A C T A O P H T H A L M O L O G I C A VOL. 5 4 1976
Department of Neuropathology Arhus Kommunehospital, University of Aarhusl) (Head: Edith Reske-Nielsen), Department of Ophthalmology, Rigshospitalet, Copenliagen2) (Head: Eilif Gregersen) and Department of Ophthalmology, Arhzrs Kommunehospital, University of Aarhus, Denmark3) (Head: Niels Ehlers)
PROGRESSIVE EXTERNAL OPHTHALMOPLEGIA Evidence for a Generalised Mitochondria1 Disease with a Defect in Pyruvate Metabolism BY
EDITH RESKE-NIELSENl), HANS C. L O U ) and MARTIN LOWESs)
Muscle biopsies from four patients with chronic progressive external ophthalmoplegia and pigmentary retinopathy with symptoms and signs from other organs were studied by means of light and electron microscopy. Examination revealed a marked proliferation of abnormal mitochondria with a degeneration of both muscle and nerve tissue. Blood levels of lactate and pyruvate were measured and abnormal values of these metabolites were found in the three patients with the most pronounced ultrastructural changes. On the basis of these findings it is suggested that there is a biochemical defect in pyruvate-lactate metabolism which could be responsible for the marked proliferation of the abnormal mitochondria.
Key words: ophthalmoplegia plus - muscle biopsy - abnormal mitochondria - metabolic defect - blood pyruvate - blood lactate.
Chronic progressive external ophthalmoplegia w a s originally considered t o b e
a disease primarily affecting.brain stem nuclei, a s first described by v o n G r a e f e (1866). However, Kiloh & Nevin'(1951) w e r e able t o demonstrable histopathoReceived June 25, 19876.
553
Edith Reske-Nielsm, H a n s C . Lou and Martin Lowes
logical changes in the extraocular muscles and concluded that the disorder was due to a myopathy. T h e neural changes reported in earlier studies were regarded as inconclusive. This view has prevailed in the majority of subsequent studies (Schwarz & Liu 1954; Beckett & Nersky 1953; Kearns & Sayre 1958; Cogan et al. 1962; Lind & Prame 1963). Koerner & Schlote (1972) stated that there was not enough evidence to consider chronic progressive external ophthalmoplegia as part of an entity of neurogenic disorders. However, it has become increasingly difficult to explain chronic progressive external ophthalmoplegia as being purely and simply a myopathy of the extraocular muscles. Involvement of other facial muscles, limb musculature, cerebellum, spinal tracts, myocardium, endocrine glands, hearing, and probably most frequent of all, retina, with pigmentary changes and chorioretinal atrophy has amply been described in the recent literature (Alfano & Berger 1957; Kearns & Sayre 1958; Jager et al. 1960; Drachman 1968; Rosenberg et al. 1968; Daroff 1969; Lowes 1975). Endocrine abnormalities have been reported by Lundberg (1962). Abnormal mitochondria in muscle tissue have been demonstrated by Gonatas (1967), Mair & Tom6 (1972), Morgan-Hughes & Mair (1973), Iamaccone et al. (1974) and Schlote & Korner (1976). I n a preliminary report by two of the authors (Lou & Reske-Nielsen 1976) a biochemical defect in pyruvate-lactate metabolism in three of the patients involved in the present study has been suggested. The purpose of the present study is to report on the ultrastructure of muscle biopsies in four cases of progressive external ophthalmoplegia and to attempt to correlate the findings with the proposed metabolic defect in pyruvate metabolism.
Material and Methods T h e material comprised four patients, two male and two female, aged from 13-50 years. The patients all had a chronic external ophthalmoplegia and pigmentary retinopathy with a variety of associated disorders including cardiac conduction defects, endocrine disturbances and central nervous system involvement. T h e main points from the case histories are summarized in Table I. Muscle biopsies were taken from the left deltoid muscle in cases 1, 2 and 3. In the fourth case biopsies were taken from the right superior rectus muscle, the right levator palpebrae muscle and the I'eft deltoid muscle. 'The biopsies were taken and prepared for histological examination as described by ReskeNielsen et al. (1969). T h e muscle specimens for electron microscopy were fixed in 2 O/o glutaraldehyde for approximately 3 h and 1 O/o osmium tetroxide for 554
Progressive Externul Ophthdmoplegia
Table I. Summary of the case histories Cases Age on presentation (years) Sex
1 50
2 23
3 17
25
M
M
F
F
+ + + +
+ +
+ + + +
-
-
+
+
+ +
+
0
+ +
4
~~
Ptosis and ophthalmoplegia Pigmentary retinopathy Cardiac conduction defects Cardiac complaints Cardiac pacemaker Frail build. Low average intelligence Deafness Facial weakness Secondary sexual abnormalities Abnormal electroencephalography Abnormal electromyography Abnormal electroretinography Abnormal colour vision Abnormal dark adaptation Enlarged blind spot Elevated CSF protein Asymptomatic diabetes Raised Sc-cholesterol Raised Se-lactate dehydrogenase Raised Se-aldolase Raised Se-creatine phosphokinase
Key:
+ present,
-
0
0
+
+
+ +
+ +
t.
0
0
0
+ +
+
0
-
+ 0
+
0 0
0
+ + + +
+ +
-
-
0 0 0
0
+ +
+
+ -
absent, 0 not recorded.
one h, then dehydrated with increasing alcohol concentrations (240/0 up to 99 o/g) and anhydrous acetone, and finally embedded in Vestopal W. Six grids from each of five blocks from each specimen were examined. T h e grids were stained with uranyl magnesium acetate and lead citrate and examined in a Zeiss EM 9 (Reske-Nielsen & Harmsen 1972). Electromyography was performed in cases 1 and 2 in the right deltoid muscle. In addition data on blood’ pyruvate and lactate concentrations were obtained. In three cases the method used fbr the estimation was that described by Lundholm et al. (1963). I n the remaining case the method described by Marbach & Weil (1967) was used. These findings are shown in Table 11. 555
Edith Reske-Nielsen, Hans C . Lou and Martin Lowes Tuble ZZ. Blood concentrations (mmolil) o l pyruvate and lactate.
Lactate (normal range 0..55-1.75) (sn 0.01)
Pyrzcvate (normal range 0.035-0.139) (SD 0.04) Patient 1 (rest) ., (rest) ,, (moderate exercise) Patient 2 (rest) Patient 3 (rest) ., (rest) ,, (moderate exercise)
0.160 0.143 0.191 0.082 0.137 0.108 0.152
2.58 2.28 4.85 1.59 2.77 1.99 3.62
Pyrzivate (normal range 0.02-0.08) Patient 4 (rest) ,, (rest)
Lactatp (normal range 0.625-1.67) 1.55 2.55
0.135 0.15
ResuIts Case 1. A 50-year-old male business executive who underwent a n operation for strabismus a t the age of 7 years. A left deltoid muscle biopsy was examined by the Department of Neurophysiology, Rigshospitalet.
Light microscopy. Increased number of internal nuclei. Several necrotic a n d basophilic fibres. In about 20’3/0 of the fibres excessive amounts of subsarcolemmal material (probably mitochondria) were present, which stained dark with lactate dehydrogenase and bright red with Gomori’s trichome stain. Electron microscopy. The cross striation of the myofibrils was well preserved but out phase in many fibres. Central myonuclei a n d the number of satellite cells were increased. Some fibres rontain subsarcolemmal accumulations of mitochondria. Intramitochondria1 crystals were abundant. Case 2. A 23-year-old male metal worker whotdeveloped ptosis of both eyes around the age of 14 years. A biopsy from the left deltoid muscle was examined.
