PATHOLOGY OF SKELETAL MUSCLE IN FIBROMYALGIA: A HISTO-IMMUNOCHEMICAL AND ULTRASTRUCTURAL STUDY

Transcription

1 British Journal of Rheumatology 1993;32:479^83 PATHOLOGY OF SKELETAL MUSCLE IN FIBROMYALGIA: A HISTO-IMMUNOCHEMICAL AND ULTRASTRUCTURAL STUDY A. M. DREWES*, A. ANDREASEN*, H. D. SCHR0DERt, B. HOGSAA* and P. JENNUM *Department of Rheumatology, Viborg County Hospital, DK-8800 Viborg; f University Department of Pathology, Odense Hospital, DK-5000 Odense; ^Department of Orthopaedic Surgery, Viborg County Hospital, DK-8800 Viborg and University Department of Neurology, Hvidovre Hospital, DK-2650 Hvidovre, Denmark SUMMARY The value of muscle biopsy infibromyalgiais still questioned. In this study we obtained 50 quadriceps biopsies from 20 patients and compared them blindly to 10 biopsies from five normal controls. Using light microscopy, histochemical and immunoenzymatic methods we found no definite evidence of muscle disease. Nevertheless, we subjected biopsies from nine of the patients andfiveother controls for further ultrastructural evaluations and demonstrated pathologicfindingse.g. empty sleeves of basement membrane, many lipofuschin bodies and other degenerative changes. We conclude that ultrastructural evaluation cannot yet be used for diagnostic purposes, but the negativefindingswith light microscopy, including histochemical and immunoenzymatic techniques, might be of importance in evaluating difficult cases. KEY WORDS: Fibromyalgia; Muscular diseases; Immunohistochemistry; Light microscopy; Electron microscopy. FIBROMYALGIA is a syndrome characterized by chronic musculoskeletal pain and multiple tenderpoints (TP) [1], but apart from the clinical examination, no diagnostic tests to confirm the diagnosis exist. The aetiology of the condition is not understood, but as myalgia and muscle weakness are central parts of the syndrome [2, 3] muscle biopsy studies should be expected to demonstrate pathologicfindings.in a few studies various light microscopical (LM) changes has been described including 'rubberband structures' [4] and 'moth-eaten' as well as ragged red fibres [5]. However the observations have not been generally accepted and needs to be confirmed. Electron microscopic (EM) studies of TP in the trapezius muscle [6] have demonstrated non-specific histologicalfindings,but as the trapezius is under continuous stress during standing and sitting, the relevance of these findings is unclear and further studies from other tender muscles have been suggested. As ultrastructural abnormalities have been seen in muscle biopsies from the quadriceps muscle in a variety of rheumatic diseases [7], this muscle was chosen for the study. Biopsies from patients with fibromyalgia were investigated using LM, EM, histochemical and immunoenzymatic techniques. Results were compared blind to biopsies from age-and sex matched controls. MATERIALS AND METHODS Twenty outpatients seen at the Department of Rheumatology, Viborg County Hospital were selected. All were suffering from fibromyalgia according to the Yunus criteria [2]. All were females with a median age of 49, range yr, and had symptoms Submitted 4 October 1991: revised version accepted 24 June for median 6 yr, range 1-25 yr including pain in the thigh muscles. None of the patients had visible atrophy of the muscles at the objective examination. In the first procedure two biopsy specimens were taken with a Bergstrom needle [8] in the quadriceps. The midpoint of a line from the major trochanter to the inside of the knee joint on the right leg was selected. With this technique two pieces of muscle belly were obtained. One was embedded in Tissue-Tec and frozen in liquid nitrogen. The other was formalin fixed, embedded in paraffin and stained with haematoxylin-eosin and modified Gomori trichrome. For histochemistry, the frozen biopsies were stained for myofibrillar adenosine triphosphatase (ATPase) preincubated at ph 4.3,4.6 and 9.4 and nicotinamid adenin dinucleotide phosphate tetrazolium reductase (NADPHT-reductase). Mouse monoclonal antibodies were directed against HLA-DR (DK 22), HLA-ABC (W6/32) and DAKO Macrophage EMB 11, all manufactured by Dakopatt, Copenhagen, Denmark. The immunohistochemical method used was a three-layer peroxidase technique. As secondary antibody DAKO P 260 rabbit anti-mouse was used and as tertiary DAKO P 217 swine antirabbit, both peroxidase conjugated. The chromogen used was 3-amino-9-ethylcarbazole. Control biopsies were obtained with the same technique from five healthy females, median age 47 yr, range yr. During the study all patients had additionally a sleep polysomnographic recording. In 11 patients there were evidence of sleep apnoea (P. Jennum et al., unpublished observations). As an interaction of low oxygen tension during sleep and muscle histology could not be excluded, the nine patients without evidence of respiratory pathology were selected for a new biopsy for ultrastructural studies. These biopsies were taken using /93/ $08.00/ British Society for Rheumatology

