How to calculate human muscle fibre areas in biopsy samples--methodological considerations

Cross-sectional muscle fibre areas (type I, II A and II B) were determined in duplicate biopsies from the left vastus lateralis (n = 11) and in biopsies from right and left vastus lateralis (n = 8). The SD for the difference in means between duplicate biopsies was 510 microns 2 for type I, 1020 microns 2 for type II A and 860 microns 2 for type II B. Expressed as coefficient of variation (CV) these SD constituted 10, 15 and 15%, respectively. The variation in fibre size within a sample was considerably less than the variation between samples on the assumption that at least 15-20 areas of each fibre type were measured per sample. No difference in mean fibre area for type I, II A and II B fibres was obtained between the right and left muscle. Several artefacts due to the sampling and preparing procedures are discussed and a method for determining muscle fibre areas in biopsy samples is suggested.     

Muscle fibre size and capillarity in Korean diving women

AIM: Effects of prolonged habitual cold-water immersion on fibre size and capillarity in vastus lateralis muscle were studied in human beings. The hypothesis tested in the present study was that cold acclimatized human skeletal muscle would have reduced muscle fibre size and higher capillarity, favouring the idea of efficacy of recruitment under cold environment. METHODS: Ten women breath-hold divers (BHDs) and 10 active women (controls CONs) participated in this study. Muscle biopsy was obtained from vastus lateralis and determined fibre type composition and capillary density. RESULTS: A major finding was that all BHDs revealed a markedly smaller cross-sectional area (CSA) in all fibre types than the CONs, or even than any other morphological data reported in previous investigations. Furthermore, mean CSA of type II fibre (range 1205-2766 microm2) was much smaller than type I fibre (2343-4327 microm2). The number of capillaries per fibre in different fibre types in the BHDs was higher than in the CONs (P < 0.001), and diffusional area was smaller in type II fibres than in type I fibres (P < 0.001). The BHDs and the CONs have similarity in the percentage of type I fibres, but type II fibre was predominant in both groups. Interestingly the proportion of type IIx fibre in the BHDs was higher (31%) than in the CONs (22%). No significant difference was found in the thigh circumference between the groups. CONCLUSION: The present study demonstrates that prolonged habitual cold-water immersion may induce a decrease in fibre size and an increase in capillarity in human skeletal muscle.     

Glycogen breakdown in different human muscle fibre types during exhaustive exercise of short duration.

The rates of glycogen breakdown during exhaustive intense exercise of three different intensities were determined in type I and subgroups of type II fibres. The exercise intensity corresponded to 122 +/- 2, 150 +/- 7 and 194 +/- 7% of VO2max. Muscle biopsies were taken from both legs before and immediately after exhaustion. Muscle lactate concentration increased by 27 +/- 1, 27 +/- 1 and 20 +/- 2 mmol kg-1 wet wt during the exercise at 122, 150 and 194% VO2max, respectively. The rates of glycogen depletion increased in all fibre types with increasing intensity, and the decline in type I fibres was 30-35% less than in type II fibres at all intensities. No differences were observed between the glycogen depletion rates in subgroups of type II fibres (IIA, IIAB and IIB). During the exercise at 194% VO2max, the rates of glycogen breakdown were 0.35 +/- 0.03 and 0.52 +/- 0.05 mmol s-1 kg-1 wet wt in type I and type II fibres, respectively. For both fibre types, the rates were 32 and 69% lower during the exercise at 150 and 122% VO2max. These data indicate that the glycolytic capacity of type I fibres is 30-35% lower than the capacity of type II fibres, in good agreement with the differences in phosphorylase and phosphofructokinase activities (Essén et al. 1975, Harris et al. 1976). The data also indicate that both fibre types contribute significantly to the anaerobic energy release at powers up till almost 200% VO2max.     

Does chronic hypoxaemia induce transformations of fibre types?

The study comprised nine patients with chronic obstructive lung disease. Quadriceps muscle biopsies were studied with respect to fibre type composition before and after haemodilution that brought haemoglobin (Hb) to within normal limits. Ten days elapsed between the two biopsy occasions. The arterial oxygen tension (PaO2) and saturation (SaO2) were depressed to 8.4 +/- 2.0 kPa and 89 +/- 11% in the patients with chronic obstructive lung disease and increased to 9.2 +/- 2.1 and 91 +/- 8% with haemodilation. The type II fibre proportion was 71 +/- 12% before haemodilation and significantly higher than normal (reference group, see Aniansson et al. 1981). Following haemodilation the proportion of type II fibres decreased significantly to 60 +/- 14%. The proportion of type II fibres was directly related to the haemoglobin content before, but not after, haemodilation and was inversely related to PaO2 and SaO2 both before and after haemodilation. In conclusion, hypoxaemia may be a factor underlying the high proportion of type II fibres found in patients with chronic obstructive lung disease.     

