Path. Res. Pract. 188, 607-611 (1992)
Morphometric Analysis of Skeletal Muscle Fibres and Capillaries in Mitochondrial Myopathies R. Scelsi Department of Human Pathology, Section of Pathology, University of Pavia, Pavia, Italy
SUMMARY 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,Il). A significant decrease in fibre diameters and preferendcd 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.
Introduction
Mitochondrial myopathies are a heterogeneous group of genetic myopathies and encephalomyopathies that can affect the skeletal muscle alone or as part of a multisystem disease. They are characterized by several specific errors of mitochondrial metabolism and are generally associated with morphological alterations of muscle mitochondria5, 7,12,.19. In recent years, patients showing bilateral ptosis, ophthalmoplegia and progressive muscular weakness have been classified as chronic progressive external ophthalmoplegia (CPEO), the commonest mitochondrial myopathy. Complete lack of histochemically demonstrable cytochrome c oxidase (COX)_.activity has been observed in individual muscle fibres of several patients with CPE03,19. © 1992 by Gustav Fischer Verlag, Stuttgart
In the present study we report on the morphological and quantitative analysis of fibres and capillary bed of quadriceps femoris muscle biopsies from 40 patients with CPEO with partial COX deficiency. Attention was focused on the fibre type distribution and morphology, on the oxidative enzyme-histochemical reactions and 011 measures of the intramuscular capillary distribution.
Material and Methods Forty patients of both sexes, their age ranging from 24-56 years, who showed muscle weakness, bilateral ptosis and external ophthalmoparesis, were studied. TIle ischaemic forearm work test revealed a pathological increase of lactate after .2 min:up to 5.78 mmoVI (normal value: max 5 mmolll). In ten patients the serum level of creatine phosphokinase (CPK) and 0344-0338/9210 18 8-0607$3.5 010
608 . R. Scelsi lactate dehydrogenase (LDH) were increased. An electromyography revealed myopathic signs, especially in the extremities. The clinical diagnosis was exercise-intolerance and myopathy due to the so-called neuromuscular mitochondriopathy. A detailed record of clinical and laboratory findings of the patients was reported in previous papers14.17.18. The biopsies were taken under local anesthesia from the quadriceps femoris muscle. One part was frozen in isopentane cooled in liquid nitrogen at -170°C and transverse cryostat sections were stained with H & E, modified Gomori trichrome stain, PAS, Oil red 0 and were prepared for the following enzyme-histochemical reactions: reduced nicotinamide adenine dinucleotide (NADH) tetrazolium oxidoreductase, ATPase after alkaline and acid preincubation, succynic dehydrogenase (SDH), cytochrome c oxidase, acid and alkaline phosphatase according to standard techniques 6 • The second part was divided into several small portions, fixed in Karnovky fluid and then embedded in epoxy resin. Semithin transverse sections were stained with p-phenylenendiamine and toluidine blue, and examined under light microscopy. Thin sections were stained with lead citrate and examined under a Zeiss EM 9 electron microscope. The last part of the biopsy was fixed in 10 % neutral formalin and transverse epoxy-resin-embedded sections were stained with H & E, and with Gomori silver impregnation for reticulin. Morphometric analysis of muscle fibre types and diameter was performed on transverse cryostat sections treated for acid and alkaline ATPase. Histometric measurements (orthogonal diameter) of 200 fibres of a random coherent area were done with an IBAS I-II automatic imaging analyzer (Kontron). Fibres with mitochondrial abnormalities were detected by the modified Gomori trichrome staining and by enzymehistochemical reactions for NADH, SDH and COX; they revealed the so-called red ragged fibres and striking disorders in the oxydative enzyme distribution in muscle fibres. Overall capillary measures were performed on transverse semithin sections of epoxy-resin-embedded material and on transverse formalin-fixed sections embedded in epoxy resin and stained with a silver impregnation for reticulin. The following values were determined for each of the biopsies: capillaries per fibre (c/F), number of capillaries surrounding a single fibre (CAF) and capillary density (CD; capillaries per sq mm of muscle fibre). The CIF ratio was determined by counting muscle fibres and capillaries on a total of 20 micrographs from four semithin transverse plastic sections. The CD was determined from the same micrographs. The mean CAF value was determined by counting the capillary number surrounding a minimum of 100 fibres. The capillaries were identified directly from the sections while viewed at 400X magnification. The various sample sizes utilized for calculating the type I fibre percentage and diameter, c/F, CD and CAF were determined by doing sequential calculations of means and SD for the above measures on five histologically normal biopsies. Analysis of variance was used to test mean differences between normal biopsies from five healthy subjects and biopsies from myopathic patients 13 .