Light microscofy. A few abnormal muscle fibres which appeared red a n d granular with Gomori’s trichome stain. Histochemistry was not performed.
556
Progressive External Ophthalmoplegiu Ekctrofz microscopy. Accumulations of mitochondria were seen close to the nucleus beneath the sarcoplasma membrane. Some showed abnormal cristae and vacuoles. Electron dense granules, presumably glycogen granules, were present. No paracrystalline inclusions could be demonstrated. Lipid vacuoles were seen between the myofibrils. Some myofibrils were atrophic and disorganized with spiky and flowing Z membranes. Case 3. A 17-year-old schoolgirl who developed a gradually progressive bilateral ptosis at the age of 8 years. A biopsy was taken from the left deltoid muscle.
Light microscopy. Several fibres in each fasicle contained loosely structured muscle fibres. These were granular and red-stained with Gomori’s trichrome stain. Furthermore, many basophilic muscle fibres with occasional necrotic fibres were seen. Histochemistry was not performed.
Fig. 1. a. Ultrastructure of the left deltoid muscle showing subsarcolemmal aggregations of abnormal mitochondria (Mi) and thinning of the myofibrils (Mf). Longitudinal section x 6000. (Case 3. E M 597).
b. Electron micrograph revealing large mitochondria (Mi) partly or totally filled by concentrically arranged membranes. Longitudinal section x 27 000. (Case 3. E M 597).
557
Edith Keske-Nielten, H U T ~C. S Lou arid M a r t i n Lowes
55s
Electroii microscopy. T h e dominating feature was a n abnormality o t the mitochondria. There were large aggregates of mitochondria beneath the sarcolcmma and between the myofibrils. T h e size of the mitochondria varied considerably, and many giant mitochondria were present. T h e configuration was also variable, and many bizarre forms were seen. T h e external membrane was double or triple and in some cases contained even more layers. T h e cristae were often absent or transversed the mitochondria. Some mitochondria were filled out by thin concentrically arranged membranes, often with two centres. Others had paracrystalline laminated inclusions and vacuoles with a single or double limiting membrane. T h e vacuoles were either empty or contained myelin bodies or electron dense granules. Furthermore, some mitochondria contained tuhular inclusions. Glycogen granules were abundant. Many giant mitochondria with a double limiting membrane were distended by closely packed electron dense granules. Between the mitochondria glycogen granules and lipid droplets were present. I n addition, many fibres were atrophic or even disrupted and the basal membranes were tolded. T h e sarcotubular system as well as the ve5sels were normal (Figs. 1 a, 1 h and 2).
Case 4. A 2.5-year-old wonian who developed ptosis of both eyes around the age of 10 years. This case has previously been published in Acta ophthal. (Kbh.) (Lowes 1975) and shows all the characteristics of the foregoing patients to a marked degree. Three muscle biopsies were taken - one from the right rectus superior muscle, one from the right levator palpebrae muscle taken a t the time of opcration for ptosis, a n d one from the left deltoid muscle. T h e ptosis operation has proved cosmetically successful with no exposure derect on closure.
Light rnicrosco/iy of superior rcctus (Neurop. No. 19131j. T h e scanty material consisted o l connective tissue with no inflammatory infiltration. There were a Jew muscle fibres of varying diameter, hut without any degenerative changes. With Gomori’s trichronie staining shiny, red granules were observed between the myofihrils. Enzyme histochemistry was not performed (Fig. 3 a, 3 b). Electron nzicroscopy of .fu/wrkir rcct2c.r. T h e muscle fibres and fibrils were small, as a result of a loss of myofilaments. T h e Z-lines showed a n extremely variable pattern with abnormally thin, thickened, spiky and flowing lines. Areas of muscle tissue were occasionally seen which had either disappeared or revealed “contraction clumps”. Folded and empty basement membranes could also be seen. T h e connective tissue was increased. T h e most striking ultrastructural changes were large aggregations of mitochondria. These were situated under the sarcoplasma membrane, either close to the nucleus or hetween the myofibrils. T h e mitochondria were much larger than normal and had a n extremely varied appearance (Fig. 3 c, 3 d). Some of these organelles were rounded possessing a single, double or triple membrane. Others had a bizarre club-shaped appearance. They werc either empty or contained a n electron dense material. Occasionally both vacuoles and granules were
. Fig. 2. Left deltoid muscle. Giant mitochondrion with ahnormally arranged cristae (Cr), vacuoles (Va) and glycogen material (G). On the left a sarcomere (Sj can be seen. Longitudinal section x 90 000. (Case 3 . EM 597). 559
F i g . 3 a, 0, c, d . (See text, p. 561) 560
Progressivt, External Ophthalmoplegiu found in the same mitochondrion. T h e cristae generally had a n abnormal arrangement with numerous concentric circles inside the organelles. Certain of the mitochondria appeared normal in one part, but abnormal in the other part. In the giant mitochondria a variable number of laminated, crystal-like inclusions could be seen within the cristae. T h e configuration of the laminated material depended on the plane of sectioning. T h e mitochondria often contained lipid bodies a n d glycogen granules. Lipid bodies were also seen in close proximity to the mitochondria (Figs. 4 a, 4 b, 5 a , 5 b). T w o end plates were successfully demonstrated. T h e axon, the primary a n d the secondary synaptic clefts were morphologically normal. T h e subneural apparatus, which is limited by the muscular sarcoplasmal membrane, contained numerous, large, closely packed, abnormal mitochondria which displaced the secondary synaptic clefts (not illustrated). (Neurop. No. 20843 prep. 3). T h e biopsy consisted of connective tissue with a few blood vessels and a scanty lymphocyte infiltration. I n the connective tissue there were occasional small groups of muscle fibres
Light microscopy of levator pal@me
Fig. 3. a. Light microscopy of the right superior rectus muscle showing muscle fibres (MF) in cross a n d longitudinal sections separated by connective tissue. x 40. (Case 4. LM 19131). b. Higher magnification of Fig. 3 a x 100. c. Electron micrograph of right superior muscle. Large collections of mitochondria (Mi) under the sarcoplasma membrane (SM) close to the nucleus (N). x 6000. (Case 4. E M 633). d. Thin myofibrils (Mf) with abnormal mitochondriia (Mi) in the interspaces. Longitudinal section x 6000.
Fig. 4. a. Right superior rectus muscle. Accumulation of bizarre and enlarged mitochondria (abn Mi) under the folded sarcoplasma membrane (SM). Lipid bodies (LB) can be seen. Longitudinal section x 27 000. (Case 4. E M 633). b. Mitochondria showing a varied appearance with a n abnormal internal structure (Mi) between thinned out myofibrils (Mf). Longitudinal section x 27 000. (Case 4. EM 633).
Fig. 5. a. Right superior rectus muscle showing a n area below the nucleus (N) which contains remnants of myofibrils (Mf) with laminated mitochondria (Mi) and lipid bodies (LB). Longitudinal section x 27 000. '(Case 4. E M 633). b. Bulging of the sarcoplasma membrane (SM) which contains large abnormal mitochondria (abn Mi) with a complex arrangement of the cristae. Longitudinal section x 27 000. (Case 4. EM 633).
56 I
Fig. 4 a, b. (See text, p. 561).
Fig. 5 u. b. (See text, p. 561).
Edith Reske-Nielsen, Huns C. Lou and Martin Lowes
Fig. 6 . a. Close-up of the adnexal region showing the marked ptosis. (Case 4). b. Biopsy from the right levator palpebrae muscle. Light microscopy shows connective tissue (COT) and fat cells (FC). No recognisable muscle fibres. x 100. (Case 4. LM 20843 prep. 3).