2 480 BRITISH JOURNAL OF RHEUMATOLOGY VOL. 32 NO. 6 TABLE 1 ULTRASTRUCTURAL FINDINGS IN QUADRICEPS BIOPSIES FROM PATIENTS WITH FIBROMYALGIA Patient no. Empty sleeves of basement membrane Angular fibres Lipofuschin inclusions Filamentous disarray Nuclear aggregates Lipid droplets Slight changes in mitochondria m h FIG. 1. Ultrastructure of quadriceps muscle from a patient with fibromyalgia showing sleeves of basement membrane () in part empty, in part with cellular debris (arrow). Uranyl acetatelead citrate, x HLA-ABC appears on muscle membranes in inflammatory myopathy [11]. DAKO Macrophage is a specific marker for macrophages. RESULTS Light microscopy Haematoxylin-eosin and modified Gomori trichrome staining showed no sign of myofibrillar or interstitial abnormalities. We found no evidence of abnormal mitchondrial deposition. In some preparations small angulated fibres were found scattered between the normal muscle fibres. Histochemical techniques The distribution of type I, Ha and lib fibres appeared normal in all biopsies. We did not find 'moth-eaten' or ragged red fibres in biopsies from the patients or from the healthy controls. Immunoenzymatic techniques No expression of HLA-DR or HLA-ABC was found on muscle fibres, but some interstitial cells were positive. The DAKO Macrophage staining revealed no evidence of macrophage infiltration. In summary no evidence of inflammatory myopathy was found and therefore lymphocyte-subclass staining was not performed. Electron microscopy In one biopsy only a few muscle fibres were found and it was unsuitable for further investigation. The remaining eight biopsies all showed various degenerative manifestations, results are summarized in Table I. The most striking finding was empty sleeves of basement membrane (Fig. 1), which was found in six of the patients. As suggested at the LM level, atrophic angularfibreswere found scattered among the normal fibres (Fig. 2). In some of the patients the membrane was duplicated (Fig. 3). Focally nuclear aggregates and filamentous disarray were seen. Manifestations of cellular damage as lipofuschin inclusions were often found (Figs 2 and 3). In two patients more lipid droplets than normally found, were scattered between the myofilaments (Fig. 4). Mitochondria with slightly irregular crista patterns were observed in six cases (Fig. 5). Abnormal mitochondrial or glycogen deposits, mononuclear cell infiltration or subcellular papillary projections were not demonstrated. In some patients the sarcolemma appeared somewhat folded and myofibrillar separation was also demonstrated, we believe the Biopty system (Biopty, Radiplast, Sweden) with a 2 mm Tru-cut needle (Biopty-cut, Radiplast, Sweden) after incision of the skin [9]. As smaller amounts of tissue were necessary we performed only one biopsy from every patient. For normal comparison, biopsies from another five healthy females, median age 35 yr, range yr were used. The biopsies were fixed immediately in 2% glutaraldehyde and then transferred to 1% osmium tetraoxide and processed for standard transmission electron microscopy with uranyl acetate and lead citrate for staining. Following light microscopical examination in 1 u.m epon sections three areas were selected for further ultrastructural examination. All assessments were performed blind. Modified Gomori trichrome is valuable for grading of some abnormal cytoplasmatic structures as 'ragged red fibres' and distribution of mitochondria in certain types of muscular disease e.g. mitochondrial and inclusion body myopathy. ATPase staining is used to visualize the distribution of type I, Ha and lib fibres. Staining with NADPHT-reductase demonstrates fibrillary organization e.g. 'moth-eaten' and mitochondrial distribution. The anti HLA-DR immunoenzymatic staining techniques have proved to be specific for inflammation as HLA-DR reactivity on the cellular surface roughly correlates with the extent of inflammation [10]. Furthermore the anti HLA-DR demonstrates B lymphocytes and activated T lymphocytes.

