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Cold exposure differentially stimulates angiogenesis in glycolytic and oxidative muscles of rats and hamsters
- D. Deveci, S. Egginton
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- Journal:
- Experimental Physiology / Volume 88 / Issue 6 / November 2003
- Published online by Cambridge University Press:
- 07 November 2003, pp. 741-746
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- November 2003
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Control (normothermic) and cold-acclimated (environmental temperature gradually reduced from 20 to 5 °C for 4 weeks) groups of male rats and hamsters were compared to elucidate the nature of angiogenesis in oxidative and glycolytic muscles of these species during progressive cold exposure. Skeletal muscle capillarity and fibre cross-sectional area were measured in the tonic soleus (SOL) and phasic tibialis anterior (TA). Cryostat sections were stained for alkaline phosphatase (ALP) activity to identify all capillaries, and proliferating cell nuclear antigen (PCNA) to localise the site of cellular proliferation. Cold-induced angiogenesis, indicated by an increase in capillary to fibre ratio (C:F), occurred in SOL of rats (~20 % increase, P < 0.05) but not hamsters (~9.5 % increase, n.s.), and in TA of hamsters (~22 % increase, P < 0.01) but not rats (~1 % increase, n.s.). The change in C:F was highest in the glycolytic cortex region of TA where fibre size is larger than in the oxidative core. Capillary-specific cell proliferation (co-localised ALP and PCNA labelling) increased in parallel with C:F. The total PCNA label density within the interstitium was some 5-fold higher than that co-localised with capillaries, but where angiogenesis occurred the relative increase in capillary labelling was 2-fold greater than for other cells of the interstitium. These data suggest a significant role for endothelial cell proliferation in the angiogenic response, indicative of the sprouting form of angiogenesis. There was a tendency for fibre hypertrophy in both SOL and TA of rats, especially in the core region of TA (P < 0.01), such that capillary density (CD) and intramuscular diffusion distances (DD) were largely unchanged following cold exposure. In contrast, fibre size was maintained in hamsters, DD reduced and CD increased compared to control TA (P < 0.01). In conclusion, cold acclimation stimulated angiogenesis in muscle of hamsters more than in rats, possibly due to a higher metabolic rate in the smaller species. Angiogenesis was also seen in SOL of rat, where oxidative capacity and muscle activity is higher than the TA. Thus, a combination of oxidative capacity, muscle activity, and fibre size may determine the degree of angiogenesis in response to low environmental temperature. Experimental Physiology (2003) 88.6, 741-746.
Muscle ischaemia in rats may be relieved by overload-induced angiogenesis
- D. Deveci, S. Egginton
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- Journal:
- Experimental Physiology / Volume 87 / Issue 4 / July 2002
- Published online by Cambridge University Press:
- 25 June 2002, pp. 479-488
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- July 2002
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Alleviation of muscle ischaemia by improving capillary supply has proved difficult, possibly reflecting the inability to substantially increase blood flow. We reasoned that muscle overload, which induces angiogenesis in the absence of altered blood flow, may be an alternative to drug therapy. Male Wistar rats underwent unilateral ligation of the common iliac artery, with or without ipsilateral extirpation of the tibialis anterior muscle. Six weeks later ischaemic (I) extensor digitorum longus (EDL) had a 10% (P< 0.05) decrease in relative muscle mass, while overloaded muscles (O) had undergone hypertrophy of 39% and 52% relative to contralateral (CL) and control (C) muscle masses, respectively (P<0.01). Muscle atrophy was prevented by the combination of overload and ischaemia (O/I), with hypertrophy of 24% (vs. CL) and 35% (vs. C), respectively (P<0.01). Changes in muscle fibre cross-sectional area paralleled the changes in muscle mass, with means of 1898 ± 59, 1531 ± 90, 2253 ± 155 and 2292 ± 80 mm2 for C, I, O and O/I, respectively (P<0.01 vs. C and I). Capillary to fibre ratio (C:F) was significantly increased in overloaded (2.58 ± 0.09) compared to contralateral (1.78 ± 0.04), control (1.61 ± 0.05) and ischaemic (1.73 ± 0.06) muscles (P<0.001). A similar increase in C:F was seen in overloaded plus ischaemic muscle (2.59 ± 0.07) compared to contralateral (1.40 ± 0.01) and control or ischaemic values (P<0.01). In both O and O/I muscle groups, C:F and capillary density (CD) increased most in the region of EDL where fibre size was largest, while hypertrophy of fibres was least in the same region for both groups. These data suggest that the microvascular deficit evident in chronic muscle ischaemia may be alleviated by angiogenesis that is induced by mechanical stimuli via chronic muscle overloaded.