and fibres showing striking disorders in the sarcoplasmic oxidative enzyme activities were observed in all cases. The COX preparations revealed complete deficiency in the enzyme content in scattered fibres (4 to 15 % of muscle fibres). Electron microscopy showed degeneration of occasional fibres as seen by fragmentation of sarcomeres, autophagic vacuoles, myelin figures and lysosomes. The most striking finding was the presence of abnormal mitochondria with variable size and abnormal cristae. Electron dense round and rectangular paracristalline inclusions were seen between mitochondrial cristae. Quantitative Analysis
The fibre type composition and diameter of quadriceps femoris muscle from CPEO patients and from 5 normal subjects are reported in Table 1. Red ragged fibres and/or fibres with oxidative enzyme changes were present in all patients. Preferential type I fibre atrophy was seen in 50 % of CPEO patients and the mean fibre diameter was significantly reduced, as compared with normal biopsies. With regard to the structural changes seen after enzyme-
Results
Fibre Morphology The routine staining showed muscle fibre hypotrophy with variability in the fibre size and occasional internal nuclei. In 10 cases, scattered necrotic fibres undergoing phagocytosis had a positive reaction in the acid phosphatase preparations. A number of red ragged fibres showing subsarcolemmal accumulation of red granular material
Fig. 1. Fibres with subsarcolemmal aggregates; they are bordered by intensely staining material after reactions for oxidative enzyme activities. CPEO patient. NADH, X 250.
MItochondrIal MyopathIes .
6U~
Table 1. Morphometric analysis of muscle fibres in CPEO patients and in normal subjects (Mean ± SD) Fibre diam- Type 1 eter (fl,) fibre (%)
Fibres with Red ragged oxidative fibres (%) alterations (%)
CPEO cases (40) Normal adults (5)
38.4 ± 4.6 58.0 ± 7.4
0
38.0 ± 2.20 18.6 ± 8.2 56.0 ± 4.0
8.2 ± 2.6
-
CPEO patients versus normal adult subjects: 0p
Morphometric Analysis of Skeletal Muscle Fibres and Capillaries in Mitochondrial Myopathies R. Scelsi Department of Human Pathology, Section of Pathology, University of Pavia, Pavia, Italy
SUMMARY 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,Il). A significant decrease in fibre diameters and preferendcd 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.
Introduction
Mitochondrial myopathies are a heterogeneous group of genetic myopathies and encephalomyopathies that can affect the skeletal muscle alone or as part of a multisystem disease. They are characterized by several specific errors of mitochondrial metabolism and are generally associated with morphological alterations of muscle mitochondria5, 7,12,.19. In recent years, patients showing bilateral ptosis, ophthalmoplegia and progressive muscular weakness have been classified as chronic progressive external ophthalmoplegia (CPEO), the commonest mitochondrial myopathy. Complete lack of histochemically demonstrable cytochrome c oxidase (COX)_.activity has been observed in individual muscle fibres of several patients with CPE03,19. © 1992 by Gustav Fischer Verlag, Stuttgart
In the present study we report on the morphological and quantitative analysis of fibres and capillary bed of quadriceps femoris muscle biopsies from 40 patients with CPEO with partial COX deficiency. Attention was focused on the fibre type distribution and morphology, on the oxidative enzyme-histochemical reactions and 011 measures of the intramuscular capillary distribution.
