Alpha adrenergic receptor blockade increases capillarization and fractional O2 extraction and lowers blood flow in contracting human skeletal muscle.

AIM: To assess the effect of elevated basal shear stress on angiogenesis in humans and the role of enhanced skeletal muscle capillarization on blood flow and O2 extraction. METHODS: Limb haemodynamics and O2 extraction were measured at rest and during one-leg knee-extensor exercise (12 and 24 W) in 10 healthy untrained young men before and after 4-week treatment with an α1 receptor-antagonist (Terazosin, 1-2 mg day-1 ). Corresponding biopsies were taken from the m. vastus lateralis. RESULTS: Resting leg blood flow was increased by 57% 6 h following Terazosin treatment (P < 0.05), while basal capillary-to-fibre ratio was 1.69 ± 0.08 and increased to 1.90 ± 0.08 after treatment (P < 0.05). Leg O2 extraction during knee-extensor exercise was higher (4-5%; P < 0.05), leg blood flow and venous lactate levels lower (6-7%; P < 0.05), while leg VO2 was not different after Terazosin treatment. CONCLUSIONS: These results demonstrate that daily treatment with an α-adrenergic receptor blocker induces capillary growth in human skeletal muscle, likely due to increased shear stress. The increase in capillarization resulted in an increased fractional O2 extraction, a lower blood flow and venous lactate levels in the exercising leg. The increase in capillarization, and concomitant functional readouts in the exercising leg, may provide a basis for novel angiotherapy.

Shear stress-induced angiogenesis in mouse muscle is independent of the vasodilator mechanism and quickly reversible.

AIM: Is modulation of skeletal muscle capillary supply by altering blood flow due to a presumptive shear stress response per se, or dependent on the vasodilator mechanism? METHODS: The response to four different vasodilators, and cotreatment with blockers of NO and prostaglandin synthesis, was compared. Femoral artery blood flow was correlated with capillary-to-fibre ratio (C:F) and protein levels of putative angiogenic compounds. RESULTS: All vasodilators induced a similar increase in blood flow after 14 days, with a similar effect on C:F (1.62 ± 0.05, 1.60 ± 0.01, 1.57 ± 0.06, 1.57 ± 0.07, respectively, all P < 0.05 vs. control 1.20 ± 0.01). Concomitant inhibitors revealed differential effects on blood flow and angiogenesis, demonstrating that a similar response may have different signalling origins. The time course of this response with the most commonly used vasodilator, prazosin, showed that blood flow increased from 0.40 mL min-1 to 0.61 mL min-1 by 28 days (P < 0.05), dropped within 1 week after the cessation of treatment (0.54 mL min-1 ; P < 0.05) and returned to control levels by 6 weeks. In parallel with FBF, capillary rarefaction began within 1 week (P < 0.05), giving C:F values similar to control by 2 weeks. Of the dominant signalling pathways, prazosin decreased muscle VEGF, but increased its cognate receptor Flk-1 (both P < 0.01); levels of eNOS varied with blood flow (P < 0.05), and Ang-1 initially increased, while its receptor Tie-2 was unchanged, with only modest changes in the antiangiogenic factor TSP-1. CONCLUSION: Hyperaemia-induced angiogenesis, likely in response to elevated shear stress, is independent of the vasodilator involved, with a rapid induction and quick regression following the stimulus withdrawal.

In Memoriam: Olga Hudlická (11.07.26-03.05.14).

Dr Olga Hudlická, Professor Emeritus in the Department of Physiology, University of Birmingham Medical School, died suddenly on 3rd May not long after a fall. She was one of the best-known vascular physiologists of the last century, investigating control of blood flow and regulation of angiogenesis in skeletal and cardiac muscle. This article is protected by copyright. All rights reserved.

Re-evaluating the use of Voronoi Tessellations in the assessment of oxygen supply from capillaries in muscle.

