Extraocular Muscle Findings in Myasthenia Gravis Associated Treatment-Resistant Ophthalmoplegia

We report the histopathological and ultrastructural tissue analysis of extraocular muscle (EOM) obtained from a patient with seronegative myasthenia gravis (MG) with treatment-resistant ophthalmoplegia for 3.5 years. The EOM demonstrated predominantly myopathic features and ultrastructural evidence of mitochondrial dysfunction, but the most striking features were increased endomysial collagen and adipocyte replacement of muscle fibers. By contrast, control EOM from a patient undergoing strabismus surgery for a sensory exotropia in a nonseeing eye and a similar duration of deviation, showed normal muscle histology. Although the histopathological and ultrastructural findings largely resemble those of limb muscle in MG, the abundant endomysial collagen may be nonspecific and secondary to poor force generation as a result of chronic ophthalmoplegia.

Clinical Observation Extraocular Muscle Findings in Myasthenia Gravis Associated Treatment-Resistant Ophthalmoplegia Robyn M. Rautenbach, MSc, Komala Pillay, MBChB, Anthony D. N. Murray, MBChB, Jeannine M. Heckmann, PhD Abstract: We report the histopathological and ultrastructural tissue analysis of extraocular muscle (EOM) obtained from a patient with seronegative myasthenia gravis (MG) with treatment-resistant ophthalmoplegia for 3.5 years. The EOM demonstrated predominantly myopathic features and ultrastructural evidence of mitochondrial dysfunction, but the most striking features were increased endomysial collagen and adipocyte replacement of muscle fibers. By contrast, control EOM from a patient undergoing strabismus surgery for a sensory exotropia in a nonseeing eye and a similar duration of deviation, showed normal muscle histology. Although the histopathological and ultrastructural findings largely resemble those of limb muscle in MG, the abundant endomysial collagen may be nonspecific and secondary to poor force generation as a result of chronic ophthalmoplegia. Journal of Neuro-Ophthalmology 2017;37:414-417 doi: 10.1097/WNO.0000000000000534 © 2017 by North American Neuro-Ophthalmology Society A lthough extraocular muscles (EOMs) are frequently involved early in the course of myasthenia gravis (MG), the resultant ophthalmoplegia usually responds to standard immunosuppressive treatment (1). In MG, the pathogenic antibodies most frequently target the acetylcho- Neurology Research Group (RR, JMH), Department of Medicine, University of Cape Town, Cape Town, South Africa; Division of Ophthalmology (RMR), Department of Surgical Sciences, University of Stellenbosch, Belville, South Africa; Division of Anatomical Pathology (KP), Department of Pathology, University of Cape Town, Cape Town, South Africa; Division of Ophthalmology (ADNM), Department of Surgery, University of Cape Town, Cape Town, South Africa; and Division of Neurology (JMH), Department of Medicine, University of Cape Town, Cape Town, South Africa. R. Rautenbach was supported by a Discovery Fellowship, J. M. Heckmann received a South African Medical Research Grant. The authors report no conflicts of interest. Address correspondence to Jeannine M. Heckmann, PhD, E8-74 Neurology, Groote Schuur Hospital, Observatory, 7925 Cape Town, South Africa; E-mail: [e-mail] 414 line receptors (AChR) and comprise IgG1 which can activate complement (2). The EOMs may be at particular risk of complement-mediated damage in MG (3). A subgroup of MG patients without detectable AChR antibodies may have antibodies directed against muscle-specific tyrosine kinase (MuSK), low-density lipoprotein receptor-related protein 4 (LRP4) or other endplate targets, some of which remain unknown (2,4). We have reported treatment-resistant ophthalmoplegia to occur in a proportion of MG patients with detectable AChR antibodies, African ancestry, and juvenileonset MG, but also among those with MuSK antibodies and triple-seronegative MG (4-6). Here we describe the histopathological and ultrastructural tissue analysis of EOM obtained from a triple-seronegative patient with generalized MG who responded to treatment in her nonocular muscles, but maintained treatment-resistant ophthalmoplegia for 3 years. We compare the histology of this patient with a control, an individual with a sensory exotropia (XT) of similar duration. CASE REPORT A 45-year-old woman developed classic features of MG-associated fatigable weakness involving extraocular, bulbar, and proximal limb muscles (MG Foundation of America Classification Grade 3B). She had been symptomatic for 6 months before the initiation of immunotherapy. Confirmation of the clinical diagnosis of MG included pyridostigmine responsiveness and .10% decremental response to repetitive nerve stimulation testing [case reported in (4)]. Although her nonocular muscles responded completely to treatment, her EOMs remained treatment resistant. Indeed, she progressed from fatigable diplopia and ptosis at presentation to persistent binocular diplopia and ptosis with little or no variability, despite immunotherapy for more than 3 years. This included pyridostigmine, prednisone, methotrexate, and intravenous immunoglobulin. Surgical Rautenbach et al: J Neuro-Ophthalmol 2017; 37: 414-417 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Clinical Observation FIG. 1. Myasthenic and control medial rectus muscles. A. In myasthenic muscle, there is increased endomysial collagen and adipose tissue replacing atrophic muscle fibers (arrows) and myofiber splitting with whorled-fiber