Sepsis increases accumulation of cell-free hemoglobin in intima of submucosal arterioles in rats

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Introduction

It was reported from in vitro studies that interruption of the endothelial barrier increases endothelial permeability for cell-free hemoglobin solutions[1]. We studied the effects of sepsis on the abluminal distribution of intravenously infused cell-free hemoglobin (DCLHb, Baxter Corp.) in submucosa of rat ileum.

Methods

Rats were randomized to sham laparotomy (n=6) or cecal ligation and perforation (CLP, n=7) to induce sepsis. Twenty-four h later, 300 mg of DCLHb labeled with Evans-Blue dye was infused. The laparotomy was reopened, and material was obtained from the ileum 90 min after DCLHb infusion for histological examination. Using fluorescence microscopy, the concentration of cell-free hemoglobin in intima and smooth muscle of submucosal arterioles and in submucosal tissue was graded by two blinded investigators using a score (0, no; 1, minor; 2, moderate; 3, strong; 4, very strong fluorescence). Additional studies (n=3) were performed to exclude that the tracer alone accounted for the observed effects.

Results

DCLHb fluorescence scores were low in submucosal tissue and in smooth muscle of arterioles for both groups (all <0.75 points.). In the intima of arterioles, considerable fluorescence was observed, with scores increased in the septic group as compared to the sham group (3.2±0.5 vs 1.4±0.4 points, mean ±SEM, P<0.05). Infusion of Evans-Blue dye alone resulted in scores <0.75 points in all tissues.

Conclusion

Sepsis increased the accumulation of cell-free hemoglobin in the intima of submucosal arterioles. This finding may be relevant for the previously documented effects of cell-free hemoglobin to modify perfusion pressure and microvascular blood flow in sepsis [2].