The ability to characterise capillary supply plays a key role in developing effective therapeutic interventions for numerous pathological conditions, such as capillary loss in skeletal or cardiac muscle. However, quantifying capillary supply is fraught with difficulties. Averaged measures such as capillary density or mean inter-capillary distance cannot account for the local geometry of the underlying capillary distribution, and thus can only highlight a tissue wide, global hypoxia. Detailed tissue geometry, such as muscle fibre size, has been incorporated into indices of capillary supply by considering the distribution of Voronoi tessellations generated from capillary locations in a plane perpendicular to muscle fibre orientation, implicitly assuming that each Voronoi polygon represents the area of supply of its enclosed capillary. Using a modelling framework to assess the capillary supply capacity under maximal sustainable conditions in muscle, we theoretically demonstrate that Voronoi tessellations often provide an accurate representation of the regions supplied by each capillary. However, we highlight that this use of Voronoi tessellations is inappropriate and inaccurate in the presence of extensive capillary rarefaction and pathological variations in oxygen tension of different capillaries. In such cases, oxygen flux trapping regions are developed to provide a more general representation of the capillary supply regions, in particular incorporating the additional influences of heterogeneity that are absent in the consideration of Voronoi tessellations.

Physiological factors influencing capillary growth.

(1) Angiogenesis (growth of new capillaries from an existing capillary bed) may result from a mismatch in microvascular supply and metabolic demand (metabolic error signal). Krogh examined the distribution and number of capillaries to explore the correlation between O(2) delivery and O(2) consumption. Subsequently, the heterogeneity in angiogenic response within a muscle has been shown to reflect either differences in fibre type composition or mechanical load. However, local control leads to targetted angiogenesis in the vicinity of glycolytic fibre types following muscle stimulation, or oxidative fibres following endurance training, while heterogeneity of capillary spacing is maintained during ontogenetic growth. (2) Despite limited microscopy resolution and lack of specific markers, Krogh's interest in the structure of the capillary wall paved the way for understanding the mechanisms of capillary growth. Angiogenesis may be influenced by the response of perivascular or stromal cells (fibroblasts, macrophages and pericytes) to altered activity, likely acting as a source for chemical signals modulating capillary growth such as vascular endothelial growth factor. In addition, haemodynamic factors such as shear stress and muscle stretch play a significant role in adaptive remodelling of the microcirculation. (3) Most indices of capillarity are highly dependent on fibre size, resulting in possible bias because of scaling. To examine the consequences of capillary distribution, it is therefore helpful to quantify the area of tissue supplied by individual capillaries. This allows the spatial limitations inherent in most models of tissue oxygenation to be overcome generating an alternative approach to Krogh's tissue cylinder, the capillary domain, to improve descriptions of intracellular oxygen diffusion.

Staining of nasal mucosa to examine remodelling.

BACKGROUND AND OBJECTIVE: The process of embedding tissue in paraffin degrades many important molecules involved in respiratory epithelial remodelling. We therefore examined alternative methods. METHODS: Inferior turbinate and nasal polyp biopsies were either placed in formalin or immediately snap-frozen in the operating theatre. Novel protocols for staining remodelling markers were compared with current methods. RESULTS: Our method, using a mixture of three lectins, stained a significantly greater proportion of samples, compared with using Ulex europeaus lectin alone (84 vs 62 per cent; p < 0.005). Comparison of different proliferation markers showed that Ki67 was more suitable than proliferating cell nuclear antigen for frozen sections. CONCLUSIONS: This study indicates that our robust, repeatable methods for examining whole mounts and for staining capillaries, cell proliferation and nuclei on the same section of nasal mucosa are superior to current methods. The use of fresh tissue that has not been paraffin-embedded would allow a greater suite of epitopes to be examined in the future.

Heart rate and ventilation in Antarctic fishes are largely determined by ecotype.