3 DREWES ETAL: SKELETAL MUSCLE PATHOLOGY IN FIBROMYALGIA 481 these changes are artefacts due to the contraction of the muscle fibres during the biopsy and fixation procedures. The vessels and the amount of collagen and elastin between muscle fibres were normal. None of the controls demonstrated abnormal findings. DISCUSSION In this study we have demonstrated various pathologic findings in the quadriceps muscle in patients with fibromyalgia. Studies of biopsies from TP in the trapezius muscle in patients with fibromyalgia [5, 6] have suggested pathologic findings using histochemistry staining methods and EM. However these findings are non-specific, and Yunus etal. [12] reported in a controlled and blind study the same ultrastructural abnormalities in healthy subjects. As the trapezius muscle is under continuous strain and contraction the findings are supposed to be a result of the normal stress on this muscle. For this reason we chose the quadriceps muscle as the histology of the muscle is very well- FIG. 3. Same muscle fibre as in Fig. 2 demonstrating duplication of the membrane (arrow). Filamentous disarray (FD) of the myofibrils is also seen. A lipofuschin inclusion (L) can be seen in the neighbouring cell, x FIG. 2. Section of quadriceps muscle showing an atrophic angular fibre with nuclear aggregates (NA) and empty sleeves of basement membrane (). Lipofuschin inclusions (L) are also seen, x 4600.

4 482 BRITISH JOURNAL OF RHEUMATOLOGY VOL. 32 NO. 6 known and most patients complain of pain in the thighs as a part of the syndrome. In addition biopsy specimens are easily obtained from the quadriceps. We added biopsies from healthy control subjects to eliminate errors due to the preparation procedure. We did not take the biopsy directly from TP as we found it more valuable to use a uniform biopsy-procedure from the same localization in order to compare the individual biopsies with similar biopsies from other materials. We used needle biopsies as this method is rapid and provides sufficient amounts of high quality tissue (approximately 300 fibres) combined with minimal patient discomfort. The technique has proved to be extremely FIG. 5. Structurally abnormal mitochondria with crista having a slightly irregular pattern, x FIG. 4. Increased number of lipid droplets in the quadriceps muscle of a patient with fibromyalgia. x reliable and has become our routine procedure for muscle biopsy [9]. Barthels et al. [4, 13] demonstrated 'rubberband' structures around the fibres using a teased fibre technique in the quadriceps after glycerination. However at the ultrastructural level we did not demonstrate elastic fibres, collagen or evidence of other pathologic connections between or around the cells. The immunoenzymatic techniques have demonstrated the non-inflammatory nature of the disease and there was no evidence of primary myopathy. The various degenerative changes at the ultrastructural level were non-specific and can be seen in neurogenic atrophy and other muscle diseases [14], in a variety of rheumatic diseases [7] as well as in older age groups. The findings might be caused by metabolic disorders e.g. reduced tissue oxygenation and alterations in high energy phosphates as demonstrated in TP of the trapezius muscle of patients with fibromyalgia [15, 16] or related to increased muscle tension as indicated by EMG studies [17]. The changes could not be a result of immobility as all our patients functioned normally, although they could be expected to exercise to a lesser degree due to muscle pain and stiffness. None of the patients used corticosteroids, but only simple analgesics which could not induce changes in the muscles. Although pain in the muscles is a primary symptom in fibromyalgia, descriptions of the pain are that it is mostly diffuse and can be localized to both tendons and joints. Nevertheless pathological findings were to be expected in muscles if in any tissue. Two patients had an increased number of lipid droplets. The number of lipid droplets in normal skeletal muscle is variable and increases when the fatty acids in the blood are elevated or in carnitine deficiency. Nevertheless no drugs which would interfere with free fatty acids were given and plasma levels of carnitine was normal. In conclusion discrete ultrastructural abnormalities are found in muscle biopsies from the quadriceps in