(maximum 3-4) with a rounded appearance a n d an intense stain with haematoxylineosin. Gomori’s trichrome stain was inconclusive. T h e small nerve branches were totally dernyelinated a n d contained only a few degenerated nerve fibres. Histochemistry was not performed (Fig. 6 a, 6 b).
564
Fig. 7 a, b. (See text, p. 5 6 6 ) .
565 Acta ophthal. 54, 5
37
Edith Reske-Nielsen, Hans C. Lou arid Martin Lowes Electron rnicrotcopy of leuator paljlebrae. All the micrographs revealed normal blood vessels, but with marked pinocytosis a n d considerable connective tissue. Abnormal mitochondria were occasionally observed in the cytoplasma of fibroblasts. Some of the mitochondria were empty, but in others a n abnormal arrangement of the cristae could be observed (Fig. 7 a, b). T h e preparation contained no recognisable muscle tissue.
Light rnzcroscojly of deltoid muscle (Neurop. No. 20843 prep. 1). Skin from the deltoid region examined by conventional technique was normal. Gomori’s trichrome stain was inconclusive. Electron microscopy was not periormed. Muscle fibres with a granular structure and single basophilic fibre segments were found in all fasicles. N o necrotic fibres were observed. I n the modilied Gomori trichrome stain almost every muscle fibre, especially the granular type, contained bright, shiny, red granules. These granules were either confined to the subsarcoleinmal regions or formed a coarse punctate pattern over the entire cross-sectional area of the fibre. There was a n increase in the number of nuclei, a n d these were occasionally centra!ly placed. Frozen sectzonc. A n intense staining of the intermyofibrillary structures was a striking feature of the haematoxylin-eosin staining. These fibrillary structures displayed a
ccarse reticular pattern when compared with normal material. Numerous small lipid droplets stained by Scharlach Rot were seen in cross-section of the muscle fibres. On histochemical examination the same areas were intensely stained with D P N H tetrazolium reductase a n d could be seen as abnormal deposits in the subsarcolemmal regions and over the entire cross-sectional area of the fibres. ATPase demonstrated a n absence of myofibrillary material in the affected regions of Fig. 7. a. Right levator palpebrae muscle. Mitochondria (Mi) containing abnormal cristae a r e to be found in the cytoplasm of a Fibroblast. x 27 000. (Case 4. E M 757). b. Higher magniiication of Fig. 8 b. x 81 000.
Fig. 8. a. Left deltoid muscle. Light microscopy (frozen section) with a n intense staining of the intermyofibrillary structures. T h e nuclei a r e marked by circles. (Haematoxylineosin). Cross-section x 400. (Case 4. L M 20843 prep. 2). b. Histochemical preparation ( D P N H ) . Both type 1 and type 2 muscle fibres are shown. Mitochondria1 enzymes a r e visualised as abnormal deposits under the sarcoplasma (marked by circles) a n d as a coarse network over the entire cross-section in both types of fibres. x 400.
Fig. 9. a. Left deltoid muscle. Section demonstrating the giant a n d peculiar mitochondria (Mi) with numerous crystalline inclusions sectiohed in various planes. Longitudinal section x 27 000. (Case 4. E M 756). b. Fingerprint-like configuration of mitochondria (Mi) lying between two myofibrils (Mf). Longitudinal section x 27 000.
566
Fig. 8 a, b. (See text, p. 566).
567
Fig. 9 a, b. (See text, p. 566). 568
Progressive External Ophthalmoplegia the muscle. The biopsy material showed a normal mosaic pattern of the type 1 and type 2 fibres. Both histochemical fibre types were afiected, but the changes were more pronounced in type 1 than in type 2 fibres (Fig. 8 a, 8 b). Electron microscopy o/ deltoid muscle. The alterations were similar to those described in the rectus superior muscle. The degenerative changes found in the muscle fibres were conspicuous with large aggregations of giant mitochondria located under the sarcoplasma membrane and between the fibrils. These organelles were often bizarre with an abnormal configuration and a varied arrangement of the cristae inside the mitochondria. Several mitochondria contained multiple crystalline inclusions within the cristae, especially the giant elongated ones. Vacuoles, glycogen, granular and osmiophilic material were also seen. These were often present in the same mitochondrion, together with the crystalline inclusions. Lipid and osmiophilic bodies often lay close to the mitochondria. Mitochondria containing a granular material and/or vacuoles were observed in a capillary vessel wall (Figs. 9 a, 9 b, 10 a. 10 b).
* Fig. 10 a and b. Left deltoid muscle. Closely packed mitochondria (Mi) showing double and triple membranes. Certain of the mitochondria contain electron dense granules, while others appear empty. Note also the club-shaped mitochondria with crystalline inclusions. x 27 000. (Case 4. EM 756).
569
Edith Reske-Nielsen, Hans C . Lou and M a r t i n Lawes
Discussion T h e material comprised four patients who had a chronic progressive external ophthalmoplegia with a variety of additional findings which are summarized in Table I. I n all of the patients abnormalities of muscle fibres were seen. Light microscopy showed the characteristic red colour of muscle fibres with Gomori’s trichrome stain. Electron microscopy revealed pronounced mitochondrial alterations. A characteristic feature in these patients was a marked proliferation of mitochondria which were much larger than normal and often quite bizarre. The internal structure of the cristae was complex with elongated crystalline inclusions. Abnormalities of muscle fibres were found, including “contraction clumps” which have also been described in families with malignant hyperthermia (Reske-Nielsen et al. 1975). T h e levator palpebrae muscle of case 4 showed an almost complete replacement of muscle fibres by connective tissue and f a t cells, with a marked loss of nerve fibres and myelin sheaths. Two end plates were observed, the secondary synaptic clefts were displaced by closely packed abnormal mitochondria. These findings were compared with normal muscle tissue obtained from healthy young individuals (Reske-Nielsen & Harmsen 1972). Similar findings in ophthalmoplegia have been described by several authors (von Wijngaarden et al. 1967; Pateisky et al. 1970; MorganHughes & Mair 1973; Karpati et al. 1973; Schneck et al. 1973; Shapira et al. 1975; Schlote & Korner 1976). In the present study the abnormal mitochondria were not only found in muscle tissue, but also, to a much lesser extent, in fibrous tissue and endothelial cells of blood vessels. The fact that other tissues are involved is in agreement with other authors. In patients with opthalmoplegia Gonates (1967) found abnormal mitochondria in liver celIs, Karpati et al. (1973) found them in sweat glands and Schneck et al. (1973) found them in cerebellum. This indicates that the morphological mitochondrial abnormality is universal. T h e cases reported in the literature and in the present study comprise a complicated collection of symptoms and signs derived from different organs which cannot immediatedly by explained in any one particular way. T h e main organs involved are the extraocular muscles, the myocardium, the retina (probably the pigment epithelium), the central nervous system and the endocrine organs. These structures characteristically have large metabolic requirments which are mainly provided by carbohydrate metabolism (Altman & Dittmer 1971). The morphological findings are considered compatible with a metabolic defect located in the mitochondria. In order to attempt to explain the abovementioned mitochondrial changes the problem was examined from a biochemical 570
l’rogrcssiue External Ophthalmoplegia
1 Loctofe
%
Pyruvote
4
Mitochondria Lactate
Pyruvote
Malate /Ace;,
Fatty Acid ond Sterodd synthesis
CoA
\
\
A
( C i t r i c Acld Cycle)
Fig. 11. A schematical representation of the biochemical pathway involving pyruvare and lactate metabolism.
point of view. Pyruvate metabolism was studied and the blood levels of pyruvate and lactate were measured. Increased levels of these metabolites were found in the three patients who had the most pronounced ultrastructural alterations in the biopsies. I n three patients (1, 3 and 4) an increase in lactate, and in two patients (1 and 4) a n increase in pyruvate were demonstrated. The findings suggest a defect in pyruvate metabolism (Fig. 11). Increased blood levels of pyruvate and lactate have not previously been demonstrated in ophthalmoplegia plus. T h e mitochondrial changes could be explained by the defect in the pyruvatelactate metabolism with a block in the metabolism of pyruvate and decreased activity of the citric acid cycle. The enzyme systems involved are located in the mitochondrial cristae. In our opinion the proposed biochemical defect could be the primary [actor responsible for the extreme proliferation of the abnormal mitochondria.