Glycogen and lactate metabolism during low-intensity exercise in man

The influence of high lactate concentration on glycogen metabolism in active type I and inactive type II fibres was investigated. High muscle lactate concentration (26.7 +/- 1.4 mmol kg-1 wet wt) was achieved by three bouts (2 min duration) of bicycle exercise at 112% Vo2 max. Exercise was continued at 40% Vo2 max for 1 h. Serial venous blood samples and biopsies from the vastus lateralis muscle were taken. Over the first 20 min of this low-intensity exercise muscle lactate concentration decreased by 22.9 +/- 0.7 mmol kg-1 wet wt, while glycogen remained unchanged in type I fibres and increased by 20 mmol kg-1 wet wt in type II fibres. During the next 40 min of low-intensity exercise lactate decreased by 1.6 +/- 1.2 mmol kg-1 wet wt, while glycogen concentration decreased by 21 +/- 7 mmol kg-1 wet wt in type I fibres but remained stable in type II fibres. In a second series of experiments, in which lactate was allowed to disappear before the light exercise was started, no changes in glycogen concentration were seen in type II fibres during the 1 h of 40% Vo2 max exercise, while a continuous reduction in glycogen of 28 +/- 8 mmol kg-1 wet wt was found in type I fibres. The results indicate that in the presence of high lactate levels muscle glycogen was resynthesized in inactive type II muscle fibres, while lactate was oxidized in preference to glycogen in type I fibres.     

Neutrophils as mediators of human skeletal muscle ischemia-reperfusion syndrome.

Nine patients with aortic aneurysm undergoing arterial reconstruction with temporary aortic occlusion were studied. Since a typical condition of ischemia-reperfusion of the muscles of the lower limbs was created during this surgery, muscle biopsies from the right femoral quadriceps as well as blood samples from the homolateral saphenous vein were taken: (1) before clamping of the aorta, (2) just before declamping, and (3) 30 minutes after reperfusion. Light microscopy revealed a consistent granulocyte infiltration in the ischemic and reperfused skeletal muscle. Ultrastructural damage to the muscle fibers was seen during ischemia and became more severe upon reperfusion. The recruitment of granulocytes into the muscle tissue paralleled the activation of the blood complement system and an increase in circulating neutrophils. Although a spontaneous superoxide anion (O2-) generation from such granulocytes cannot be proved, upon stimulation with formyl-methionyl-leucyl-phenylalanine neutrophils showed a reduced ability in O2 free radical production at the end of ischemia and enhanced O2- generation at reperfusion as compared with the controls. All these findings indicate an active role of granulocytes in the genesis of reperfusion-induced tissue injuries.     

Influence of reduced muscle temperature on metabolism in type I and type II human muscle fibres during intensive exercise

Six male subjects performed intensive cycle exercise to exhaustion after cooling their legs in water at 10-12 degrees C (muscle temperature (Tm) 28 +/- 2.6 degrees C, mean +/- SD). Exercise at exactly the same rate and duration (370 +/- 34 W, 1.5 +/- 0.2 min) was then repeated by each subject 2-5 weeks later at normal Tm (35 +/- 1.0 degrees C). Muscle biopsies were taken from the vastus lateralis muscle at rest and after exercise. The muscle tissue was freeze-dried and fragments of single fibres were dissected out. The fibres were classified and pooled into groups of type I and type II. Analyses of glycogen, glucose 6-phosphate, lactate and phosphagens were performed on pools of type-identified fibres. After exercise at reduced Tm, all subjects had higher concentrations of glucose 6-phosphate and lactate in both type I and type II fibres, and in most subjects the concentrations of ATP and phosphocreatine were lower as compared with the findings after exercise at normal Tm. During exercise the glycogen content of both fibre types decreased to a greater extent at reduced than at normal Tm in most subjects. The results suggest that during intensive dynamic exercise at reduced Tm there is a higher degree of glycolysis from glycogen in the muscle than in the normal situation. In some subjects the cause of fatigue may be related to a more rapid accumulation of lactate in the cold muscle, while in others fatigue may be related to alternative factors, e.g. low levels of ATP and phosphocreatine.     

