Evaluation with endothelial nitric oxide synthase (eNOS) immunoreactivity of the protective role of astaxanthin on hepatorenal injury of remote organs caused by ischaemia reperfusion of the lower extremities

Introduction
Ischemia and following reperfusion triggers local and systemic damage with the involvement of free oxygen radicals and inflammatory mediators. Although blood flow saves extremity from necrosis,multi organ dysfunction may progress and cause death of the patient.

Aim
The study aims to examine the effect of astaxanthin (AST) on the prevention of remote tissue injury resulting from lower extremity ischaemia–reperfusion (I/R). To elucidate the potential hepatoprotective and renoprotective effects of AST, in addition to histopathological findings, the intrahepatic and intrarenal kinetics of endothelial nitric oxide synthase (eNOS) during I/R were determined by using the immunohistochemical method.

Material and methods
Twenty-eight male Wistar albino rats were divided into four groups. For the control group, only the anaesthesia procedure (2 h) was conducted without I/R. In the I/R group, 2 h of reperfusion was conducted following ischaemia under anaesthesia. For the I/R group + AST, 7 days prior to ischaemia, 125 mg/kg AST was given with gavage, and 2 h of ischaemia and 2 h of reperfusion were conducted under anaesthesia. Following necropsy, liver and kidney tissue samples were fixed in 10% buffered formalin for 48 h for histopathological and immunohistochemical investigation.

Results
The histological analysis revealed that severe I/R hepatorenal injury such as inflammatory cell infiltration, dilatation in sinusoids and lumen of tubuli, congestion in glomerular capillaries, degeneration in hepatocyte and epithelial cells of tubuli, and necrosis was ameliorated by AST. Immunohistochemical studies showed that the I/R-induced elevation in eNOS expression was reduced by AST treatment.

Conclusions
In the case of acute lower extremity I/R, AST decreased the ischaemic injury in liver and renal tissues by protecting the microcirculation and providing a cytoprotective effect with vasodilatation.