Acknowledgment T h e authors wish to thank the staff of the Neuropathological Department, Arhus Kommunehospital for their invaluable technical assistance. This study was supported by “Muskelsvindfonden”, Arhus, Denmark.
References Alfano J. E. PS Berger J. P. (1957) Retinitis pigmentosa, ophthalmoplegia and spastic quadriplegia. Anaer. /. Ophthal. 43, 231-240. Altman P. L. PS Dittmer D. S. (1971) Biologicnl Handbooks. Rrspirn~ioia and Circulation, pp. 0-9.30. Fed. Amer. Soc. Exp. B i d . Bethesrln, Mcl.
571
Edith Rcske-Nielsen, HanF C. Lori and Martin Lowes Beckett R. & Netsky M. (1953) Familial ocular myopathy and external ophthalmoplegia. Arch. Nrurol. Psychiat. (Chicago) 69. 64-72, Cogan F., Kubarawa T. & Richardson F,. (1962) Pathology of abiotrophic ophthalmoplegia externa. Bull. Johns Hoiik. Hosp. 111, 42-56. Daroff R. B. (1969) Chronic progressive cxtcrnal ophthalmoplegia. Arch. Ophthal. (Chicago) 82, 845-850. Drachman D . A. (1968) Ophthalmoplegia plus. Arch. Nrzirol. 18, 654-673. Gonatas N. K. (1967) A generalized disorder of nervous system, skeletal muscle and heart resembling Relsum’s disease and Hurler’s syndrome. A m e r . J . Mcd. $2, 169-178. Iamaccone S. T., Griggs R. C., Markesbery W . R. & Joynt R. J. (1974) Familial progressive external ophthalinoplegia and ragged rcd libres. Nerrrology 24, 1033-1038. Jager B. V., Fred H . L., Butler R. B. & Carnes W. H. (1960) Occurrence of retinal pigmentation, ophthalmoplegia, ataxia, deafness a n d heart block. A ~ n e r ./. Mrd. 29, 888-892. Karpati G., Carpenter S., Larbrisseau A. & Latontaine R. (1973) T h e Kearns-Shy syndrome. A multisystem disease with mitochondria1 abnormality demonstrated in skeletal muscle and skin. /. n e w . Scicncc.s 19, 133-151. Kearns T. P. & Sayre G. P. (1958) Retinitis pignientosa, external ophthalmoplegia and complete heart block. Arch. Ophthnl. (Chicago) 60, 280-289. Kiloh L . G. & Nevin S. (19.51) Progressive dystrophy of the external muscles (ocular myopathy). Brain 74, 115-143. Koerner F. & Schlote W. (1972) Chronic progressive external ophthalmoplegia. Arch. Ophthal. (Chicagcl 88, 155-156. Lind I. & Prame G. (1963) Chronic progressive external ophthalmoplegia and muscular dystrophy. Acta ophthnl. (Kbh.) 41, 497-507. Lou H. C. & Reske-Nielsen E. (1976) Progressive external ophthalmoplegia. Evidence for a disorder in pyruvate-lactate metabolism. Arch. Nczirol. (in press). Lowes M. (1975) Chronic progressive external ophthalmoplegia, pigmentary retinopathy a n d heart block (Kearns-Sayrc syndrome). A c t n o { ~ h t h r r l .(Kbh.) 53, 610-619. Lundberg P. 0. (1962) Observations on endocrine [unction in ocular myopathy. Acta neurol. srnnd. 38, 142-155. Lundholm L., Mohne-Lundholm E. & Vamos N. (1963) Lactic acid assay L(+) lactic acid dehydrogenase from rabbit muscle. Acta fJhysioZ. s c u d . 58, 243-249. Mair W . G. P. & Tom6 F. M. S. (1972) Atlas o{ iiltrustrrirtrtra o / discasrd hzimm muscle. Churchill Livingstone, Edinhurgh a n d London. Marbach E. P. & Weil M. H . (1967) Rapid Enzymatic measurement of Blood Lactate and Pyruvate. Cliti. Chem. 13, 314-325. Morgan-Hughes J. A. t?: Mair W. G. P. (1973) Atypical muscle mitochondria in oculoskeletal myopathy. Bruin 96. 215-224. Pateisky K., Sluga E. & Summer K. (1970) Clinical report on 4 cases of oculocardiac myopathy with giant mitochondria and glycogen storage disease. In: Walton J. N., Canal N. a n d Scarlato G., Eds. Muscle discusrs,, pp. 633-634. Excerpta med. (Amst.). Reske-Nielsen E., Harmsen A . & Hcljgaard J. (1969), Modified technique of muscle biopsy. Actn path. nzicrobiol. scutid. 77, 578-588. Reske-Nielsen E., Haase J. & Kelstrup J. (1975) Malignant hyperthermia in a family. Actn path. microbiol. scnnd. 8 3 , 651-660.
512
Progressive External Ophthalmoplegia Reske-Nielsen E. & Harmsen A. (1972) Electronmicroscopal study of muscle biopsies from healthy young people. A c t a path. microhiol. scnrid. 80, 449-467. Rosenberg R. N., Schotland D. L., Lovelace R.E. & Rowland L. P. (1968) Progressive ophthalmoplegia. Arch. Neurol. 19, 362-376. Schlote W . & Korner F. (1976) Chronic progressive external ophthalmoplegia - a neuromuscular disorder? In: Lennerstrand G. and Bach-y-Rita P., Eds. Proceedings
the International Symposium on Basic Mechanisms Clinical Implications, pp. 549-533. Pergamon Press.
of
of
Ocular Motility and their
Schneck L., Adachi M., Briet P., Wolintz A. & Volk B. W . (1973) Ophthalmoplegia plus with morphological and chemical studies of cerebellar and muscle tissue. J . neur. Sciences 19, 37-44. Schwarz G. & Liu C. (1954) Chronic progressive ophthalmoplegia. Arch. Neurol. Psychiat. (Chicago) 71, 31-53. Shapira Y., Cederbaum S. D., Concilla P. A,, Nielsen D. & Lippe B. M. (1975) Familial poliodystrophy, mitochondria1 myopathy and lactate acidemia. Neurology (Minneap.) 25, 614-626. van Wijngaarden G. K., Bethlem J., Meijer A. E. F. H., Hulsmann W. C. & Feltkamp s. A. (1967) Skeletal muscle disease with abnormal mitochondria. Brain 90, 577-592. von Graefe A. (1 866) Bemerkungen iiber doppelseitige Augenmuskellahmungen basilaren Ursprungs. Alhrccht 'u. Graefes Arch. klin. exp. Ophtfial. l.?, 265-277.
Author's address: Dr. Edith Reske-Nielsen, Department of Neuropathology, Arhus Kommunehospital, DK-8000 Arhus C, Denmark.