Metabolism changes in single human fibres during brief maximal exercise

Changes in high-energy phosphate levels in single human skeletal muscle fibres after 10 s of maximal (all-out) dynamic exercise were investigated. Muscle biopsies from vastus lateralis of two volunteers were collected at rest and immediately post exercise. Single muscle fibres were dissected from dry muscle and were assigned into one of four groups according to their myosin heavy chain (MyHC) isoform content: that is type I, IIA, IIAx and IIXa (the latter two groups containing either less or more than 50% IIX MyHC). Fragments of characterised fibres were analysed by HPLC for ATP, inosine-monophosphate (IMP), phosphocreatine (PCr) and creatine levels. After 10 s of exercise, PCr content ([PCr]) declined by approximately 46, 53, 62 and 59 % in type I, IIA, IIAx and IIXa fibres, respectively (P < 0.01 from rest). [ATP] declined only in type II fibres, especially in IIAx and IIXa fibres in which [IMP] reached mean values of 16 +/- 1 and 18 +/- 4 mmol (kg dry mass)(-1), respectively. While [PCr] was reduced in all fibre types during the brief maximal dynamic exercise, it was apparent that type II fibres expressing the IIX myosin heavy chain isoform were under a greatest metabolic stress as indicated by the reductions in [ATP].     

Muscle glycogen depletion patterns in type I and subgroups of type II fibres during prolonged severe exercise in man

Glycogen depletion of muscle fibre types I, II A, II AB and II B was studied using a histochemical method to quantify glycogen content in individual fibres. The reliability was examined in 29 muscle biopsies, in which total glycogen content was compared to average periodic acid Schiff (PAS) stain intensity in sections from the same samples. Over a wide range of glycogen content (1-252 mmole glucosyl units . kg-1 wet weight) a linear relationship (r = 0.93) was found between the two methods for quantification of muscle glycogen. Glycogen depletion patterns in type I, II A, II AB and II B fibres were studied in 5 subjects during exhaustive bicycle exercise at 75% of VO2 max. At rest before exercise glycogen content was 16% higher in type II subgroups than in type I (p less than 0.05). From start of exercise the same glycogen depletion rate was observed in type I and II A. Glycogen content of Type II AB and II B was unchanged during the first part of exercise. Later a decrease was observed, first in type II AB and finally in II B, suggesting a decrease in threshold force of these fibre types. The results indicate physiological differences between the 3 subgroups of type II fibres in man, whereas at the present exercise intensity type I and II A fibres were recruited simultaneously from start.     

Enzyme activities in type I and II muscle fibres of human skeletal muscle in relation to age and torque development

The quadriceps muscles from 20- 30- and 70-year-old clinically healthy men and women were studied regarding maximal isometric and isokinetic muscle torque in Newton metres (Nm), morphology and enzyme activity. Biopsy specimens were taken from the vastus lateralis muscle and freeze-dried, and individual fibres were dissected out and identified as type I or type II. The activities of citrate synthase (CS), 3-OHacyl-coA dehydrogenase (HAD), lactate dehydrogenase (LDH), myokinase (MK) and creatine phosphokinase (CPK) were determined in pools of type I and type II fibres. In both age groups a higher oxidative (CS, HAD, 1.3-1.5 x) and a lower glycolytic (LDH, 0.7 x) capacity was found in type I than in type II fibres. The myokinase activity was higher in type II (2 x) than in type I, whereas CPK activity was similar. The young men showed higher CS activity in both type I and type II fibres (1.5 x) and higher CPK activity in type I fibres (1.4 x) than the young women. There were only minor changes in oxidative or glycolytic capacities in relation to age. Myokinase was the only enzyme that decreased markedly with age in both pools of fibre types. Type II fibre area and mean fibre area correlated significantly to muscle torque in both sexes. In men, myokinase activity in type II fibres was significantly correlated to type II fibre area and to maximal muscle torque.     

