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

Cross-linked hemoglobin (alpha alpha-Hb) and polyethylene glycol (PEG)-conj ugated Hb have both been considered as possible "blood substitutes." Previo usly, we showed that PEG-Hb extravasates rapidly in the intestinal mucosa a nd 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 microvascul ar leakage to albumin and to investigate possible changes in the integrity of the interendothelial cell junctions and the endothelial actin cytoskelet on. In anesthetized Sprague-Dawley rats, the microvasculature of a mesenter ic 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 mi n. Controls (n = 4) just received HBS-BSA. In some experiments (n = 9 for a lpha alpha-Hb; n = 5 for PEG-Hb), the perfusate was then replaced by FITC-a lbumin in HBS-BSA for the next 3 min. The vasculature was then perfusion fi xed, stained for filamentous actin and for mast cells, and viewed microscop ically. In the remaining experiments, the mesenteric microvasculature was s tained with silver nitrate to determine the number of endothelial junctiona l gaps per length of venules. Both Hbs increased the number and area of lea ks per micrometer of venular length compared with control, but alpha alpha- Kb increased to a greater extent than PEG-Hb. Formation of leaks was accomp anied by changes in the endothelial actin cytoskeleton and by an increased number of endothelial gaps. Mast cell degranulation was significantly great er (P < 0.05) in Hb-treated preparations compared with controls, but there was no direct correlation between sites of degranulation and albumin leakag e. These Hbs appear to induce venular leakage in the mesentery by mechanism s similar to those previously observed after treatment with histamine or ni tric oxide synthase inhibitors.