573
Department of Neuropathology Arhus Kommunehospital, University of Aarhusl) (Head: Edith Reske-Nielsen), Department of Ophthalmology, Rigshospitalet, Copenliagen2) (Head: Eilif Gregersen) and Department of Ophthalmology, Arhzrs Kommunehospital, University of Aarhus, Denmark3) (Head: Niels Ehlers)
PROGRESSIVE EXTERNAL OPHTHALMOPLEGIA Evidence for a Generalised Mitochondria1 Disease with a Defect in Pyruvate Metabolism BY
EDITH RESKE-NIELSENl), HANS C. L O U ) and MARTIN LOWESs)
Muscle biopsies from four patients with chronic progressive external ophthalmoplegia and pigmentary retinopathy with symptoms and signs from other organs were studied by means of light and electron microscopy. Examination revealed a marked proliferation of abnormal mitochondria with a degeneration of both muscle and nerve tissue. Blood levels of lactate and pyruvate were measured and abnormal values of these metabolites were found in the three patients with the most pronounced ultrastructural changes. On the basis of these findings it is suggested that there is a biochemical defect in pyruvate-lactate metabolism which could be responsible for the marked proliferation of the abnormal mitochondria.
Key words: ophthalmoplegia plus - muscle biopsy - abnormal mitochondria - metabolic defect - blood pyruvate - blood lactate.
Chronic progressive external ophthalmoplegia w a s originally considered t o b e
a disease primarily affecting.brain stem nuclei, a s first described by v o n G r a e f e (1866). However, Kiloh & Nevin'(1951) w e r e able t o demonstrable histopathoReceived June 25, 19876.
553
Edith Reske-Nielsm, H a n s C . Lou and Martin Lowes
logical changes in the extraocular muscles and concluded that the disorder was due to a myopathy. T h e neural changes reported in earlier studies were regarded as inconclusive. This view has prevailed in the majority of subsequent studies (Schwarz & Liu 1954; Beckett & Nersky 1953; Kearns & Sayre 1958; Cogan et al. 1962; Lind & Prame 1963). Koerner & Schlote (1972) stated that there was not enough evidence to consider chronic progressive external ophthalmoplegia as part of an entity of neurogenic disorders. However, it has become increasingly difficult to explain chronic progressive external ophthalmoplegia as being purely and simply a myopathy of the extraocular muscles. Involvement of other facial muscles, limb musculature, cerebellum, spinal tracts, myocardium, endocrine glands, hearing, and probably most frequent of all, retina, with pigmentary changes and chorioretinal atrophy has amply been described in the recent literature (Alfano & Berger 1957; Kearns & Sayre 1958; Jager et al. 1960; Drachman 1968; Rosenberg et al. 1968; Daroff 1969; Lowes 1975). Endocrine abnormalities have been reported by Lundberg (1962). Abnormal mitochondria in muscle tissue have been demonstrated by Gonatas (1967), Mair & Tom6 (1972), Morgan-Hughes & Mair (1973), Iamaccone et al. (1974) and Schlote & Korner (1976). I n a preliminary report by two of the authors (Lou & Reske-Nielsen 1976) a biochemical defect in pyruvate-lactate metabolism in three of the patients involved in the present study has been suggested. The purpose of the present study is to report on the ultrastructure of muscle biopsies in four cases of progressive external ophthalmoplegia and to attempt to correlate the findings with the proposed metabolic defect in pyruvate metabolism.
Material and Methods T h e material comprised four patients, two male and two female, aged from 13-50 years. The patients all had a chronic external ophthalmoplegia and pigmentary retinopathy with a variety of associated disorders including cardiac conduction defects, endocrine disturbances and central nervous system involvement. T h e main points from the case histories are summarized in Table I. Muscle biopsies were taken from the left deltoid muscle in cases 1, 2 and 3. In the fourth case biopsies were taken from the right superior rectus muscle, the right levator palpebrae muscle and the I'eft deltoid muscle. 'The biopsies were taken and prepared for histological examination as described by ReskeNielsen et al. (1969). T h e muscle specimens for electron microscopy were fixed in 2 O/o glutaraldehyde for approximately 3 h and 1 O/o osmium tetroxide for 554
Progressive Externul Ophthdmoplegia
Table I. Summary of the case histories Cases Age on presentation (years) Sex
1 50
2 23
3 17
25
M
M
F
F
+ + + +
+ +
+ + + +
-
-
+
+
+ +
+
0
+ +
4
~~
Ptosis and ophthalmoplegia Pigmentary retinopathy Cardiac conduction defects Cardiac complaints Cardiac pacemaker Frail build. Low average intelligence Deafness Facial weakness Secondary sexual abnormalities Abnormal electroencephalography Abnormal electromyography Abnormal electroretinography Abnormal colour vision Abnormal dark adaptation Enlarged blind spot Elevated CSF protein Asymptomatic diabetes Raised Sc-cholesterol Raised Se-lactate dehydrogenase Raised Se-aldolase Raised Se-creatine phosphokinase
Key:
+ present,
-
0
0
+
+
+ +
+ +
t.
0
0
0
+ +
+
0
-
+ 0
+
0 0
0
+ + + +
+ +
-
-
0 0 0
0
+ +
+
+ -
absent, 0 not recorded.
one h, then dehydrated with increasing alcohol concentrations (240/0 up to 99 o/g) and anhydrous acetone, and finally embedded in Vestopal W. Six grids from each of five blocks from each specimen were examined. T h e grids were stained with uranyl magnesium acetate and lead citrate and examined in a Zeiss EM 9 (Reske-Nielsen & Harmsen 1972). Electromyography was performed in cases 1 and 2 in the right deltoid muscle. In addition data on blood’ pyruvate and lactate concentrations were obtained. In three cases the method used fbr the estimation was that described by Lundholm et al. (1963). I n the remaining case the method described by Marbach & Weil (1967) was used. These findings are shown in Table 11. 555
Edith Reske-Nielsen, Hans C . Lou and Martin Lowes Tuble ZZ. Blood concentrations (mmolil) o l pyruvate and lactate.
Lactate (normal range 0..55-1.75) (sn 0.01)
Pyrzcvate (normal range 0.035-0.139) (SD 0.04) Patient 1 (rest) ., (rest) ,, (moderate exercise) Patient 2 (rest) Patient 3 (rest) ., (rest) ,, (moderate exercise)
0.160 0.143 0.191 0.082 0.137 0.108 0.152
2.58 2.28 4.85 1.59 2.77 1.99 3.62
Pyrzivate (normal range 0.02-0.08) Patient 4 (rest) ,, (rest)
Lactatp (normal range 0.625-1.67) 1.55 2.55
0.135 0.15
ResuIts Case 1. A 50-year-old male business executive who underwent a n operation for strabismus a t the age of 7 years. A left deltoid muscle biopsy was examined by the Department of Neurophysiology, Rigshospitalet.
Light microscopy. Increased number of internal nuclei. Several necrotic a n d basophilic fibres. In about 20’3/0 of the fibres excessive amounts of subsarcolemmal material (probably mitochondria) were present, which stained dark with lactate dehydrogenase and bright red with Gomori’s trichome stain. Electron microscopy. The cross striation of the myofibrils was well preserved but out phase in many fibres. Central myonuclei a n d the number of satellite cells were increased. Some fibres rontain subsarcolemmal accumulations of mitochondria. Intramitochondria1 crystals were abundant. Case 2. A 23-year-old male metal worker whotdeveloped ptosis of both eyes around the age of 14 years. A biopsy from the left deltoid muscle was examined.
