Muscle Fiber Viability, a Novel Method for the Fast Detection of Ischemic Muscle Injury in Rats

Zsolt Turóczi,Péter Arányi,Ákos Lukáts,László Harsányi,Attila Szijártó,Gábor Lotz,Dávid Garbaisz,Peter Rosenberger

doi:10.1371/journal.pone.0084783

Abstract

Acute lower extremity ischemia is a limb- and life-threatening clinical problem. Rapid detection of the degree of injury is crucial, however at present there are no exact diagnostic tests available to achieve this purpose. Our goal was to examine a novel technique - which has the potential to accurately assess the degree of ischemic muscle injury within a short period of time - in a clinically relevant rodent model. Male Wistar rats were exposed to 4, 6, 8 and 9 hours of bilateral lower limb ischemia induced by the occlusion of the infrarenal aorta. Additional animals underwent 8 and 9 hours of ischemia followed by 2 hours of reperfusion to examine the effects of revascularization. Muscle samples were collected from the left anterior tibial muscle for viability assessment. The degree of muscle damage (muscle fiber viability) was assessed by morphometric evaluation of NADH-tetrazolium reductase reaction on frozen sections. Right hind limbs were perfusion-fixed with paraformaldehyde and glutaraldehyde for light and electron microscopic examinations. Muscle fiber viability decreased progressively over the time of ischemia, with significant differences found between the consecutive times. High correlation was detected between the length of ischemia and the values of muscle fiber viability. After reperfusion, viability showed significant reduction in the 8-hour-ischemia and 2-hour-reperfusion group compared to the 8-hour-ischemia-only group, and decreased further after 9 hours of ischemia and 2 hours of reperfusion. Light- and electron microscopic findings correlated strongly with the values of muscle fiber viability: lesser viability values represented higher degree of ultrastructural injury while similar viability results corresponded to similar morphological injury. Muscle fiber viability was capable of accurately determining the degree of muscle injury in our rat model. Our method might therefore be useful in clinical settings in the diagnostics of acute ischemic muscle injury.

Highlights

Acute long-lasting arterial occlusions represent serious clinical problems due to their frequent occurrence and severe complications [1]
Microcirculation Microcirculatory flow was assessed throughout the ischemic period in all groups to evaluate whether this model is able to produce continuous ischemia without significant variations in flow
The flow dropped to 17.367.3% of the baseline flow and remained constant during the course of ischemia even 9 hours after the occlusion (16.569.2% of baseline)

Summary

Introduction

Acute long-lasting arterial occlusions represent serious clinical problems due to their frequent occurrence (incidence: 15/100000) and severe complications [1]. Even recent studies put postoperative limb loss between 10 and 30%, and post-operative mortality between 10–20% [1,2,3], both of which are direct consequences of the severe ischemic-reperfusion injury to the extremities. The Rutherford classification is widely used for staging (Stages I– III) the severity of acute ischemic injury of the limbs in the clinical practice [4], designed to determine the urgency of a revascularization procedure [5]. Assessment of the degree of ischemic injury within a clinically relevant time-frame still remains unsolved. Rapid determination of the precise degree of ischemic injury is of great clinical importance [6], whereas revascularization of a severely injured extremity might aggravate complication rates and mortality. In case of irreversible injury amputation is the only solution to avoid serious life threatening complications [7]

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Discussion

Conclusion