Material and Methods Forty patients of both sexes, their age ranging from 24-56 years, who showed muscle weakness, bilateral ptosis and external ophthalmoparesis, were studied. TIle ischaemic forearm work test revealed a pathological increase of lactate after .2 min:up to 5.78 mmoVI (normal value: max 5 mmolll). In ten patients the serum level of creatine phosphokinase (CPK) and 0344-0338/9210 18 8-0607$3.5 010
608 . R. Scelsi lactate dehydrogenase (LDH) were increased. An electromyography revealed myopathic signs, especially in the extremities. The clinical diagnosis was exercise-intolerance and myopathy due to the so-called neuromuscular mitochondriopathy. A detailed record of clinical and laboratory findings of the patients was reported in previous papers14.17.18. The biopsies were taken under local anesthesia from the quadriceps femoris muscle. One part was frozen in isopentane cooled in liquid nitrogen at -170°C and transverse cryostat sections were stained with H & E, modified Gomori trichrome stain, PAS, Oil red 0 and were prepared for the following enzyme-histochemical reactions: reduced nicotinamide adenine dinucleotide (NADH) tetrazolium oxidoreductase, ATPase after alkaline and acid preincubation, succynic dehydrogenase (SDH), cytochrome c oxidase, acid and alkaline phosphatase according to standard techniques 6 • The second part was divided into several small portions, fixed in Karnovky fluid and then embedded in epoxy resin. Semithin transverse sections were stained with p-phenylenendiamine and toluidine blue, and examined under light microscopy. Thin sections were stained with lead citrate and examined under a Zeiss EM 9 electron microscope. The last part of the biopsy was fixed in 10 % neutral formalin and transverse epoxy-resin-embedded sections were stained with H & E, and with Gomori silver impregnation for reticulin. Morphometric analysis of muscle fibre types and diameter was performed on transverse cryostat sections treated for acid and alkaline ATPase. Histometric measurements (orthogonal diameter) of 200 fibres of a random coherent area were done with an IBAS I-II automatic imaging analyzer (Kontron). Fibres with mitochondrial abnormalities were detected by the modified Gomori trichrome staining and by enzymehistochemical reactions for NADH, SDH and COX; they revealed the so-called red ragged fibres and striking disorders in the oxydative enzyme distribution in muscle fibres. Overall capillary measures were performed on transverse semithin sections of epoxy-resin-embedded material and on transverse formalin-fixed sections embedded in epoxy resin and stained with a silver impregnation for reticulin. The following values were determined for each of the biopsies: capillaries per fibre (c/F), number of capillaries surrounding a single fibre (CAF) and capillary density (CD; capillaries per sq mm of muscle fibre). The CIF ratio was determined by counting muscle fibres and capillaries on a total of 20 micrographs from four semithin transverse plastic sections. The CD was determined from the same micrographs. The mean CAF value was determined by counting the capillary number surrounding a minimum of 100 fibres. The capillaries were identified directly from the sections while viewed at 400X magnification. The various sample sizes utilized for calculating the type I fibre percentage and diameter, c/F, CD and CAF were determined by doing sequential calculations of means and SD for the above measures on five histologically normal biopsies. Analysis of variance was used to test mean differences between normal biopsies from five healthy subjects and biopsies from myopathic patients 13 .
and fibres showing striking disorders in the sarcoplasmic oxidative enzyme activities were observed in all cases. The COX preparations revealed complete deficiency in the enzyme content in scattered fibres (4 to 15 % of muscle fibres). Electron microscopy showed degeneration of occasional fibres as seen by fragmentation of sarcomeres, autophagic vacuoles, myelin figures and lysosomes. The most striking finding was the presence of abnormal mitochondria with variable size and abnormal cristae. Electron dense round and rectangular paracristalline inclusions were seen between mitochondrial cristae. Quantitative Analysis
The fibre type composition and diameter of quadriceps femoris muscle from CPEO patients and from 5 normal subjects are reported in Table 1. Red ragged fibres and/or fibres with oxidative enzyme changes were present in all patients. Preferential type I fibre atrophy was seen in 50 % of CPEO patients and the mean fibre diameter was significantly reduced, as compared with normal biopsies. With regard to the structural changes seen after enzyme-
Results
Fibre Morphology The routine staining showed muscle fibre hypotrophy with variability in the fibre size and occasional internal nuclei. In 10 cases, scattered necrotic fibres undergoing phagocytosis had a positive reaction in the acid phosphatase preparations. A number of red ragged fibres showing subsarcolemmal accumulation of red granular material
Fig. 1. Fibres with subsarcolemmal aggregates; they are bordered by intensely staining material after reactions for oxidative enzyme activities. CPEO patient. NADH, X 250.
MItochondrIal MyopathIes .
6U~
Table 1. Morphometric analysis of muscle fibres in CPEO patients and in normal subjects (Mean ± SD) Fibre diam- Type 1 eter (fl,) fibre (%)
Fibres with Red ragged oxidative fibres (%) alterations (%)
CPEO cases (40) Normal adults (5)
38.4 ± 4.6 58.0 ± 7.4
0
38.0 ± 2.20 18.6 ± 8.2 56.0 ± 4.0
8.2 ± 2.6
-
CPEO patients versus normal adult subjects: 0p