Light microscofy. A few abnormal muscle fibres which appeared red a n d granular with Gomori’s trichome stain. Histochemistry was not performed.
556
Progressive External Ophthalmoplegiu Ekctrofz microscopy. Accumulations of mitochondria were seen close to the nucleus beneath the sarcoplasma membrane. Some showed abnormal cristae and vacuoles. Electron dense granules, presumably glycogen granules, were present. No paracrystalline inclusions could be demonstrated. Lipid vacuoles were seen between the myofibrils. Some myofibrils were atrophic and disorganized with spiky and flowing Z membranes. Case 3. A 17-year-old schoolgirl who developed a gradually progressive bilateral ptosis at the age of 8 years. A biopsy was taken from the left deltoid muscle.
Light microscopy. Several fibres in each fasicle contained loosely structured muscle fibres. These were granular and red-stained with Gomori’s trichrome stain. Furthermore, many basophilic muscle fibres with occasional necrotic fibres were seen. Histochemistry was not performed.
Fig. 1. a. Ultrastructure of the left deltoid muscle showing subsarcolemmal aggregations of abnormal mitochondria (Mi) and thinning of the myofibrils (Mf). Longitudinal section x 6000. (Case 3. E M 597).
b. Electron micrograph revealing large mitochondria (Mi) partly or totally filled by concentrically arranged membranes. Longitudinal section x 27 000. (Case 3. E M 597).
557
Edith Keske-Nielten, H U T ~C. S Lou arid M a r t i n Lowes
55s
Electroii microscopy. T h e dominating feature was a n abnormality o t the mitochondria. There were large aggregates of mitochondria beneath the sarcolcmma and between the myofibrils. T h e size of the mitochondria varied considerably, and many giant mitochondria were present. T h e configuration was also variable, and many bizarre forms were seen. T h e external membrane was double or triple and in some cases contained even more layers. T h e cristae were often absent or transversed the mitochondria. Some mitochondria were filled out by thin concentrically arranged membranes, often with two centres. Others had paracrystalline laminated inclusions and vacuoles with a single or double limiting membrane. T h e vacuoles were either empty or contained myelin bodies or electron dense granules. Furthermore, some mitochondria contained tuhular inclusions. Glycogen granules were abundant. Many giant mitochondria with a double limiting membrane were distended by closely packed electron dense granules. Between the mitochondria glycogen granules and lipid droplets were present. I n addition, many fibres were atrophic or even disrupted and the basal membranes were tolded. T h e sarcotubular system as well as the ve5sels were normal (Figs. 1 a, 1 h and 2).
Case 4. A 2.5-year-old wonian who developed ptosis of both eyes around the age of 10 years. This case has previously been published in Acta ophthal. (Kbh.) (Lowes 1975) and shows all the characteristics of the foregoing patients to a marked degree. Three muscle biopsies were taken - one from the right rectus superior muscle, one from the right levator palpebrae muscle taken a t the time of opcration for ptosis, a n d one from the left deltoid muscle. T h e ptosis operation has proved cosmetically successful with no exposure derect on closure.
Light rnicrosco/iy of superior rcctus (Neurop. No. 19131j. T h e scanty material consisted o l connective tissue with no inflammatory infiltration. There were a Jew muscle fibres of varying diameter, hut without any degenerative changes. With Gomori’s trichronie staining shiny, red granules were observed between the myofihrils. Enzyme histochemistry was not performed (Fig. 3 a, 3 b). Electron nzicroscopy of .fu/wrkir rcct2c.r. T h e muscle fibres and fibrils were small, as a result of a loss of myofilaments. T h e Z-lines showed a n extremely variable pattern with abnormally thin, thickened, spiky and flowing lines. Areas of muscle tissue were occasionally seen which had either disappeared or revealed “contraction clumps”. Folded and empty basement membranes could also be seen. T h e connective tissue was increased. T h e most striking ultrastructural changes were large aggregations of mitochondria. These were situated under the sarcoplasma membrane, either close to the nucleus or hetween the myofibrils. T h e mitochondria were much larger than normal and had a n extremely varied appearance (Fig. 3 c, 3 d). Some of these organelles were rounded possessing a single, double or triple membrane. Others had a bizarre club-shaped appearance. They werc either empty or contained a n electron dense material. Occasionally both vacuoles and granules were
. Fig. 2. Left deltoid muscle. Giant mitochondrion with ahnormally arranged cristae (Cr), vacuoles (Va) and glycogen material (G). On the left a sarcomere (Sj can be seen. Longitudinal section x 90 000. (Case 3 . EM 597). 559
F i g . 3 a, 0, c, d . (See text, p. 561) 560
Progressivt, External Ophthalmoplegiu found in the same mitochondrion. T h e cristae generally had a n abnormal arrangement with numerous concentric circles inside the organelles. Certain of the mitochondria appeared normal in one part, but abnormal in the other part. In the giant mitochondria a variable number of laminated, crystal-like inclusions could be seen within the cristae. T h e configuration of the laminated material depended on the plane of sectioning. T h e mitochondria often contained lipid bodies a n d glycogen granules. Lipid bodies were also seen in close proximity to the mitochondria (Figs. 4 a, 4 b, 5 a , 5 b). T w o end plates were successfully demonstrated. T h e axon, the primary a n d the secondary synaptic clefts were morphologically normal. T h e subneural apparatus, which is limited by the muscular sarcoplasmal membrane, contained numerous, large, closely packed, abnormal mitochondria which displaced the secondary synaptic clefts (not illustrated). (Neurop. No. 20843 prep. 3). T h e biopsy consisted of connective tissue with a few blood vessels and a scanty lymphocyte infiltration. I n the connective tissue there were occasional small groups of muscle fibres
Light microscopy of levator pal@me
Fig. 3. a. Light microscopy of the right superior rectus muscle showing muscle fibres (MF) in cross a n d longitudinal sections separated by connective tissue. x 40. (Case 4. LM 19131). b. Higher magnification of Fig. 3 a x 100. c. Electron micrograph of right superior muscle. Large collections of mitochondria (Mi) under the sarcoplasma membrane (SM) close to the nucleus (N). x 6000. (Case 4. E M 633). d. Thin myofibrils (Mf) with abnormal mitochondriia (Mi) in the interspaces. Longitudinal section x 6000.
Fig. 4. a. Right superior rectus muscle. Accumulation of bizarre and enlarged mitochondria (abn Mi) under the folded sarcoplasma membrane (SM). Lipid bodies (LB) can be seen. Longitudinal section x 27 000. (Case 4. E M 633). b. Mitochondria showing a varied appearance with a n abnormal internal structure (Mi) between thinned out myofibrils (Mf). Longitudinal section x 27 000. (Case 4. EM 633).
Fig. 5. a. Right superior rectus muscle showing a n area below the nucleus (N) which contains remnants of myofibrils (Mf) with laminated mitochondria (Mi) and lipid bodies (LB). Longitudinal section x 27 000. '(Case 4. E M 633). b. Bulging of the sarcoplasma membrane (SM) which contains large abnormal mitochondria (abn Mi) with a complex arrangement of the cristae. Longitudinal section x 27 000. (Case 4. EM 633).
56 I
Fig. 4 a, b. (See text, p. 561).
Fig. 5 u. b. (See text, p. 561).
Edith Reske-Nielsen, Huns C. Lou and Martin Lowes
Fig. 6 . a. Close-up of the adnexal region showing the marked ptosis. (Case 4). b. Biopsy from the right levator palpebrae muscle. Light microscopy shows connective tissue (COT) and fat cells (FC). No recognisable muscle fibres. x 100. (Case 4. LM 20843 prep. 3).