Extrinsic neural and humoral influences on heart rate (fH) and ventilation frequency (fV) were examined following varying periods of post-surgical recovery in eight related Antarctic fish species inhabiting an array of inshore niches. Resting fH after recovery from handling was lower than previous reports, and the novel measurement of routine fH in free-swimming Dissostichus mawsoni (6.14 beats min(-1), bpm) is the lowest recorded for any fish. The extent of cardio-depressive cholinergic (vagal) tonus explained the large range of fH among species and varied with behavioural repertoire, being lower in the more active species, apart from Notothenia coriiceps. Adrenergic tonus was low compared with cholinergic tonus, with the exception of Trematomus newnesi. Hence, high cardiac cholinergic tonus may be a genotypic trait of the notothenioids that diverged with ecotype. Power spectral analysis showed that the vagal influence produced comparable spectra among species of similar morphology and ecotype. Removal of autonomic tonus resulted in a remarkably similar intrinsic fH between species. Simultaneous measurements of cardio-respiratory variables and oxygen consumption (M(O(2))) were made in the benthic Trematomus bernacchii and cryopelagic Pagothenia borchgrevinki. The slopes of the relationship between fH and M(O(2)) were similar. Trematomus bernacchii, however, had a higher M(O(2)) for a given fH than P. borchgrevinki, and P. borchgrevinki required a two-fold larger range in fH to reach a similar maximum M(O(2)), suggesting that there is a difference in cardiovascular fitness between the two species. Overall, the data suggest that cardio-respiratory control in Antarctic nototheniids is largely determined by activity levels associated with a given ecotype.

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

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.

Polar cod, Boreogadus saida (Gadidae), show an intermediate stress response between Antarctic and temperate fishes.

To examine whether the attenuated stress response observed in Antarctic notothenioid fishes is a specialism for life in sub-zero waters, the polar cod, Boreogadus saida, and the temperate shorthorned sculpin, Myoxocephalus scorpius, were subjected to various stress treatments. Activity stress in both species had no effect on plasma catecholamine and cortisol levels, splenic mass, and on the haematological variables in B. saida. In contrast, heat stress caused a significant rise in circulating noradrenaline and adrenaline levels in B. saida, accompanied by a significant increase in haematocrit and haemoglobin concentrations, at constant plasma cortisol levels, red blood cell count and splenic mass. A concomitant rise in blood lactate concentrations indicated that heat-stressed B. saida were hypoxaemic. The capacity to synthesise catecholamines in B. saida was 38% of the value in M. scorpius, but similar to the values for Antarctic notothenioids. The lack of any adrenergic response to activity stress suggests that dominance of cholinergic control of the cardiovascular system may not be restricted to Antarctic notothenioids. Rather, the stress response in B. saida appears to be intermediate between Antarctic and temperate teleosts, in keeping with their relatively recent occupation of cold Arctic waters.

A new method in applying power spectral statistics to examine cardio-respiratory interactions in fish.

Power spectral analysis (PSA) provides a powerful tool for determining frequency oscillations in time signals, and it is accepted that mammals can show distinct components in the heart rate (fH) spectrum that are synchronous with ventilatory frequency (fV). Using similar signal processing techniques, these fundamental components at fV are not apparent in the spectrum calculated from fish fH. Here we compare conventional PSA on the R-R interval tachogram generated from ECG traces recorded in rats and fish, with PSA on the raw ECG waveform. The rat R-R tachogram showed a defined sigmoidal component, whereas the fish R-R tachogram was a more chaotic waveform. In agreement with the literature, PSA of these respective waveforms produced a component at the same frequency as ventilation in the rat, but of lower frequency than ventilation for the fish. Applying PSA to the rat ECG produced a spectrum with a fundamental component of similar frequency to that observed in the R-R tachogram spectrum, indicating that the latter adequately contained heart rate variability (HRV) oscillations. However, PSA of the ECG in fish contrasted with that from the R-R tachogram, with components observed in the latter spectrum being absent from the former. This suggests that the frequency components determined by PSA on the fish R-R tachogram were not true components, but were aliased (or folded-back) from higher up in the spectrum. Using established aliasing equations, recalculation of these peaks showed that their true frequency was similar to that of the ventilatory frequency for individual fish. The extent of cardio-respiratory interaction, resulting in fV < f(H/2) in rats but fV > f(H/2) in fish, is suggested to be the origin of the differences observed.

The effects of sustained exercise and hypoxia upon oxygen tensions in the red muscle of rainbow trout.

