscular deficits which an uncontrolled crushing force can cause

Methods

Results

Discussion

Conclusion