Age and sex affect human muscle fibre adaptations to heavy-resistance strength training

This study assessed age and sex effects on muscle fibre adaptations to heavy-resistance strength training (ST). Twenty-two young men and women (20-30 years old) and 18 older men and women (65-75 years old) completed 9 weeks of heavy-resistance knee extension exercises with the dominant leg 3 days week(-1); the non-dominant leg served as a within-subject, untrained control. Bilateral vastus lateralis muscle biopsies were obtained before and after ST for analysis of type I, IIa and IIx muscle fibre cross-sectional area (CSA) and fibre type distribution. One-repetition maximum (1-RM) strength was also assessed before and after ST. ST resulted in increased CSA of type I, IIa and IIx muscle fibres in the trained leg of young men, type I and IIa fibres in young women, type IIa fibres in older men, and type IIx fibres in older women (all P<0.05). Analysis of fibre type distribution revealed a significant increase in the percentage of type I fibres (P<0.05) along with a decrease in type IIx fibres (P=0.054) after ST only in young women. There were no significant changes in muscle fibre CSA or fibre type distribution in the untrained leg for any group. All groups displayed significant increases in 1-RM (27-39%; all P<0.01). In summary, ST led to significant increases in 1-RM and type II fibre CSA in all groups; however, age and sex influence specific muscle fibre subtype responses to ST.     

How to calculate human muscle fibre areas in biopsy samples—methodological considerations

Cross-sectional muscle fibre areas (type I, IIA and IIB) were determined in duplicate biopsies from the left vastus lateralis (n=11) and in biopsies from right and left vastus lateralis (n=8).The SD for the difference in means between duplicate biopsies was 510 μm² for type I, 1020 μm² for type II A and 860 μm² for type II B.Expressed as coefficient of variation (CV) these SD constituted 10, 15 and 15%, respectively. The variation in fibre size within a sample was considerably less than the variation between samples on the assumption that at least 15–20 areas of each fibre type were measured per sample. No difference in mean fibre area for type I, IIA and IIB fibres was obtained between the right and left muscle. Several artefacts due to the sampling and preparing procedures are discussed and a method for determining muscle fibre areas in biopsy samples is suggested.     

Influence of the interval between removal and freezing of muscle samples on muscle fibre size with special reference to sample size and fibre type

To study the influences of the interval between removal and freezing of a muscle sample on fibre size, rat hindlimb muscles (soleus and extensor digitorum longus, EDL) were cut into three or five pieces and frozen at different intervals, cross-sectioned, stained and analysed morphometrically. As in a previous study (Larsson & Skogsberg 1988), a pronounced interval-dependent change in soleus muscle fibre size was observed, the fibres in soleus muscle samples frozen immediately (within 15 s) being significantly (P less than 0.01-0.001) larger than those samples frozen 4 or 10 min after removal. However, the interval-dependent change was only noted in small muscle samples, i.e. those where the maximum muscle fibre length was 6.5 mm, while samples in which the fibres were approximately twice this length showed no change. In the slow-twitch soleus, type I fibres predominate, and type II fibres were often too few in one or more cross-sections in each animal to allow reliable measurements of fibre size. The fast-twitch EDL was therefore studied, and the results indicated an interval-dependent change in fibre size which was differentiated according to enzyme-histochemical type, i.e. there was a significant change in type I and IIA fibres but not in type IIB. It is concluded that the interval between removal and freezing of small muscle samples, as in percutaneous muscle biopsies in man, is of vital importance for fibre size, and that this interval should be at least 2-4 min.     

Differential effects of cold exposure on muscle fibre composition and capillary supply in hibernator and non-hibernator rodents

Changes in the composition of fibre types and the capillary supply of skeletal muscle (tibialis anterior) were quantified in rats and hamsters subjected to 8-10 weeks of cold exposure and reduced photoperiod (10 degrees C, 1 h light-23 h dark). Muscle mass decreased in both species (by 12% and 17%, respectively). Following acclimation to cold there were no specific changes in fibre cross-sectional area (FCSA) in rats, whereas in hamsters there was a substantial atrophy of Type II, but not Type I fibres. In rat muscle there was little difference between the two groups in average capillary to fibre ratio (C:F) (1.76 +/- 0.15, normothermia, N; 1.69 +/- 0.05, hypothermia, H) and average capillary density (CD) (188 +/- 14 mm(-2), N; 201 +/- 12 mm(-2,) H). Similarly, the average C:F was unaltered in hamsters (2.75 +/- 0.11, N; 2.72 +/- 0.15, H), although the 30 % smaller fibre size observed with hypothermia resulted in a corresponding increase in average CD, to 1539 +/- 80 mm(-2) (P < 0.01). However, there was a coordinated regional adaptation to cold exposure in hamsters resulting in capillary rarefaction in the glycolytic cortex and angiogenesis in the oxidative core. Following acclimation of rats to cold there was a reduction in the supply area of individual vessels (capillary domain), particularly in the cortex (9310, N; 8938 microm2, H; P < 0.05). In contrast, hypothermic hamsters showed only a small decrease in mean domain area in the cortex (948 microm2, N; 846 microm2, H; n.s.) but a marked reduction in the core (871 microm2, N; 604 microm2, H; P < 0.01). Rats showed little or no change in local capillary supply (LCFR) to fast fibres on acclimation to cold, while in hamsters the LCFR of Type IIb fibres showed a decrease in the cortex (2.7, N; 2.3, H) and an increase in the core (3.0, N; 3.3, H) during acclimation to cold. These data suggest that during a simulated onset of winter rats maintain FCSA and capillary supply as part of an avoidance strategy, whereas hamsters increase muscle capillarity in part as a consequence of disuse atrophy.     

