Ultrastructural demonstration of endothelial glycocalyx disruption in the reperfused rat heart. Involvement of oxygen free radicals.
To determine the effect of post-ischaemic reperfusion on the ultrastructure of the endothelial glycocalyx and the role of oxygen free radicals, isolated working rat hearts were subjected to 20 min ischaemia followed by 3 or 30 min of reperfusion. Ruthenium red and lanthanum chloride were used to delineate the endothelial glycocalyx, and histochemical manganese/diaminobenzidine (Mn+2/DAB) or iron/diaminobenzidine (Fe+2/DAB) techniques were applied to visualize superoxide and hydrogen peroxide in myocardial capillaries. We found that ischaemia alone led to only a slightly flocculent appearance of the glycocalyx and its disruption was not observed until the onset of reperfusion. Prolongation of reperfusion to 30 min had no further effect on the ultrastructure of the glycocalyx. The ultrastructure of endothelial cells was normal. The disruption of the glycocalyx correlated in time and place with the appearance of Mn+2/DAB and Fe+2/DAB reaction products on the luminal surface of endothelial cells. Treatment with 5 mM N-(2-mercaptopropionyl)-glycine (MPG), an .OH radical scavenger, starting before ischaemia prevented the disruption of the glycocalyx, while 100 mM 3-morpholinosydnonimine (SIN-1), capable of generating both NO and -O2 simultaneously when applied at the time of reperfusion, increased the mean density of capillaries positively stained with Mn+2/DAB and Fe+2/DAB, and caused substantial disruption of the glycocalyx and damage to endothelial cells, which was not prevented by MPG. Our results suggest that the onset of reperfusion is critical for injury to the endothelial glycocalyx. Most probably the hydroxyl radical derived from the Fenton reaction is responsible for this injury. Peroxynitrite and/or nitric dioxide, if present upon reperfusion, may also account for damage of the endothelial glycocalyx.
Czarnowska
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Department of Pathomorphology Child Health Center, Warsaw, Poland.
Karwatowska-Prokopczuk
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D000818:Animals; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000648:ultrastructure; D006023:Glycoproteins / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism*; D066298:In Vitro Techniques; D008297:Male; D008854:Microscopy, Electron; D009206:Myocardium / Q000378:metabolism* / Q000648:ultrastructure; D011134:Polysaccharides / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D015424:Reperfusion; D013481:Superoxides / Q000378:metabolism*
D006023:Glycoproteins; D011134:Polysaccharides; D013481:Superoxides; D006861:Hydrogen Peroxide