Teleost fish possess discrete blocks of oxidative red muscle (RM) and glycolytic white muscle, whereas tetrapod skeletal muscles are mixed oxidative/glycolytic. It has been suggested that the anatomy of RM in teleost fish could lead to higher intramuscular O2 partial pressures (PO2) than in mammalian skeletal muscles. This study provides the first direct experimental support for this suggestion by using novel optical fibre sensors to discover a mean (+/- S.E.M., N=6) normoxic steady-state red muscle PO2 (PrmO2) of 61+/-10 mmHg (1 mmHg=133.3 Pa) in free-swimming rainbow trout Oncorhynchus mykiss. This is significantly higher than literature reports for mammalian muscles, where the PO2 never exceeds 40 mmHg. Aerobic RM powers sustained swimming in rainbow trout. During graded incremental exercise, PrmO2 declined from 62+/-5 mmHg at the lowest swim speed down to 45+/-3 mmHg at maximum rates of aerobic work, but then rose again to 51+/-5 mmHg at exhaustion. These measurements of PrmO2 during exercise indicated, therefore, that O2 supply to the RM was not a major limiting factor at exhaustion in trout. The current study found no evidence that teleost haemoglobins with a Root effect cause extremely elevated O2 tensions in aerobic tissues. Under normoxic conditions, PrmO2 was significantly lower than arterial PO2 (119+/-5 mmHg), and remained lower when the arterial to tissue PO2 gradient was reduced by exposure to mild hypoxia. When two sequential levels of mild hypoxia (30 min at a water PO2 of 100 mmHg then 30 min at 75 mmHg) caused PaO2 to fall to 84+/-2 mmHg then 61+/-3 mmHg, respectively, this elicited simultaneous reductions in PrmO2,to 51+/-6 mmHg then 41+/-5 mmHg, respectively. Although these hypoxic reductions in PrmO2 were significantly smaller than those in PaO2, the effect could be attributed to the sigmoid shape of the trout haemoglobin-O2 dissociation curve.

The influence of acute hypothermia on renal function of anaesthetized euthermic and acclimatized rats.

Acute hypothermia has a major impact on cardiovascular control and renal function, but the extent to which these can be correlated with and influenced by changes in the altered pattern of sympathetic outflow to the kidneys is unclear. Moreover, it is unknown whether these responses to acute hypothermia are altered by chronic cold exposure and this study aimed to examine these factors. Renal function and renal sympathetic nerve activity (RSNA) were measured in male Wistar rats, euthermic (control) or acclimatized (exposed to progressively lower environmental temperature and photoperiod over 8 weeks), anaesthetized with chloralose/urethane. Reduction of core temperature (Tc) to 25 degrees C caused approximately 40% reduction in heart rate (HR), approximately 10% fall in mean arterial blood pressure (MABP), and decreased glomerular filtration rate (GFR) by approximately 50% and approximately 5% in euthermic and acclimatized rats, respectively. At 25 degrees C, urine flow increased some two-fold and absolute and fractional sodium excretions by 4- to 6-fold in the euthermic rats and to a lesser extent in the cold acclimatized rats, while basal levels of fluid excretion were higher in the acclimatized rats. A loss of pulsatility in the RSNA signal with cooling was seen in both groups. One of the factors contributing to modest hypotension during acute hypothermia is a reduction in RSNA. There was a progressive fall in the proportion of RSNA power at HR frequency with cooling of 20% in euthermic and 80% in acclimatized rats. All variables were restored to basal levels on rewarming in both groups of rats. We conclude that natriuresis and diuresis in euthermic rats during hypothermia is a consequence of a reduction in nephron reabsorption, reduced urine osmolality and possibly altered patterning of RSNA. In acclimatized rats, the response was modified by altered renal haemodynamics and/or hormonal influences induced by chronic cold exposure to minimize the hypothermic stress on renal function.

The use of power spectral analysis to determine cardiorespiratory control in the short-horned sculpin Myoxocephalus scorpius.

Anaesthesia and minor surgery to place electrocardiogram recording electrodes in the short-horned sculpin caused a decrease in mean normal beat (R-R) interval and heart rate variability (HRV), measured as the standard deviation in the R-R interval (SDRR). Mean R-R interval increased to a steady state value (1.9+/-2.9 s) 72 h post-surgery, but SDRR took 120 h to stabilise (0.56+/-0.09 s). Power spectral analysis applied to recordings of instantaneous heart rate showed no spectral peaks immediately after surgery, with the development of twin peaks (at 0.02 and 0.05 Hz) that also became stable 120 h post surgery. Bilateral cardiac vagotomy abolished the variability in beat-to-beat interval, and both the high and low frequency peaks, suggesting that much of the regulation of heart rate and HRV in sculpin was under parasympathetic, cholinergic control that was withdrawn as a result of surgical and handling stress. Rate of oxygen consumption and heart rate (f(H)) were monitored simultaneously and showed a good correlation with both mean R-R interval (r(2)=-0.89) and SDRR (r(2)=0.93), although a more significant (ANCOVA, P=0.02) covariance existed between the post-surgical decrease in and increase in SDRR. These data suggest that sculpin use f(H) as a way of moderating oxygen consumption, fine-tuned on a beat-to-beat basis by cholinergic control. We conclude that power spectral analysis is a useful method of determining HRV in fish, and that HRV is a more sensitive measure of recovery from disturbance than f(H) alone.

