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Am J Physiol Heart Circ Physiol 277: H650-H659, 1999;
0363-6135/99 $5.00
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Vol. 277, Issue 2, H650-H659, August 1999

Modified hemoglobins produce venular interendothelial gaps and albumin leakage in the rat mesentery

Ann L. Baldwin

Department of Physiology, College of Medicine, University of Arizona, Tucson, Arizona 85724-5051

Cross-linked hemoglobin (alpha alpha -Hb) and polyethylene glycol (PEG)-conjugated Hb have both been considered as possible "blood substitutes." Previously, we showed that PEG-Hb extravasates rapidly in the intestinal mucosa and causes transient epithelial sloughing, resulting in temporary opening of the intestinal epithelial barrier. In the present study, the rat mesenteric preparation was used to quantify the effects of the two Hbs on microvascular leakage to albumin and to investigate possible changes in the integrity of the interendothelial cell junctions and the endothelial actin cytoskeleton. In anesthetized Sprague-Dawley rats, the microvasculature of a mesenteric window was perfused with HEPES-buffered saline (HBS) containing 0.5 mg/ml BSA and 2 mg/ml alpha alpha -Hb (n = 16) or PEG-Hb (n = 5) for 2 or 10 min. Controls (n = 4) just received HBS-BSA. In some experiments (n = 9 for alpha alpha -Hb ; n = 5 for PEG-Hb), the perfusate was then replaced by FITC-albumin in HBS-BSA for the next 3 min. The vasculature was then perfusion fixed, stained for filamentous actin and for mast cells, and viewed microscopically. In the remaining experiments, the mesenteric microvasculature was stained with silver nitrate to determine the number of endothelial junctional gaps per length of venules. Both Hbs increased the number and area of leaks per micrometer of venular length compared with control, but alpha alpha -Hb increased to a greater extent than PEG-Hb. Formation of leaks was accompanied by changes in the endothelial actin cytoskeleton and by an increased number of endothelial gaps. Mast cell degranulation was significantly greater (P < 0.05) in Hb-treated preparations compared with controls, but there was no direct correlation between sites of degranulation and albumin leakage. These Hbs appear to induce venular leakage in the mesentery by mechanisms similar to those previously observed after treatment with histamine or nitric oxide synthase inhibitors.

blood substitutes; actin cytoskeleton; silver nitrate; microscopy


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