Increased expression of inducible nitric oxide synthase (iNOS) has been repeatedly demonstrated in rodent models of sepsis, and is thought to account for the marked increase in nitric oxide metabolites also seen in these models. In human sepsis nitric oxide metabolites show only a modest increase, and to date only one report of a study of three septic patients (Stoclet et al. 1999) has demonstrated iNOS expression in arterial tissue. In this study iNOS was demonstrated in the intima, media and adventitia. We wished to confirm or refute these findings.

Specimens of mesenteric artery were obtained from four patients undergoing resection of perforated large bowel. These patients were suffering from septic shock (as defined by the American Society of Chest Physicians/Society of Critical Care Medicine) due to peritonitis and required admission to the ICU post-operatively. Control specimens were obtained from three non-septic patients undergoing resection of colonic neoplasms. The need for specific patient consent was waived by the local ethics committee.

The arterial specimens were washed in 10 mM PBS and fixed in 4 % paraformaldehyde in 10 mM PBS. Following overnight desiccation in 20 % sucrose in 10 mM PBS, specimens were frozen in OCT at -70°C. Subsequently 6 µm sections were cut onto electrostatically coated slides. Immunohistochemistry was performed using a standard technique (Buttery & Polak, 2000) with the following modifications. Non-specific binding was blocked with 10 % goat serum. Polyclonal rabbit anti-iNOS (Santa Cruz) was used as the primary antibody, and TRITC-labelled polyclonal goat anti-rabbit IgG (Santa Cruz) was used as the secondary antibody. Fluorescence was stimulated at 568 nm. Control tissue was processed in the absence of the primary and/or the secondary antibody. Microscopy settings were identical for each batch of tissue and its relevant controls.

Autofluorescence was seen in the internal elastic lamina and adventitia of control slides. In all four patients with septic shock, inducible nitric oxide synthase (iNOS) was identified in the media (Fig. 1). At higher magnification, iNOS appeared to be present throughout the cytoplasm of the smooth muscle cells. Unfortunately, endothelium was not well preserved in these tissue specimens. In two out of three non-septic control patients, no iNOS was visualised in the media. In the other control patient, iNOS was visualised in the media. This patient had received pre-operative chemotherapy.

In conclusion, iNOS is strongly expressed in vascular smooth muscle in human septic shock. Future work will quantify protein expression using immunoblotting.

Figure 1. Confocal image of iNOS staining in human vascular smooth muscle. Scalebar = 100 µm.

Buttery, L.D.K. & Polak, J.M. (2000). Septic Shock Methods and Protocols, pp. 145-157. Humana Press Inc., Totowa, NJ, USA.

Stoclet, J.C., Martinez, M.C., Ohlmann, P., Chasserot, S., Schott, C., Kleschyov, A.L., Schneider, F. & Andriantsitohaina, R. (1999). Circulation 100, 107-112.