Effect of hypothermia on baroreflex control of heart rate and renal sympathetic nerve activity in anaesthetized rats.

The present study investigated the effect of acute hypothermia on baroreflex control of heart rate (HR) and renal sympathetic nerve activity (RSNA) by generating baroreflex logistic function curves, using bolus doses of phenylephrine and sodium nitroprusside, in anaesthetized male Wistar rats at a core temperature (T(b)) of 37 degrees C, during acute severe hypothermia at T(b)= 25 degrees C and on rewarming to 37 degrees C. Comparisons were made between rats without (euthermic, n= 6) and with (acclimated, n= 7) prior exposure to lower ambient temperatures and shorter photoperiod, simulating adaptation to winter conditions. In both groups of rats, acute hypothermia to T(b)= 25 degrees C shifted the baroreflex-RSNA curve slightly leftwards and downwards with decreases in the setpoint pressure and maximal gain, whereas it markedly impaired the baroreflex-HR curve characterized by decreases in response range by approximately 90% (P < 0.001), minimum response by approximately 10% (P < 0.05) and maximum gain by approximately 95% (P < 0.001), from that at T(b)= 37 degrees C. All parameters were restored to precooling levels on rewarming. Electrical stimulation of cardiac vagal efferents induced a voltage-related bradycardia, the magnitude of which was partially reduced during acute hypothermia, and there was a significant prolongation of the electrocardiogram intervals indicating a delay in cardiac conduction. Mild suppression of baroreflex control of RSNA could contribute to hypothermic hypotension and may primarily reflect an effect of T(b) on central drive. The marked attenuation of the baroreflex control of HR during hypothermia was likely to be due to an impairment of both the central and peripheral components of the reflex arc. Baroreflex control of RSNA and HR was similar between both groups of rats, which implied that the control was non-adaptive on chronic cold exposure.

Cold exposure differentially stimulates angiogenesis in glycolytic and oxidative muscles of rats and hamsters.

Control (normothermic) and cold-acclimated (environmental temperature gradually reduced from 20 to 5 degrees 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 (approximately 20 % increase, P < 0.05) but not hamsters (approximately 9.5 % increase, n.s.), and in TA of hamsters (approximately 22 % increase, P < 0.01) but not rats (approximately 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.

Differences in local environment determine the site of physiological angiogenesis in rat skeletal muscle.

The specificity in location of angiogenesis to either glycolytic or oxidative fibre types, or muscle regions, was examined in the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles of rat. Angiogenesis was induced by mechanical means either with (chronic muscle stimulation) or without (muscle stretch by overload) changes in blood flow, treatments which invoked only minor changes in fibre type and fibre size. Proliferation estimated by PCNA labelling of cells co-localised with capillaries was very rare in control muscles, where it occurred mainly in the glycolytic regions, but was increased in both models of angiogenesis. However, when labelled capillaries were scored according to the type of surrounding fibres, only muscle stimulation significantly accentuated proliferation of capillaries surrounded by glycolytic fibres. We conclude that while mechanical stimuli are important for proliferation in glycolytic regions in both models, capillary growth occurs specifically around glycolytic fibres in that region when the angiogenic stimulus includes increased blood flow and/or increased metabolic demand.

The effect of chronic skeletal muscle stimulation on capillary growth in the rat: are sensory nerve fibres involved?