Effect of exercise on concentrations of free amino acids in pools of type I and type II fibres in human muscle with reduced glycogen stores

A few animal studies have shown that some amino acid concentrations vary between different muscle fibre types. In the present study, amino acid concentrations were measured in separate pools of different fibre types in human skeletal muscle, with reduced glycogen stores, before and after sustained exercise. Five subjects exercised at a submaximal work rate for 60 min and then at a maximal rate for 20 min. Biopsy samples were taken from the vastus lateralis muscle before and after exercise; they were freeze-dried and individual fibres were dissected out. Fragments of these fibres were stained for myosin-adenosine triphosphatase (ATPase) and identified as type I or type II fibres. The concentrations of free amino acids were measured by high performance liquid chromatography (HPLC) in perchloric acid (PCA) extracts containing pools of either type of fibre. After exercise, glycogen was decreased in type I fibres (53%) and in four subjects also in type II fibres. The concentrations of most amino acids were similar in the two fibre types before exercise, but the glutamate, aspartate and arginine levels were 10% higher in type II than in type I fibres. After exercise, the glutamate concentration was decreased by 45% in both fibre types and the branched-chain amino acids (BCAA) were decreased in type II fibres (14%). Exercise caused an increase by 25-30% in tyrosine concentration in both type I and type II fibres. The results show that amino acids can be measured in pools of fibre fragments and suggest that amino acid metabolism play an important role in both type I and type II fibres during exercise.     

Resynthesis of glycogen in different muscle fibre types after prolonged exhaustive exercise in man

The rate of glycogen resynthesis was examined in different muscle fibre types after prolonged exhaustive exercise. Six subjects exercised to exhaustion at 75% of VO2 max, and muscle biopsies were taken after 0, 90 and 180 min of recovery. Glucose drinks (1.4, 0.7 and 0.7 g kg-1 body wt) were taken at time 0, 60 and 120 min. Photometric determination of periodic acid-Schiff stain intensity revealed a 65% faster rate of glycogen resynthesis in type IIA and IIAB as compared to type I fibres during the first 90 min. Thereafter no differences between the various fibre types were detected. No differences in the rate of glycogen resynthesis were observed between the subgroups of type II muscle fibres. These results suggest that there was a slower acceleration of glycogen resynthesis in type I compared to type II fibres. In all fibre types a positive relationship between rate of synthesis and glycogen concentration was observed. It is suggested that the size of the glycogen molecule and hence the number of available terminal glucosyl units is a major determinant of rate of resynthesis.     

Effect of hyperthyroidism on fibre-type composition, fibre area, glycogen content and enzyme activity in human skeletal muscle

Seven hyperthyroid patients were studied by repeated muscle biopsies (vastus lateralis) before and after a period of medical treatment which averaged 10 months. The biopsies were analysed with regard to fibre-type composition, fibre area, capillary density, glycogen content and enzyme activities representing the glycolytic capacity (hexokinase, 6-phosphofructokinase), oxidative capacity (oxoglutarate dehydrogenase, citrate synthase) and Ca2+- and Mg2+-stimulated ATPase in muscle. In the pretreatment biopsy (hyperthyroid state), there was a significantly lower proportion of type I fibres (30% vs. 41%), a higher capillary density (23%), lower glycogen content (33%), and higher hexokinase activity (32%) compared with the post-treatment biopsy. No significant changes in the activity of the remaining enzymes were observed. The present study indicates that hyperthyroidism induces a transformation from type I to type II fibres in human skeletal muscle. The increase in hexokinase activity probably reflects a higher glucose utilization by skeletal muscle in order to compensate partially for the reduced glycogen content.     