5 DREWES ETAL.: SKELETAL MUSCLE PATHOLOGY IN FIBROMYALGIA 483 patients with fibromyalgia compared to normals. However, EM cannot yet be used for diagnostic purposes, and the relevance of the findings must await further studies. The negative findings at LM, including histochemical and immuno-enzymatic techniques, might be of importance in evaluating difficult cases. ACKNOWLEDGEMENTS We wish to thank Drj0rgen Buhl for taking the biopsies for electron microscopy and Dr Thorsten Ingemann- Hansen for advice and assistance. REFERENCES 1. The American College of Rheumatology Criteria for the classification of fibromyalgia. Arthritis Rheum 1990;33: Yunus MB, Masi AT, Calabro JJ, Miller KA, Feigenbaum SL. Primary fibromyalgia (fibrositis): clinical study of 50 patients with matched normal controls. Semin Arthritis Rheum 1981;11: Jacobsen S, Danneskiold-Sams0e B. Isometric and isokinetic muscle strength in patients with fibrositis syndrome. Scand J Rheumatol 1987;16: Bartels EM, Danneskiold-Sams0e B. Histological abnormalities in muscle from patients with certain types of fibrositis. Lancet 1986;i: Bengtsson A, Henriksson KG, Larsson J. Muscle biopsy in primary fibromyalgia. Light-microscopical and histochemical findings. Scand J Rheumatol 1986;15:l Kalyan-Raman UP, Kalyan-Raman, Yunus MB, Masi AT. Muscle pathology in primary fibromyalgia syndrome: a light microscopic, histochemical and ultrastructural study. J Rheumatol 1984;ll: Russel ML, Hanna WM. Ultrastructural pathology of skeletal muscle in various rheumatic diseases. J Rheumatol 1988;15: Bergstrom J. Muscle electrolytes in man. Scand J Clin Lab Invest 1963;suppl 68. ANNOUNCEMENT 9. Lindequist S, Larsen C, Schr0der HD. Ultrasound guided needle biopsy of skeletal muscle in neuromuscular disease. Acta Radiologica 1990;31: Olsson T, Henriksson KG, Klareskog L, Forsum U. HLA-DR pression, T-lymphocyte phenotypes, OKM1 and OKT9 reactive cells in inflammatory myopathy. Muscle and Nerve 1985;8: Appleyard ST, Dubowitz V, Dunn MJ, Rose ML. Increased expression of HLA ABC class I antigens by muscle fibres in Duchenne muscular dystrophy, inflammatory myopathy, and other neuromuscular disorders. Lancet 1985;ii: Yunus MB, Kalyan-Raman P, Masi AT, Aldag JC. Electron microscopic studies of muscle biopsy in primary fibromyalgia syndrome: a controlled and blinded study. J Rheumatol 1989;16: Jakobsen S, Barthels EM, Danneskiold-Sams0e B. Single cell morphology of muscle in patients with chronic muscle pain. Scand./ Rheumatol 1991 ;20: Carpenter S, Karpati G. Normal organelles and constituents of skeletal muscle cells and their pathological reactions. In: Carpenter S, Karpati G, eds Pathology of skeletal muscle. New York: Churchill Livingstone, 1984: Lund N, Bengtsson A, Thorborg P. Muscle tissue oxygen pressure in primary fibromyalgia. Scand J Rheumatol 1986;15: Bengtsson A, Henriksson KG, Larsson J. Reduced high-energy phosphate levels in the painful muscles of patients with primary fibromyalgia. Arthritis Rheum 1986;29: Elert JE, Rantapaa SB, Henrikson-Larsen K, Gerdle B. Increased EMG activity during short pauses in patients with primary fibromyalgia. Scand J Rheumatol 1989;18: ROYAL SOCIETY OF MEDICINE, SECTION OF RHEUMATOLOGY AND REHABILITATION MEETING Date: June Combined meeting with the East Anglian Society for Rheumatology and Rehabilitation. Further details available from: Miss Louisa Raine, The Royal Society of Medicine, 1 Wimpole Street, London W1M 8AE. Tel: ext. 211.