Indirect chronic electrical stimulation of skeletal muscle activates not only efferent but also afferent nerve fibres. To investigate effects specific to this on capillary growth, one of the earliest changes, cell proliferation and capillary ultrastructure were studied in ankle flexors of rats with and without deafferentation of the stimulated side. Two weeks after preganglionic section of dorsal roots L4-L6, the peroneal nerve was stimulated (10 Hz, 8 h day(-1)) for 2 or 7 days. Proliferating nuclei labelled by bromodeoxyuridine or proliferating cell nuclear antigen staining were colocalized to alkaline phosphatase-stained capillaries (Lc) or other interstitial nuclei (Li) in frozen sections of extensor digitorum longus. Capillary fine structure was examined in extensor hallucis proprius by transmission electron microscopy. The stimulation-induced increase in capillary and interstitial proliferation (Lc 9.9 +/- 1.9 %, Li 8.8 +/- 2.1 % vs. Lc 2.6 +/- 0.4 %, Li 1.9 +/- 0.3 % in controls, P < 0.05) was depressed at 2 days by dorsal root section (Lc 4.8 +/- 0.7 %, Li 3.2 +/- 0.9 %, P < 0.05), an effect likely to be mainly on fibroblasts; no depression was seen at 7 days. Dorsal root section reduced stimulation-induced capillary endothelial swelling at both time points. In contralateral muscles of intact rats, stimulation increased interstitial cell proliferation and capillary swelling, both effects being eliminated by dorsal root section. Capillary growth induced by stimulation (24 % increase in capillary : fibre ratio at 7 days) was unaffected by deafferentation. The reduction in capillary ultrastructural changes and interstitial proliferation in both stimulated and contralateral muscles implies that stimulation of afferent fibres leads directly to release of humoral factors and/or activation via dorsal roots of fibres that release humoral substances. Contralateral muscles are an inadequate control for the effects of chronic stimulation in the intact animal.

Muscle fine structure may maintain the function of oxidative fibres in haemoglobinless Antarctic fishes.

Muscle fine structure and metabolism were examined in four species of Antarctic fishes that vary in their expression of haemoglobin (Hb). To determine how locomotory pectoral muscles maintain function, metabolic capacity, capillary supply and fibre ultrastructure were examined in two nototheniid species that express Hb (Notothenia coriiceps and Gobionotothen gibberifrons) and two species of channichthyid icefish that lack Hb (Chaenocephalus aceratus and Chionodraco rastrospinosus). Surprisingly, icefish have higher densities of mitochondria than red-blooded species (C. aceratus, 53+/-3% of cell volume; C. rastrospinosus, 39+/-3%; N. coriiceps, 29+/-3%; G. gibberifrons, 25+/-1%). Despite higher mitochondrial densities the aerobic metabolic capacities per g wet mass, estimated from measurements of maximal activities of key metabolic enzymes, are lower in icefish compared to red-blooded species. This apparent incongruity can be explained by the significantly lower mitochondrial cristae surface area per unit mitochondrion volume in icefishes (C. aceratus, 20.8+/-1.6 microm(-1); C. rastrospinosus, 25.5+/-1.8 microm(-1)) compared to red-blooded species (N. coriiceps, 33.6+/-3.0 microm(-1); G. gibberifrons, 37.7+/-3.6 microm(-1)). Consequently, the cristae surface area per unit muscle mass is conserved at approximately 9 m(2)g(-1). Although high mitochondrial densities in icefish muscle do not enhance aerobic metabolic capacity, they may facilitate intracellular oxygen movement because oxygen is more soluble in lipid, including the hydrocarbon core of intracellular membrane systems, than in aqueous cytoplasm. This may be particularly vital in icefish, which have larger oxidative muscle fibres compared to red-blooded nototheniods (C. aceratus, 2932+/-428 microm(2); C. rastrospinosus, 9352+/-318 microm(2); N. coriiceps, 1843+/-312 microm(2); G. gibberifrons, 2103+/-194 microm(2)). These large fibres contribute to a relatively low capillary density, which is partially compensated for in icefish by a high index of tortuosity in the capillary bed (C. aceratus=1.4, N. coriiceps=1.1).

Muscle ischaemia in rats may be relieved by overload-induced angiogenesis.

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 overload.

Chronic hypoxia induces prolonged angiogenesis in skeletal muscles of rat.

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 % O(2) for 6 weeks) adult male Wistar rats (final body mass approximately 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.