Glycogen depletion of human skeletal muscle fibers in response to high-frequency electrical stimulation.

The purpose of the present study was to evaluate the pattern of change in muscular glycogen content in response to high-frequency electrical stimulation (HFES). Muscle biopsies were taken from the vastus lateralis muscle of 7 healthy young men before, 15 min after, and 30 min after electrical stimulation delivered at a 50-Hz frequency (15 s on, 45 s off) at an intensity of 100 mA. The glycogen content of type I, IIA, and IIB muscle fibres was evaluated using microphotometry of periodic acid Schiff (PAS) stained fibres. After 15 min of electrical stimulation, the glycogen content in type I, IIA, and IIB muscle fibres significantly decreased from 113 +/- 10 (mean +/- SE) to 103 +/- 10 (p < or = 0.05), 129 +/- 9 to 102 +/- 12 (p < or = 0.01), and 118 +/- 8 to 90 +/- 13 (p < or = 0.01) arbitrary relative units, respectively. No further decrement in glycogen content was observed in all three fibre types following an additional 15 min of HFES. In addition, isometric force decreased by approximately 50%, from 125.9 +/- 20.0 N to 64.2 +/- 7.7 N (p < or = 0.01), during the first 15 contractions. No further decrease in isometric force was observed following an additional 15 contractions of HFES. These results reveal that significant reductions in isometric force of knee extensor muscles and glycogen content of all human skeletal muscle fibre types in vastus lateralis muscle are observable after 15 min of neuromuscular high-frequency transcutaneous electrical stimulation.     

Differences of morphometrical parameters in hind limb muscle fibres between ovarectomized and sexually intact female dogs.

Slow and fast twitch fibres of the Mm. tibialis cranialis, semitendinosus and sartorius of seven sexually intact and seven ovarectomized female beagles were histochemically and morphometrically analysed. Along with type I and type IIA fibres, another main type II fibre (IIS), which seems to be peculiar to the dog, was found in the Mm. semitendinosus and tibialis cranialis. Type I fibers comprised 26% and type II fibres 74% of all recorded muscle fibres in the M. tibialis cranialis, 29% (type I) and 71% (type II) in the M. semitendinosus and 51% (type I) and 49% (type II) in the M. sartorius, respectively. The average single profile area and the corresponding mean diameter of fibre types I and II in the investigated hind limb muscles were generally larger in ovarectomized than in sexually intact animals. This was more evident in type II than in type I fibres. However only the type II fibres of the M. tibialis cranialis and sartorius exhibited a statistically significant increase in diameter (p < 0.01 and p < 0.05, respectively). Accordingly, the mean density (number of fibres/mm2) of both fibre types in the hind limb muscles of spayed dogs was generally reduced. Again, this reduction attained statistical relevance in the type I and II fibres of the tibialis cranialis. In addition, the fibre densities of type I in the semitendinosus and type II in the sartorius muscles were also significantly reduced in ovarectomized dogs. In conclusion, ovarectomized beagles showed a generally increased mean diameter of the investigated type I and II hind limb muscle fibres and a concomitant decreased average fibre density of the respective types when compared to sexually intact animals.     

Morphometric analysis of skeletal muscle fibres and capillaries in mitochondrial myopathies.

A morphological and quantitative study of skeletal muscle fibres and of capillary supply was performed on needle and open biopsies of quadriceps femoris muscle from 40 patients with chronic progressive external ophthalmoplegia (CPEO) with partial deficiency of cytochrome c oxidase (COX). Muscle biopsies from 5 healthy adult subjects were used as control. The study was carried out by light and electron microscopy, using an Automatic Interactive Image Analysis System (IBAS I,II). A significant decrease in fibre diameters and preferential type I fibre atrophy was seen. Red ragged fibres and fibres with cytoarchitectural changes after enzyme-histochemical reactions for detection of oxidative activities were also observed. Seventy per cent of affected fibres showed an intense subsarcolemmal rim of oxidative activity corresponding to ultrastructural accumulation of enlarged, polymorphous mitochondria in subsarcolemmal areas. The study of the capillary distribution in muscle revealed a reduction of the number of capillaries per fibre and surrounding a fibre. The primary metabolic error of the disease with defects in the oxygen utilisation, the fibre atrophy and the muscle disuse are the possible variables influencing the capillary number in CPEO patients.