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

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

Attenuation of Skeletal Muscle and Renal Injury to the Lower Limb following Ischemia-Reperfusion Using mPTP Inhibitor NIM-811.

INTRODUCTION: Operation on the infrarenal aorta and large arteries of the lower extremities may cause rhabdomyolysis of the skeletal muscle, which in turn may induce remote kidney injury. NIM-811 (N-metyl-4-isoleucine-cyclosporine) is a mitochondria specific drug, which can prevent ischemic-reperfusion (IR) injury, by inhibiting mitochondrial permeability transition pores (mPTP). OBJECTIVES: Our aim was to reduce damages in the skeletal muscle and the kidney after IR of the lower limb with NIM-811. MATERIALS AND METHODS: Wistar rats underwent 180 minutes of bilateral lower limb ischemia and 240 minutes of reperfusion. Four animal groups were formed called Sham (receiving vehicle and sham surgery), NIM-Sham (receiving NIM-811 and sham surgery), IR (receiving vehicle and surgery), and NIM-IR (receiving NIM-811 and surgery). Serum, urine and histological samples were taken at the end of reperfusion. NADH-tetrazolium staining, muscle Wet/Dry (W/D) ratio calculations, laser Doppler-flowmetry (LDF) and mean arterial pressure (MAP) monitoring were performed. Renal peroxynitrite concentration, serum TNF-alpha and IL-6 levels were measured. RESULTS: Less significant histopathological changes were observable in the NIM-IR group as compared with the IR group. Serum K+ and necroenzyme levels were significantly lower in the NIM-IR group than in the IR group (LDH: p<0.001; CK: p<0.001; K+: p = 0.017). Muscle mitochondrial viability proved to be significantly higher (p = 0.001) and renal function parameters were significantly better (creatinine: p = 0.016; FENa: p<0.001) in the NIM-IR group in comparison to the IR group. Serum TNF-alpha and IL-6 levels were significantly lower (TNF-alpha: p = 0.003, IL-6: p = 0.040) as well as W/D ratio and peroxynitrite concentration were significantly lower (p = 0.014; p<0.001) in the NIM-IR group than in the IR group. CONCLUSION: NIM-811 could have the potential of reducing rhabdomyolysis and impairment of the kidney after lower limb IR injury

Cryopreservation moderates the thrombogenicity of arterial allografts during storage

Introduction: Management of vascular infections represents a major challenge in vascular surgery. The use of cryopreserved vascular allografts could be a feasible therapeutic option, but the optimal conditions for their production and use are not precisely defined. Aims To evaluate the effects of cryopreservation and the duration of storage on the thrombogenicity of femoral artery allografts. Methods In our prospective study, eleven multi-organ-donation-harvested human femoral arteries were examined at five time points during storage at -80C: before cryopreservation as a fresh native sample and immediately, one, twelve and twenty-four weeks after the cryopreservation. Cross-sections of allografts were perfused with heparin-anticoagulated blood at shear-rates relevant to medium-sized arteries. The deposited platelets and fibrin were immunostained. The thrombogenicity of the intima, media and adventitia layers of the artery grafts was assessed quantitatively from the relative area covered by fibrin- and platelet-related fluorescent signal in the confocal micrographs. Results Regression analysis of the fibrin and platelet coverage in the course of the 24-week storage excluded the possibility for increase in the graft thrombogenicity in the course of time and supported the hypothesis for a descending trend in fibrin generation and platelet deposition on the arterial wall. The fibrin deposition in the cryopreserved samples did not exceed the level detected in any of the three layers of the native graft. However, an early (up to week 12) shift above the native sample level was observed in the platelet adhesion to the media. Conclusions The hemostatic potential of cryopreserved arterial allografts was retained, whereas their thrombogenic potential declined during the 6-month storage. The only transient prothrombotic change was observed in the media layer, where the platelet deposition exceeded that of the fresh native grafts in the initial twelve weeks after cryopreservation, suggesting a potential clinical benefit from antiplatelet therapy in this time-window

Postconditioning Protects Skeletal Muscle Against a Long-Lasting Vascular Occlusion

ABSTRACT Purpose/Aim of the Study: Long-lasting lower limb arterial occlusion is a condition with high incidence and complication rates. With the absence of appropriate treatment to cure advanced complications, mortality rates are high. Postconditioning (PC) might be capable of limiting the degree of ischemic-reperfusion (IR) injuries, thus reducing complications and mortality rates. The aim of this study was to evaluate the impact of postconditioning during the first postoperative day on skeletal muscle after a long-lasting arterial occlusion. Materials and Methods: Male Wistar rats (n = 72) underwent 8 hr of infrarenal aortic occlusion followed by 2, 6, 12, or 24 hr of reperfusion. In one group of each reperfusion period, postconditioning was applied. Muscle samples were collected for histological examinations. Furthermore, muscle fiber viability and muscle wet-to-dry ratio were assessed. Blood samples were taken for creatine-kinase measurements. Results: Postconditioning strongly reduced morphological injury compared to the corresponding ischemic-reperfusion group (p < .001). Serum creatine-kinase levels showed a peak at 6 hr post-ischemia (IR: 6702.2 +/- 797.5; PC: 5523.3 +/- 769.3 IU/l) and decreased to normal level by the end of the experiment (Sham: 171.5 +/- 71.6; IR: 186.2 +/- 82.7; PC: 174.2 +/- 72.4 IU/l). Creatine-kinase levels were significantly reduced by postconditioning (p2hr = .028; p6hr = .06; p12hr = .042). A marked decrease in viability was observed in the ischemic-reperfusion groups (2 hr: 11.0 +/- 4.1; 6 hr: 10.3 +/- 3.6; 12 hr: 9.4 +/- 3.3; 24 hr: 8.6 +/- 2.8%), whereas with postconditioning, viability was preserved (2 hr: 26.4 +/- 5.5; 6 hr: 24.6 +/- 4.5; 12 hr: 24.5 +/- 6.8; 24 hr: 26.2 +/- 6.1%; p < .001); moreover, a significant decrease in the wet-to-dry ratio was achieved (p < .001). Conclusion: Postconditioning was able to reduce local complications after a long-lasting lower limb vascular occlusion