Acta Neuropathol (1990) 80:118 - 122 Acta Neuropathologca 9 Springer-Verlag 1990 Involvement of extraocular muscle in mitochondrial encephalomyopathy S. Takeda, E. Ohama, and F. Ikuta Department of Pathology, Brain Research Institute,Niigata University, Niigata 951, Japan Received June 20, 1989/Revised August 28, 1989/Revised, accepted January 8, 1990 Summary. We carried out a histological examination o f the extraocular muscles (EOMs) in a case o f m y o c l o n u s epilepsy associated with ragged-red fibers ( M E R R F ) and two cases o f mitochondrial m y o p a t h y , encephalopathy, lactic acidosis, and stroke-like episodes ( M E L A S ) , which did n o t manifest external o p h t h a l m o p l e g i a clinically. By light microscopy, m a n y granular and vesicular fibers were seen associated with endomysial fibrosis. Electron mic r o s c o p y revealed that the fibers showed p r o m i n e n t accumulation o f a b n o r m a l m i t o c h o n d r i a , extensive loss o f myofibrils, proliferation o f free sarcoplasmic reticulum and an increased a m o u n t o f lipid vacuoles. These changes were m o r e p r o n o u n c e d in M E L A S than in M E R R F . H i r a n o bodies were often seen in the subsarcolemmal area o f muscle fibers and also in the intramuscular myelinated nerve fibers and a x o n terminals. These findings suggest the presence o f m i t o c h o n d r i a l m y o p a t h y o f the E O M s in cases o f M E L A S and M E R R F . Keywords" M i t o c h o n d r i a l e n c e p h a l o m y o p a t h y - Extraocular muscle - Ultrastructure - M i t o c h o n d r i a l m y o p athy M i t o c h o n d r i a l e n c e p h a l o m y o p a t h y is a heterogeneous disorder encompassing three distinctive clinical syndromes, Kearns-Sayre s y n d r o m e (KSS), m y o c l o n u s epilepsy associated with ragged-red fibers ( M E R R F ) , and m i t o c h o n d r i a l m y o p a t h y , encephalopathy, lactic acidosis and stroke-like episodes ( M E L A S ) , and other disorders. KSS shows progressive external ophthalmoplegia, the extracellular muscle ( E O M s ) u n d e r g o i n g severe degeneration with endomysial fibrosis and containing m a n y ragged-red fibers ( R R F s ) [3, 4, 12, 25]. O n the other hand, M E R R F and M E L A S do not manifest external ophthalmoplegia, and this is one o f the i m p o r t a n t clinical features which differentiate them f r o m KSS [5, 21]. The E O M in M E R R F and M E L A S has n o t been examined in previously reported a u t o p s y cases [6, Offprint requests to: S. Takeda (address see above) 7, 10, 13, 15, 23, 24]. In this report, we describe the histological features o f the E O M in M E R R F and M E L A S . The nervous system c o m p o n e n t s concerned with ocular m o v e m e n t were also examined. Materials and methods All of the present three cases examined (one case of MERRF and two cases of MELAS) have been reported elsewhere with regard to their detailed clinical histories and histopathological findings [7, 17-20, 28, 29]. The MERRF patient was a 30-year-old woman who died after a clinical com'se of 16 years. She was found to have saccadic eye movement at the age of 25 years. However, the limitation of extraocular movement was not followed. The postmortem examination was performed ] t h after death. MELAS patient 1 was a t4-year-old boy who died after a 13-year clinical course. He was found to have alternate external strabismus at the age of 2 years. MELAS patient 2 was a 16-yearold girl who died after a total clinical course of 10 years. She did not show limitation of extraocular movement. They were autopsied 2 h after death. The superior and medial rectus muscles, superior colliculus, periaqueductal gray matter, medial longitudinal fasciculus, and nuclei and nerve roots of the IIIrd, IVth and VIth cranial nerves in these three cases were examined by light microscopy. Specimens of the EOM were taken at autopsy and fixed in 3% glutaraldehyde1% paraformaldehyde. They were then processed for electron microscopy and examined using a Hitachi HSI1-B electron microscope. Four cases were selected for the control study. They died aged 41 to 46, and had no abnormal ocular movement. They included a case of liver cirrhosis, a case of multiple cerebral infarcts and two cases of subarachnoid hemorrhage. They were autopsied 2 to 8 h after death. The superior rectus muscles were also taken at autopsy and fixed in 3% glutaraldehyde-l% paraformaldehyde, embedded in epoxy resin and examined by the light microscope, Results Extraocular muscles ( E O M ) The superior and medial rectus muscles in M E R R F and M E L A S showed essentially the same histological 119 e. Extraocular muscle (EOM) by light microscopy, a Many muscle fibers appear fine granulare and vesicular. Moderate endomysial fibrosis is evident. Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS), Masson trichrome stain; x 250. b Abundant vesicular structures and extensive loss of myofibrils are evident. MELAS, Epon section, toluidine blue and saffranin (TBS) staining; x 500. e Homogeneous rod-like structures (arrow)are sometimes present in the subsarcolemmal area where vesicular structures have accumulated. Myoclonus epilepsy associated with ragged-red fibers (MERRF), Epon section. TBS staining; x 500 Fig. 1 a - findings. Many muscle fibers showed a granular and clear appearance in ordinary paraffin sections (Fig. i a). Moderate endomysial fibrosis was noted in many fasciculi. In epoxy resin-embedded sections, all of the granular and clear fibers were revealed to have abundant vesicular structures, which were markedly accumulated in the subsarcolemmal regions, and variable loss of myofibrils (Figs. 1 b, c). Almost all of the muscle fibers had many vesicular structures. The surface of these fibers was often irregular. These features were more pronounced in MELAS. However, no neurogenic changes were observed. Homogeneous rod-like structures were often seen in the subsarcolemmal regions of the vesicular fibers (Fig. 1 c). They were eosinophilic and appeared red with Masson's trichrome stain, gray with K1/iver-Barrera stain and purple with phosphotungstic acid-hematoxylin stain. They showed round, oval or spindle-like profiles. No significant abnormalities were noted in the nerve fibers within the muscle (Fig. I c). The control superior rectus muscles preserved their sarcoplasm even in the two cases, who were autopsied 8 h after death. We observed only a small number of the vesicular structures, which were seen abundant in the muscle fibers in M E R R F and MELAS. Rod-like struc-. tures were not seen in the fibers. In M E R R F and MELAS, electron microscopy revealed that the vesicular appearance of muscle fibers by light microscopy was due to prominent accumulation of abnormal mitochondria (Fig. 2a). These mitochondria were variable in size and shape, having tubulo-vesicular cristae or a proliferated inner membrane. Giant mitochondria were often seen, especially in MELAS. The morphological changes in the mitochondria were more prominent in M E L A S than in M E R R F (Fig. 2b). We could not see the crystalline or globular mitochondrial inclusions in EOMs. The granular and sharply demarcated mitochondrial inclusions which were seen sometimes (Fig. 2b) were considered to be glycogen particles. Lipid droplets were often seen in the fibers filled by mitochondria (Fig, 2c). There was variable toss of myofibrils in muscle fibers. Some fibers showed extensive myofibril loss and prominent accumulation of glycogen particles (Fig. 2d). Even fragments of the remaining myofibrils in these fibers retained their cross-striations. Disorganization of myofibrils was sometimes observed, and some fibers showed almost complete myofibril loss. They were filled with abnormal mitochondria and sarcoplasmic reticulum (Fig. 2 e). The homogeneous rod-like structures observed by light microscopy (Fig. 1 c) were revealed to be filamentous inclusions, of which three types were evident. The first type showed association with sarcoplasmic reticulum-like structures (Fig. 3a). The second consisted of a regular array of filaments which were sometimes arranged in perpendicular planes (Fig. 3 b). The third type showed a peculiar herring-bone or ladder-like pattern (Fig. 3 c). A few zebra bodies were seen in the subsarcolemmal regions without any apparent connection with myofibrils. In M E L A S cases, smooth muscle cells of a few arterioles among the muscle fibers showed accumulation of mitochondria, some of which had abnormal cristae. Nervous system The superior colliculus from the M E L A S patient 1 showed two foci of minute necrotic lesions associated with macrophages and capillary proliferations, and from the M E L A S patient 2 and the patient with M E R R F showed mild neuronal loss with gliosis. The periaqueductal gray matter showed mild gliosis in all three cases. In the axon terminals and myelinated axons of the EOM, Hirano bodies were often seen. The IIIrd, IVth and VIth cranial nerve trunks and nuclei showed no Fig. 2 a - e . Ultrastructural changes in the EOM. a Prominent accumulation of mitochondria is evident in a muscle fiber. M E R R F , • 7,200~ b Accumulated abnormal mitochondria show proliferation of the inner membrane, tubulo-vesicular cristae and a variable size and shape. The granular mitochondrial inclusions were seen sometimes. MELAS, x 6,800. c An increased amount oflipid droplets is often present among accumulated mitochondria in muscle fibers. M E R R F , x 6,700. d Extensive loss of myofibrils and prominent accumulation of glycogen are evident. The remaining myofibrils retain their cross-striations. M E R R F , x 5,000. e Myofibrils have almost disappeared and the sarcoplasmic reticulum is