Chronic hypoxia induces prolonged angiogenesis in skeletal muscles of rat
- D. Deveci, J. M. Marshall, S. Egginton
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- Journal:
- Experimental Physiology / Volume 87 / Issue 3 / May 2002
- Published online by Cambridge University Press:
- 21 May 2002, pp. 287-291
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- May 2002
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Skeletal muscle capillarity and fibre cross-sectional area were investigated within and between diaphragm (Diaph), extensor digitorum longus (EDL), soleus (SOL) and tibialis anterior (TA) muscles of control and chronic hypoxic (12 % O2 for 6 weeks) adult male Wistar rats (final body mass ~355 g). Cryostat sections were stained for alkaline phosphatase activity to depict all capillaries, and for succinic dehydrogenase to demonstrate regional differences in oxidative capacity within the muscles. Hypoxia-induced angiogenesis occurred in all muscles (P < 0.01), with capillary-to-fibre ratio (C:F) being higher in the more active and oxidative muscles, Diaph (27 %) and SOL (26 %), than phasically active and glycolytic muscles, TA (21 %) and EDL (15 %). Diaph, SOL and EDL maintained fibre size, and hence showed an increased capillary density (CD) and reduced intramuscular diffusion distance (DD), whereas TA showed fibre hypertrophy and maintained CD and DD compared to control muscles. The extent of angiogenesis among different regions of muscle varied so as to suggest that muscle fibre size has an additional influence on capillary growth during chronic systemic hypoxia, which is progressive over an extended period of systemic hypoxia. Experimental Physiology (2002) 87.3, 287-291.
DEVELOPMENT OF THE FLUORESCENT MICROSPHERE TECHNIQUE FOR QUANTIFYING REGIONAL BLOOD FLOW IN SMALL MAMMALS
- D. DEVECI, S. EGGINTON
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- Journal:
- Experimental Physiology / Volume 84 / Issue 4 / July 1999
- Published online by Cambridge University Press:
- 04 January 2001, pp. 615-630
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- July 1999
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1.Tissues weighing from 0·006 to 3·0 g were dissected and put directly into 15 ml screw cap polypropylene tubes with a conical base (maximum of 3 g per tube). It proved unnecessary to mince tissue, even though smaller pieces may aid quicker digestion. Five millilitres of 2 M KOH in 99 % (IMS) ethanol with 0·5 % Tween-80 was then added. Tissue digestion was usually completed in 2-4 h using a dry heating block held at 60°C, with intermittent shaking. Samples were routinely processed using fresh tissues, although storage of frozen tissue (in the dark at -20°C) introduced no detectable error in BF estimation and tended to aid tissue maceration.2.After digestion was completed the tubes were centrifuged at 3000 r.p.m. (1500 g) for 15 min.3.The supernatant was carefully aspirated until < 500 µl was left, thereby reducing the possibility of accidental loss of microspheres.4.After 1 ml dH2O was added the tubes were quickly vortexed to prevent microsphere flocculation and aid resuspension of remaining pellets, while the subsequent addition of ethanoic Tween (100 % ethanol + 0·5 % Tween-80) allowed complete sedimentation by centrifugation.5.Nine millilitres of ethanoic Tween-80 was added, and the tubes were vortexed and spun at 1500 g for 15 min.6.The supernatant was aspirated as above (Step 3).7.Five millilitres of 100 mM phosphate buffer (pH 7·00) was added to neutralize the pellet as alkaline solutions quench fluorescence. Using aqueous solutions increased the possibility of microsphere loss by adhesion to the surface of tubes or aggregation, which could readily be seen by eye when tubes were examined under bright light, and hence the buffer was followed by addition of 4 ml absolute ethanol and further vortexing, before spinning (1500 g) for 20 min.8.The supernatant was aspirated, leaving up to 300 µl depending on the amount of tissue residue, and the remaining microspheres and pellet were quickly vortexed to ensure complete resuspension.9.The tubes were left to evaporate in an oven at 60°C and then briefly vortexed during this period to disperse more of the flocculent, until around 100 µl fluid remained. This improves solvent extraction of microspheres, which may be less efficient in a completely dry pellet.10.Two or three millilitres of solvent (di(ethylene glycol) ethyl ether acetate, 98 %; Aldrich Chemical Co., Poole, Dorset, UK) was added according to the expected fluorescence intensity, and vortexed several times over 3-5 min. After 30 min the tubes were sonicated in a water bath for 5 min to complete dye extraction by the solvent. The tubes were kept as far as possible in the dark after the solvent was added to avoid photo-bleaching of the fluorescent dyes.11.After sonication, the tubes were occasionally spun (1500 g) once more to sediment any undigested/undissolved material, though this was rarely necessary in our experiments. Readings should be completed within 1 h to avoid loss of signal intensity.