Demonstration of the temporal evolution of serum markers of skeletal muscle ischaemia reperfusion injury - An observational clinical study.

Objective The study aimed to investigate the temporal evolution of markers of skeletal muscle ischaemia reperfusion injury (IRI) to shed more light into its pathogenesis towards finding a clinical therapeutic intervention. P-selectin, Myeloperoxidase and TNF-alpha were selected as markers of IRI based on their role in its pathogenesis. An observational study was conducted on patients with skeletal muscle ischaemia requiring revascularization. Loco-regional venous sampling was performed at 0, +2, +4, +6, +12 and +24 hours post-reperfusion to demonstrate the temporal evolution of surrogate markers of IRI. Results Levels of P-Selectin, Myeloperoxidase and TNF-alpha demonstrated a significant peak elevation during 2h - 4h following reperfusion when compared to baseline values. (P-selectin 59.48pg/ml to 125.91pg/ml, p=0.008; Myeloperoxidase 78.84pg/ml to 116.86pg/ml, p=0.051; TNF-alpha 1.80pg/ml to 34.53pg/ml, p=0.002) All three markers gradually subsided with time and reached baseline pre-perfusion values between 6h – 8h from time of reperfusion. The study suggests that skeletal muscle IRI in humans is associated with an early exaggerated pro-inflammatory response, as supported by increased expression of reperfusion injury markers in the venous effluent. They demonstrate potential for use as markers of IRI for future clinical trials.


Introduction
Acute limb ischemia remains one of the most common peripheral vascular emergencies and its management continues to pose significant challenges 1  3 The pathogenesis of skeletal muscle ischaemia reperfusion injury(IRI) is multifactorial, and not completely unravelled. Accumulation of hypoxanthine, imbalance of calcium homeostasis, no-reflow phenomenon, increased leucocyte activation has been suggested.
Amongst these the polymorphonuclear leukocyte-endothelial interaction forms an integral component. Leukocyte activation is a multistep process involving rolling, firm adhesion and transmigration mediated by a complex interaction of cell adhesion molecules 4 . Once activated, the neutrophils release highly toxic reactive oxygen species, resulting in cellular damage 5 .
The systematic search for serological markers of skeletal muscle ischemia reperfusion has identified the leucocyte-endothelial interaction as a key step in its pathogenesis, with potential targets for therapeutic interventions. TNF-alpha, Myeloperoxidase (MPO) and soluble P-Selectin are highly sensitive surrogate markers of IRI that are involved in the enhanced leucocyte activity and pro-inflammatory pathogenesis. However, despite the strong preclinical data on neutrophil-endothelial blockade, this success hasn't yet translated to a clinically valid outcome. To the best of our knowledge there are no studies to-date that have attempted to establish whether these markers of reperfusion injury actually do increase following reperfusion of ischaemic skeletal muscle in a clinical setting. Nor do we have any evidence to suggest the timeframe that these markers are observed in the reperfusate.
The identification of the temporal evolution of highly sensitive surrogate markers of skeletal muscle IRI will shed more light into dissecting the pathogenesis of IRI and would be essential towards finding a clinical therapeutic intervention.
The study involves, demonstration of the temporal evolution of P-Selectin, myeloperoxidase and TNF-alpha in the venous effluent following reperfusion. We therefore postulate that P-selectin, Myeloperoxidase and TNF-alpha are suitable markers of reperfusion injury in skeletal muscle ischaemia reperfusion injury in a clinical setting.

Methodology
An observational study was carried out on patients admitted to the University of Colombo, Vascular Unit at the National Hospital of Sri Lanka with acute lower limb arterial occlusion requiring revascularization.
Patients for whom amputation rather than revascularization was indicated and patients below the age of 18 years, were excluded. A peripheral venous sample was obtained immediately prior to surgery as baseline. Prior to revascularization an indwelling femoral venous catheter was inserted and placed for a period of 24 hours for sampling the venous effluent. Subsequently, loco-regional venous sampling was done using the indwelling femoral venous catheter at 0, +2, +4, +6, +12 and +24 hours post-reperfusion to establish a temporal evolution of the reperfusion injury markers in the venous effluent. Loco-regional venous sampling with an indwelling venous catheter as opposed to peripheral venous sampling for 24 hours has been shown to be safe and advantageous during previous studies. Rowlands T et al 13 Table 2.
There was a significant increase in P-selectin level over the time frame, reaching a peak  Table 3. This P-selectin is one of three structural families of adhesion molecules that is responsible for leukocyte adhesion, penetration of the vessel wall, and trans-endothelial migration into tissue 20 . Myeloperoxidase, an enzyme present in leukocytes, is a marker of tissue leukocyte activity and infiltration 21 . Both P-selectin and Myeloperoxidase levels were elevated in the venous effluent following reperfusion of skeletal muscle. A peak level was 8 noted at 2h-4h after reperfusion, before it gradually subsided after 6h -8 hours. The results confirm the exaggeration of endothelial-leukocyte interaction and subsequent leukocyte activation in human skeletal muscle reperfusion injury.
Tumor necrosis factor is an inflammatory cytokine that is expressed by macrophages and other tissue early during ischemia-reperfusion injury 22 . Unlike the two previous markers, the role of TNF-alpha in skeletal muscle reperfusion injury is controversial. While some groups report beneficial effects, others find a detrimental role 23 .
TNF-alpha clearly initiates expression of an entire spectrum of inflammatory cytokines that is crucial towards activation of neutrophils as well as the complement cascade. Levels of TNF-alpha in the venous effluent increased dramatically on reperfusion, with a peak value at two hours after blood flow was restored. Results affirm, that lower extremityreperfusion in humans is associated with an exaggerated inflammatory response, as supported by increased expression TNF alpha, P-Selectin and Myeloperoxidase in the venous effluent.
The results of the study, have demonstrated that lower extremity-reperfusion in humans is associated with an exaggerated inflammatory response, as supported by increased expression of soluble P-selectin , myeloperoxidase and TNF alpha in the venous effluent. This is the first clinical study to demonstrate the temporal evolution of these markers in the venous effluent after skeletal muscle ischaemia reperfusion injury. Whilst their utility as a diagnostic tool is limited, they reflect injury severity and potential for use as markers of IRI in a more robust clinical trial.

Limitations
Our study has several limitations. There was heterogeneity in the patient population with patients with both thromboembolism induced and clamp-induced acute lower limb arterial 9 occlusions included in the study. This is reflected in the variation of baseline levels of the surrogate markers. The three markers reflect key steps in the pathogenesis of IRI, but remain indirect evidence. We were unable to obtain consent for immunohistochemical assessment of skeletal muscle to correlate with the temporal evolution of markers. Having histological samples would have aided in understanding the disease pathogenesis better. Informed, written consent was obtained from all participants prior to inclusion in the study.

Consent for publication
Not applicable.