present freely in the muscte fiber. MELAS, • t 3,300 121 Fig. 3 a - c . Filamentous inclusions within the muscle fiber, a Inclusion associated with a sarcoplasmic reticulum-like structure. MERRF, x 21,600. b Inclusion consisting of a regular array of filaments. MERRF, x 64,000. c Inclusion showing a herring-bone or ladder-like pattern. MERRF, x 21,600 degeneration. The medial longitudinal fasciculus was well preserved. [17, 27] and defects of the complex IV in the M E R R F patient (Yoneda et al., personal communication). It is still unclear whether the difference in the deficient enzyme in M E L A S and M E R R F is correlated with the severity of the histopathological changes in the EOMs. We have already performed histological examinations of the limb muscles from our present patients [17, 27, 28], and RRFs were less frequent than in the EOMs. This situation is the same as in KSS cases, in which degeneration of muscle fibers associated with RRFs is more marked in the EOMs than in limb muscles [3, 4, 8, 11, 12, 22]. The filamentous structures observed in the muscle fibers were identical with those described in the EOMs from a patient with exotropia [14] and from aged men [30] as intramuscular Hirano bodies. Our present examination revealed that these structures were associated with a sarcoplasmic reticulum-like membrane. The development of these structures might be related in some way to myofibrillar degeneration. Zebra bodies have been reported in skeletal muscles and the EOMs in various conditions. The significance of zebra bodies present in M E R R F and M E L A S is not known. Hirano bodies within myelinated nerve fibers and axon terminals was described previously in a case of progressive external ophthalmoplegia [1]. Although myelinated nerve fibers in the EOMs and the IIIrd, IVth and VIth cranial nerve trunks of our three patients were well preserved, Hirano bodies within the nerve fibers and axon terminals in the EOMs might indicate some degeneration of the intramuscular nerve fibers. Discussion In the autopsy cases of M E R R F and M E L A S reported previously, mitochondrial myopathy was noted in the limb muscles [6, 7, 10,13, 16, 23, 26, 28], or in the heart muscle [15, 18, 19]. In the present study, we carried out a histological examination of the EOMs in M E R R F and MELAS. Although frozen tissues of the muscles for modified Gomori trichrome staining were not obtained, the features o f the EOMs by light and electron microscopy corresponded to RRFs. We also observed the vesicular structures in the control muscle fibers. However, the number of these structures was smaller than that in M E R R F or MELAS. A few fibers showed R R F s or contained increased a m o u n t o f mitochondria in the normal EOMs [2, 9, 22]. Ultrastructurally, however, the EOMs in the normal cases showed normal mitochondria which were increased in number [9]. We considered that the high frequency of R R F s and the abnormal profiles of increased mitochondria in M E R R F and M E L A S were significant. The frequency of RRFs, myofibrillar degeneration and endomysial fibrosis o f the EOMs were more pronounced in M E L A S than in M E R R F . In the present cases, defects of the complex I in the mitochondrial respiratory chain have been revealed in the M E L A S patient 2 122 D a r o f f et al. [4] c o n s i d e r e d t h a t the external o p h t h a l m o p l e g i a in K S S was neurogenic. H o w e v e r , in s o m e p a t i e n t s with K S S w h o s h o w e d e x t e r n a l o p h t h a l m o p l e g i a a n d m a r k e d d e g e n e r a t i o n o f the E O M s a s s o c i a t e d w i t h R R F s , the a n a t o m i c a l structures in the b r a i n c o n c e r n e d with o c u l a r m o v e m e n t i n c l u d i n g the I I I r d , I V t h a n d V I t h c r a n i a l nerves a n d nuclei were well p r e s e r v e d [3, 12, 26]. T h e presence o f R R F s , loss o f m y o f i b r i l s a n d o t h e r a l t e r a t i o n s o f the E O M s d e s c r i b e d here m a y cause e x t e r n a l o p h t h a l m o p l e g i a in cases o f MERRF and MELAS. A c c u m u l a t i o n a n d s t r u c t u r a l c h a n g e s o f the m i t o c h o n d r i a were seen in the s m o o t h muscle cells o f the arterioles w i t h i n the E O M s in o u r M E L A S p a t i e n t s , a l t h o u g h m u c h m i l d e r t h a n t h o s e in the b r a i n [18]. This indicates t h a t the b l o o d vessels in the E O M s are also i n v o l v e d b y m i t o c h o n d r i a l a n g i o p a t h y in M E L A S [18]. Acknowledgements. We are grateful to Mr. T. Ichikawa, Mr. K. Kobayashi and Mr. S. Egawa for their technical assistance. We also thank Ms A. Sasaki, Ms K. Murayama, Ms Y. Tanahashi, Ms K. Hasegawa and Ms S. Shimakura for their assistance in preparing this manuscript. References 1. 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