Attenuation of changes in capillary fine structure and leukocyte adhesion improves muscle performance following chronic ischaemia in rats

Abstract

Acute ischaemia-reperfusion disrupts capillary fine structure and increases leukocyte adhesion in postcapillary venules. We determined whether chronic muscle ischaemia has similar consequences, and whether it is possible to ameliorate its effect on muscle performance. Following ischaemia (unilateral ligation, common iliac artery) rat hindlimb muscles were examined without other intervention or following treatment with an xanthine oxidase inhibitor (allopurinol), a Na(+)/H(+) exchange blocker (amiloride), or an oxygen free radical scavenger (vitamin E). No significant leukocyte adhesion or rolling, nor changes in capillary fine structure were observed 3 days postsurgery, when limb use was limited. However, leukocyte rolling and adhesion almost trebled by 7 days (P < 0.001), when normal gait was largely restored. Capillary fine structure was disturbed over a similar time course, e.g. relative endothelial volume (control 46%, 7 days 61%; P < 0.05), that resolved by 5 weeks. Where activity was increased by mild electrical stimulation 3 days after ligation muscles showed enhanced capillary swelling (endothelial volume 66% versus 50%, P < 0.005), but improved fatigue index (52% versus 16%, P < 0.001) as a result of greater blood flow. Muscle fatigue after ligation was related to the extent of contraction-induced hyperaemia (R(2) = 0.725), but not capillary swelling. Amiloride, and to a lesser extent allopurinol but not vitamin E, significantly decreased leukocyte rolling and adhesion, as well as capillary endothelial swelling. We conclude that increased activity of ischaemic muscles on recovery is likely to accentuate acidosis accompanying changes in microcirculation and contribute to enhanced muscle fatigue, whereas formation of oxygen free radicals may be attenuated by endogenous protective mechanisms.

Figure 1. Muscle blood flow (MBF) and fatigue in combined EDL + TA muscles at rest and at the end of 5 min isometric contractions in 3 days ischaemic (3d lig) and 3 days ischaemic with 2 days stimulation (3d SL)

Fatigue index (FI) is the percentage of the peak tension (PT) at the end of the 5 min period of contractions. * denotes values significantly different from 3 days lig (P < 0.05). For control data, please refer to Table 1.

Figure 2. Leukocyte rolling and adhesion in untreated 7 days ischaemic muscles, and ischaemic muscles in animals treated with amiloride, allopurinol or vitamin

*P < 0.05, **P < 0.01 versus ischaemia alone. For comparison, in untreated control muscles the corresponding vales are: V₃ rolling 3.0 ± 1.7, stationary 0.4 ± 0.4; V₂ rolling 9.3 ± 3.0, stationary 1.4 ± 0.6 min⁻¹ (Anderson et al. 2006).

Figure 3. Changes in capillary fine structure with ischaemia

A, electron micrograph illustrating the normal capillary phenotype from a control muscle showing the thin endothelium and smooth luminal and abluminal surfaces. B, a capillary with a swollen endothelium from a muscle 7 days after ligation of the iliac artery, where endothelial protrusions obscure the lumen. C, a capillary with a swollen endothelium from a muscle after 7 days ligation treated with amiloride, where the endothelial swelling is reduced sufficiently to allow likely passage of an erythrocyte. Abbreviations: E, erythrocyte; EC, endothelial cell; L, lumen; P, pericyte. Scale bar = 1.0 μm.

Figure 4. Capillary indices used to evaluate endothelial swelling on the basis of quantitative electron microscopy in control and ischaemic rat EDL

Abbreviations: cyto, endothelial cytoplasm; S/V, surface to volume ratio; Vv, volume density. *P < 0.05, **P < 0.01 versus control; †P < 0.05 versus 3d stimulation alone.

Figure 5. Several parameters used for evaluation of capillary endothelial swelling expressed as a ratio between the values for contralateral and ischaemic muscles following drug treatment

*P < 0.05 versus control; †P < 0.05 versus vitamin E.

Figure 6. Capillary indices used to evaluate endothelial swelling in papillary muscle

*P < 0.05, **P < 0.01 versus control.

Figure 7. The relationship between muscle fatigue resistance and muscle blood flow during contractions

Mean values are given for groups of animals at different times after ligation. Circles, ipsilateral muscle; triangles, contralateral muscle. Filled symbols represent 3d SL. Linear regression of group means give FI = 31.482 + 0.301 × MBF, R²= 0.725 (ipsilateral), and FI = 63.645 + 0.008 × MBF, R²= 0.017 (contralateral).