(maximum 3-4) with a rounded appearance a n d an intense stain with haematoxylineosin. Gomori’s trichrome stain was inconclusive. T h e small nerve branches were totally dernyelinated a n d contained only a few degenerated nerve fibres. Histochemistry was not performed (Fig. 6 a, 6 b).
564
Fig. 7 a, b. (See text, p. 5 6 6 ) .
565 Acta ophthal. 54, 5
37
Edith Reske-Nielsen, Hans C. Lou arid Martin Lowes Electron rnicrotcopy of leuator paljlebrae. All the micrographs revealed normal blood vessels, but with marked pinocytosis a n d considerable connective tissue. Abnormal mitochondria were occasionally observed in the cytoplasma of fibroblasts. Some of the mitochondria were empty, but in others a n abnormal arrangement of the cristae could be observed (Fig. 7 a, b). T h e preparation contained no recognisable muscle tissue.
Light rnzcroscojly of deltoid muscle (Neurop. No. 20843 prep. 1). Skin from the deltoid region examined by conventional technique was normal. Gomori’s trichrome stain was inconclusive. Electron microscopy was not periormed. Muscle fibres with a granular structure and single basophilic fibre segments were found in all fasicles. N o necrotic fibres were observed. I n the modilied Gomori trichrome stain almost every muscle fibre, especially the granular type, contained bright, shiny, red granules. These granules were either confined to the subsarcoleinmal regions or formed a coarse punctate pattern over the entire cross-sectional area of the fibre. There was a n increase in the number of nuclei, a n d these were occasionally centra!ly placed. Frozen sectzonc. A n intense staining of the intermyofibrillary structures was a striking feature of the haematoxylin-eosin staining. These fibrillary structures displayed a
ccarse reticular pattern when compared with normal material. Numerous small lipid droplets stained by Scharlach Rot were seen in cross-section of the muscle fibres. On histochemical examination the same areas were intensely stained with D P N H tetrazolium reductase a n d could be seen as abnormal deposits in the subsarcolemmal regions and over the entire cross-sectional area of the fibres. ATPase demonstrated a n absence of myofibrillary material in the affected regions of Fig. 7. a. Right levator palpebrae muscle. Mitochondria (Mi) containing abnormal cristae a r e to be found in the cytoplasm of a Fibroblast. x 27 000. (Case 4. E M 757). b. Higher magniiication of Fig. 8 b. x 81 000.
Fig. 8. a. Left deltoid muscle. Light microscopy (frozen section) with a n intense staining of the intermyofibrillary structures. T h e nuclei a r e marked by circles. (Haematoxylineosin). Cross-section x 400. (Case 4. L M 20843 prep. 2). b. Histochemical preparation ( D P N H ) . Both type 1 and type 2 muscle fibres are shown. Mitochondria1 enzymes a r e visualised as abnormal deposits under the sarcoplasma (marked by circles) a n d as a coarse network over the entire cross-section in both types of fibres. x 400.
Fig. 9. a. Left deltoid muscle. Section demonstrating the giant a n d peculiar mitochondria (Mi) with numerous crystalline inclusions sectiohed in various planes. Longitudinal section x 27 000. (Case 4. E M 756). b. Fingerprint-like configuration of mitochondria (Mi) lying between two myofibrils (Mf). Longitudinal section x 27 000.
566
Fig. 8 a, b. (See text, p. 566).
567
Fig. 9 a, b. (See text, p. 566). 568
Progressive External Ophthalmoplegia the muscle. The biopsy material showed a normal mosaic pattern of the type 1 and type 2 fibres. Both histochemical fibre types were afiected, but the changes were more pronounced in type 1 than in type 2 fibres (Fig. 8 a, 8 b). Electron microscopy o/ deltoid muscle. The alterations were similar to those described in the rectus superior muscle. The degenerative changes found in the muscle fibres were conspicuous with large aggregations of giant mitochondria located under the sarcoplasma membrane and between the fibrils. These organelles were often bizarre with an abnormal configuration and a varied arrangement of the cristae inside the mitochondria. Several mitochondria contained multiple crystalline inclusions within the cristae, especially the giant elongated ones. Vacuoles, glycogen, granular and osmiophilic material were also seen. These were often present in the same mitochondrion, together with the crystalline inclusions. Lipid and osmiophilic bodies often lay close to the mitochondria. Mitochondria containing a granular material and/or vacuoles were observed in a capillary vessel wall (Figs. 9 a, 9 b, 10 a. 10 b).
* Fig. 10 a and b. Left deltoid muscle. Closely packed mitochondria (Mi) showing double and triple membranes. Certain of the mitochondria contain electron dense granules, while others appear empty. Note also the club-shaped mitochondria with crystalline inclusions. x 27 000. (Case 4. EM 756).
569
Edith Reske-Nielsen, Hans C . Lou and M a r t i n Lawes
Discussion T h e material comprised four patients who had a chronic progressive external ophthalmoplegia with a variety of additional findings which are summarized in Table I. I n all of the patients abnormalities of muscle fibres were seen. Light microscopy showed the characteristic red colour of muscle fibres with Gomori’s trichrome stain. Electron microscopy revealed pronounced mitochondrial alterations. A characteristic feature in these patients was a marked proliferation of mitochondria which were much larger than normal and often quite bizarre. The internal structure of the cristae was complex with elongated crystalline inclusions. Abnormalities of muscle fibres were found, including “contraction clumps” which have also been described in families with malignant hyperthermia (Reske-Nielsen et al. 1975). T h e levator palpebrae muscle of case 4 showed an almost complete replacement of muscle fibres by connective tissue and f a t cells, with a marked loss of nerve fibres and myelin sheaths. Two end plates were observed, the secondary synaptic clefts were displaced by closely packed abnormal mitochondria. These findings were compared with normal muscle tissue obtained from healthy young individuals (Reske-Nielsen & Harmsen 1972). Similar findings in ophthalmoplegia have been described by several authors (von Wijngaarden et al. 1967; Pateisky et al. 1970; MorganHughes & Mair 1973; Karpati et al. 1973; Schneck et al. 1973; Shapira et al. 1975; Schlote & Korner 1976). In the present study the abnormal mitochondria were not only found in muscle tissue, but also, to a much lesser extent, in fibrous tissue and endothelial cells of blood vessels. The fact that other tissues are involved is in agreement with other authors. In patients with opthalmoplegia Gonates (1967) found abnormal mitochondria in liver celIs, Karpati et al. (1973) found them in sweat glands and Schneck et al. (1973) found them in cerebellum. This indicates that the morphological mitochondrial abnormality is universal. T h e cases reported in the literature and in the present study comprise a complicated collection of symptoms and signs derived from different organs which cannot immediatedly by explained in any one particular way. T h e main organs involved are the extraocular muscles, the myocardium, the retina (probably the pigment epithelium), the central nervous system and the endocrine organs. These structures characteristically have large metabolic requirments which are mainly provided by carbohydrate metabolism (Altman & Dittmer 1971). The morphological findings are considered compatible with a metabolic defect located in the mitochondria. In order to attempt to explain the abovementioned mitochondrial changes the problem was examined from a biochemical 570
l’rogrcssiue External Ophthalmoplegia
1 Loctofe
%
Pyruvote
4
Mitochondria Lactate
Pyruvote
Malate /Ace;,
Fatty Acid ond Sterodd synthesis
CoA
\
\
A
( C i t r i c Acld Cycle)
Fig. 11. A schematical representation of the biochemical pathway involving pyruvare and lactate metabolism.
point of view. Pyruvate metabolism was studied and the blood levels of pyruvate and lactate were measured. Increased levels of these metabolites were found in the three patients who had the most pronounced ultrastructural alterations in the biopsies. I n three patients (1, 3 and 4) an increase in lactate, and in two patients (1 and 4) a n increase in pyruvate were demonstrated. The findings suggest a defect in pyruvate metabolism (Fig. 11). Increased blood levels of pyruvate and lactate have not previously been demonstrated in ophthalmoplegia plus. T h e mitochondrial changes could be explained by the defect in the pyruvatelactate metabolism with a block in the metabolism of pyruvate and decreased activity of the citric acid cycle. The enzyme systems involved are located in the mitochondrial cristae. In our opinion the proposed biochemical defect could be the primary [actor responsible for the extreme proliferation of the abnormal mitochondria.
