Ischaemia and reperfusion effects on skeletal muscle tissue: morphological and histochemical studies

Abstract

This was a study on the oxidative stress due to ischaemia (I) and reperfusion (R) in skeletal muscle tissue. Using a tourniquet, groups of rats were submitted to ischaemia for 4 h, followed by different reperfusion periods. The animals were divided in four groups: control; 4 h of ischaemia (IR); 4 h of ischaemia plus 1 h reperfusion (IR-1 h); 4 h of ischaemia plus 24 h reperfusion (IR-24 h); and 4 h of ischaemia plus 72 h reperfusion (IR-72 h). At the end of the procedures, samples of soleus muscle were collected and frozen in n-hexane at -70 degrees C. Cryostat sections were submitted to haematoxylin-eosin, succinate dehydrogenase (SDH) and nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR) stains. An additional muscle sample was processed for electron microscopy. No alterations were found in control animals. IR group showed fibres had normal aspect besides some round, acidophilic and hypertrophic fibres. There were several fibres with angular outlines and smaller diameters in this group compared with control group. NADH-TR/SDH reaction was moderately intense in most fibres. In some fibres, cytoplasm showed areas without activity and other fibres had very intense reactivity. IR-1 h group showed oedema hypercontracted fibres with disorganized myofibrils, mitochondria with focal lesions and dilated sarcoplasmic reticulum. NADH-TR/SDH reaction was moderate to weak. IR-24 h showed intense inflammatory infiltrate in the endomysium and perimysium. NADH-TR/SDH reaction was similar to IR-1 h. IR-72 h showed necrotic fibres, areas with inflammatory infiltrate, reduced muscle fibres at different stages of necrosis and phagocytosis, and many small round and basophilic fibres characterizing a regeneration process. NADH-TR/SDH reaction was weak to negative. Our results suggest that ischaemia and the subsequent 1-, 24- and 72-h reperfusions induced progressive histological damage. Although progressive, it may be reversible because there were ultrastructural signs of recovery after 72-h reperfusion. This recovery could in part be due to the low oxidative stress identified by the morphological and histochemical analysis.

Figure 1

Soleus muscle: a and b (control). Normal muscle fibres (F). Peripheral nucleus (N). Endomysium (E). Haematoxylin–eosin (HE) 40×; b, oxidative (O) and glycolytic fibres (G). SDH 40×; c (ischaemia of 4 h). Round hypertrophic fibre (H). Nucleus (N). Endomysium (E). HE 40×; d (ischaemia of 4 h). Irregular distribution of oxidative metabolism enzymes: formazan rough granulations (arrows). Oedema (E), NADH-TR, 40×.

Figure 2

Soleus muscle. a (IR-1 h). Round (F). Hypercontracted fibres (h). Fibre with myofibril disorganization (arrows). Oedema (E). Haematoxylin–eosin (HE) 40×; b (IR-1 h). Round fibres with irregular distribution of oxidative metabolism enzymes (granulations) (arrows). Lack of enzyme activity (*). NADH-TR 40×; c (IR-24 h). Intense inflammatory infiltrate (I). Oedema (E). Necrotic fibres (*). HE, 40×, d (IR-72 h). Necrotic fibre (F). Small basophilic fibres with central nucleus (arrows). HE, 100×.

Figure 3

a, b. Soleus muscle (control). Muscle fibres with normal aspects. Nucleus (N). Mitochondria (m). Myofibrils showing sarcomers with Z, A and I lines. a: 21,000× and b: 27,000×.

Figure 4

Soleus muscle. a (ischaemia of 4 h). Myofibrils (Mi). Normal mitochondria (m). Edematous mitochondria with reduced cristae (*). 21,000×. b (IR-1 h). Disorganized myofibrils (Mi). Mitochondria with electron-dense material clusters in their interior (m). Fibre area with no myofibrils (*). Swelling vesicles (V). 27,000×. c (IR-24 h). Disorganized myofibrils (Mi). Electron-dense material clusters in mitochondria (m). Mitochondria with reduced cristae (*). 14,500×. d (IR-72 h). Multinucleated muscle fibre in regeneration. Nuclei (N), mitochondria (arrows). Contractile myofilaments (arrow head), 7250×.