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Cardiovascular Research 1993 27(3):371-376; doi:10.1093/cvr/27.3.371
© 1993 by European Society of Cardiology
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Copyright © 1993, European Society of Cardiology

Ultrastructural demonstration of peroxidative activity and peroxidation in ischaemic and ischaemic-reperfused rabbit hearts

Christopher P Mack, Kimberly M Brosamer and Marshal Shlafer

Department of Pharmacology, University of Michigan Medical School, M-6322 Medical Sciences Building 1, Ann Arbor, MI 48109-0626, USA: C P Mack, K M Brosamer, M Shlafer.

Correspondence to Dr Shlafer.

Objective: The aim was to characterise subcellular histochemical evidence of the involvement of peroxidation and peroxidases in myocardial reperfusion injury. The histochemical technique involved the use of 3,3'-diaminobenzidine (DAB), which reacts with peroxides and proteins with peroxidase activity to form an electron dense polymer. Methods: Isolated rabbit hearts were perfused (Langendorff method) for 30 min with oxygenated physiological saline solution. Some were subjected to 30 min of normothermic global ischaemia, with or without 30 min reperfusion. Non-ischaemic control hearts were perfused continuously for 90 min. Hearts were fixed with glutaraldehyde and cut into 100-150 µm sections that were incubated for 1 h in buffered DAB (1 mg·ml–1) with or without added KCN or H2O2. They were processed further for transmission electron microscopy. Planimetry was done on micrographs taken from random fields ({approx}500 photos). Results: The total amount of DAB polymer in non-ischaemic control heart sections incubated with DAB alone occupied l.19(SEM 0.44) (µ2·1000 µ–2 total cell area. For ischaemic-nonreperfused hearts, the value was 2.32(0.90) µ2·1000 µ–2 (p=0.223 v control); DAB occupied 7.49(1.42) µ2·1000 µ–2 in ischaemic-reperfused hearts (p=0.001 v control). DAB positive staining of mitochondria and lipid droplets, but not of peroxisomes, was significantly increased in reperfused hearts compared with non-ischaemic controls. Conclusions: Reperfusion, but not ischaemia, was associated with increased DAB staining. This suggests a reperfusion induced increase in myocyte peroxidation. Increased staining may be due to the actions of haem proteins with peroxidase activity on peroxidised lipid.

Cardiovascular Research 1993;27:371-376

KEYWORDS diaminobenzidine; histochemistry; ischaemia-reperfusion; lipids; myocardium; oxidants; peroxidases; peroxides; rabbit; ultrastructure

This work was supported by grant HL-29499 from the National Institutes of Health. We thank Mr Steven Schneider and the Encotec Company of Ann Arbor for assaying the iron contents of our buffers.


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