Acknowledgment T h e authors wish to thank the staff of the Neuropathological Department, Arhus Kommunehospital for their invaluable technical assistance. This study was supported by “Muskelsvindfonden”, Arhus, Denmark.
References Alfano J. E. PS Berger J. P. (1957) Retinitis pigmentosa, ophthalmoplegia and spastic quadriplegia. Anaer. /. Ophthal. 43, 231-240. Altman P. L. PS Dittmer D. S. (1971) Biologicnl Handbooks. Rrspirn~ioia and Circulation, pp. 0-9.30. Fed. Amer. Soc. Exp. B i d . Bethesrln, Mcl.
571
Edith Rcske-Nielsen, HanF C. Lori and Martin Lowes Beckett R. & Netsky M. (1953) Familial ocular myopathy and external ophthalmoplegia. Arch. Nrurol. Psychiat. (Chicago) 69. 64-72, Cogan F., Kubarawa T. & Richardson F,. (1962) Pathology of abiotrophic ophthalmoplegia externa. Bull. Johns Hoiik. Hosp. 111, 42-56. Daroff R. B. (1969) Chronic progressive cxtcrnal ophthalmoplegia. Arch. Ophthal. (Chicago) 82, 845-850. Drachman D . A. (1968) Ophthalmoplegia plus. Arch. Nrzirol. 18, 654-673. Gonatas N. K. (1967) A generalized disorder of nervous system, skeletal muscle and heart resembling Relsum’s disease and Hurler’s syndrome. A m e r . J . Mcd. $2, 169-178. Iamaccone S. T., Griggs R. C., Markesbery W . R. & Joynt R. J. (1974) Familial progressive external ophthalinoplegia and ragged rcd libres. Nerrrology 24, 1033-1038. Jager B. V., Fred H . L., Butler R. B. & Carnes W. H. (1960) Occurrence of retinal pigmentation, ophthalmoplegia, ataxia, deafness a n d heart block. A ~ n e r ./. Mrd. 29, 888-892. Karpati G., Carpenter S., Larbrisseau A. & Latontaine R. (1973) T h e Kearns-Shy syndrome. A multisystem disease with mitochondria1 abnormality demonstrated in skeletal muscle and skin. /. n e w . Scicncc.s 19, 133-151. Kearns T. P. & Sayre G. P. (1958) Retinitis pignientosa, external ophthalmoplegia and complete heart block. Arch. Ophthnl. (Chicago) 60, 280-289. Kiloh L . G. & Nevin S. (19.51) Progressive dystrophy of the external muscles (ocular myopathy). Brain 74, 115-143. Koerner F. & Schlote W. (1972) Chronic progressive external ophthalmoplegia. Arch. Ophthal. (Chicagcl 88, 155-156. Lind I. & Prame G. (1963) Chronic progressive external ophthalmoplegia and muscular dystrophy. Acta ophthnl. (Kbh.) 41, 497-507. Lou H. C. & Reske-Nielsen E. (1976) Progressive external ophthalmoplegia. Evidence for a disorder in pyruvate-lactate metabolism. Arch. Nczirol. (in press). Lowes M. (1975) Chronic progressive external ophthalmoplegia, pigmentary retinopathy a n d heart block (Kearns-Sayrc syndrome). A c t n o { ~ h t h r r l .(Kbh.) 53, 610-619. Lundberg P. 0. (1962) Observations on endocrine [unction in ocular myopathy. Acta neurol. srnnd. 38, 142-155. Lundholm L., Mohne-Lundholm E. & Vamos N. (1963) Lactic acid assay L(+) lactic acid dehydrogenase from rabbit muscle. Acta fJhysioZ. s c u d . 58, 243-249. Mair W . G. P. & Tom6 F. M. S. (1972) Atlas o{ iiltrustrrirtrtra o / discasrd hzimm muscle. Churchill Livingstone, Edinhurgh a n d London. Marbach E. P. & Weil M. H . (1967) Rapid Enzymatic measurement of Blood Lactate and Pyruvate. Cliti. Chem. 13, 314-325. Morgan-Hughes J. A. t?: Mair W. G. P. (1973) Atypical muscle mitochondria in oculoskeletal myopathy. Bruin 96. 215-224. Pateisky K., Sluga E. & Summer K. (1970) Clinical report on 4 cases of oculocardiac myopathy with giant mitochondria and glycogen storage disease. In: Walton J. N., Canal N. a n d Scarlato G., Eds. Muscle discusrs,, pp. 633-634. Excerpta med. (Amst.). Reske-Nielsen E., Harmsen A . & Hcljgaard J. (1969), Modified technique of muscle biopsy. Actn path. nzicrobiol. scutid. 77, 578-588. Reske-Nielsen E., Haase J. & Kelstrup J. (1975) Malignant hyperthermia in a family. Actn path. microbiol. scnnd. 8 3 , 651-660.
512
Progressive External Ophthalmoplegia Reske-Nielsen E. & Harmsen A. (1972) Electronmicroscopal study of muscle biopsies from healthy young people. A c t a path. microhiol. scnrid. 80, 449-467. Rosenberg R. N., Schotland D. L., Lovelace R.E. & Rowland L. P. (1968) Progressive ophthalmoplegia. Arch. Neurol. 19, 362-376. Schlote W . & Korner F. (1976) Chronic progressive external ophthalmoplegia - a neuromuscular disorder? In: Lennerstrand G. and Bach-y-Rita P., Eds. Proceedings
the International Symposium on Basic Mechanisms Clinical Implications, pp. 549-533. Pergamon Press.
of
of
Ocular Motility and their
Schneck L., Adachi M., Briet P., Wolintz A. & Volk B. W . (1973) Ophthalmoplegia plus with morphological and chemical studies of cerebellar and muscle tissue. J . neur. Sciences 19, 37-44. Schwarz G. & Liu C. (1954) Chronic progressive ophthalmoplegia. Arch. Neurol. Psychiat. (Chicago) 71, 31-53. Shapira Y., Cederbaum S. D., Concilla P. A,, Nielsen D. & Lippe B. M. (1975) Familial poliodystrophy, mitochondria1 myopathy and lactate acidemia. Neurology (Minneap.) 25, 614-626. van Wijngaarden G. K., Bethlem J., Meijer A. E. F. H., Hulsmann W. C. & Feltkamp s. A. (1967) Skeletal muscle disease with abnormal mitochondria. Brain 90, 577-592. von Graefe A. (1 866) Bemerkungen iiber doppelseitige Augenmuskellahmungen basilaren Ursprungs. Alhrccht 'u. Graefes Arch. klin. exp. Ophtfial. l.?, 265-277.
Author's address: Dr. Edith Reske-Nielsen, Department of Neuropathology, Arhus Kommunehospital, DK-8000 Arhus C, Denmark.
573