changes (arrowheads) in the muscle (hematoxylin & eosin, ·200). Inset demonstrates that these changes are present throughout the muscle (hematoxylin & eosin, ·40). B. There is increased endomysial collagen (arrow) replacing myofibers (modified trichrome stain, ·200). C. These changes are not present in the control medial rectus (modified trichrome stain, ·200). D. Ultrastructural analysis reveals myofibrillar disarray with Z-band streaming (arrowheads), subsarcolemmal aggregates of swollen mitochondria (white arrow) with adjacent and apparently intranuclear lipid droplets and collagen deposition (black arrow) between myofibers (·7000). E. Ultrastructure of control medial rectus muscle demonstrates the normal myofibrillar pattern and a central aggregate of normal appearing mitochondria (·3000). correction of her ocular alignment was performed while she remained on her maintenance therapy (pyridostigmine, methotrexate). Initial laboratory testing detected no circulating AChR, MuSK, or LRP4 antibodies by standard radioimmunoassay as well as cell-based assay (4). Thyroid eye disease and mitochondrial myopathy were excluded by means of biochemical testing and orbital imaging, as well as a long range polymerase chain reaction assay for mitochondrial DNA mutations, respectively (5). Examination before surgery revealed bilateral upper eyelid ptosis, and an XT of 25 prism diopters and left hypertropia of 5 prism diopters, with very limited motility Rautenbach et al: J Neuro-Ophthalmol 2017; 37: 414-417 in all directions of gaze. Forced duction testing was negative, and active force generation testing demonstrated marked paresis with virtually no clinically detectable force in all the extraocular rectus muscles. Histopathological and ultrastructural analysis was performed on 6 mm of medial rectus muscle and tendon. Care was taken to ensure that the sample was orientated to allow for tissue to be sectioned from the sample closest to the muscle belly and therefore farthest away from the tendinous insertion. There was increased endomysial collagen deposition and the presence of adipocytes replacing atrophic muscle fibers (Fig. 1A). These findings were present diffusely throughout the muscle tissue sections. 415 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Clinical Observation There was increased myofiber size variation, myofiber whorling and splitting. The modified trichrome stain highlighted the increase in endomysial collagen (Fig. 1B) compared with the control (Fig. 1C). Most of the fibers were rounded with no group atrophy. Focal nuclear clumping was noted in atrophic fibers. Ultrastructural analysis was performed on high magnification images produced by transmission electron microscopy (JEOL JEM1011 TEM) and captured with an Olympus camera using iTEM software (version 5; Olympus, Tokyo, Japan). This analysis revealed myofibrillar disarray with Z-band streaming and subsarcolemmal aggregates of swollen mitochondria (Fig. 1D). No paracrystalline bodies were identified. In comparison, we analyzed a similar sample of EOM, 6.5 mm of medial rectus muscle and tendon, obtained from a healthy 32-year-old man with a sensory exotropia of 3.5 years duration. Before surgery, this patient had full range of eye movements with no features of paresis or paralysis evident on motility testing or forced duction. This control EOM demonstrated normal histological appearance of the myofibers with the absence of collagen deposition between myofibers (Fig. 1C). On electron microscopy, we noted a normal appearing myofibrillar pattern, central aggregation of normal appearing mitochondria, and no collagenous material (Fig. 1E). mild atrophy and myopathic features of mitochondrial stress with subsarcolemmal accumulation of mitochondria and swollen/giant mitochondria. These morphological findings in muscle mitochondria have been described as nonspecific changes in muscle in which there is loss of force generation (11), and may explain why myasthenic muscles show mitochondrial changes even in very mildly affected muscles. However, it is unclear whether the extensive collagenous connective and fatty tissue replacement of atrophic fibers observed here in the EOMs as a consequence of ophthalmoplegia are specific to the treatment-resistant ophthalmoplegic MG subphenotype or the consequence of complete paralysis and an inability to elicit a generated force. Although the histopathological and ultrastructural features in our patient resemble those of limb muscle in MG, the severity of these findings may be secondary to chronic ophthalmoplegia. STATEMENT OF AUTHORSHIP Category 1: a. Conception and design: R. M. Rautenbach and J. M. Heckmann; b. Acquisition of data: R. M. Rautenbach, J. M. Heckmann, K. Pillay, and A. D. N. Murray; c. Analysis and interpretation of data: R. M. Rautenbach, J. M. Heckmann, and K. Pillay. Category 2: a. Drafting the manuscript: R. M. Rautenbach and J. M. Heckmann; b. Revising it for intellectual content: R. M. Rautenbach, J. M. Heckmann, K. Pillay, and A. D. N. Murray. Category 3: a. Final approval of the completed manuscript: R. M. Rautenbach, J. M. Heckmann, K. Pillay, and A. D. N. Murray. DISCUSSION Gratton et al (7) described the histology of EOMs in a 66year-old man with AChR antibody-positive MG after 14 months of treatment-resistant ophthalmoplegia. Although ultrastructural analysis was not performed, histology of the superior rectus muscle showed features similar to our patient with atrophy and near-complete replacement of EOM by fibrocellular material and connective tissue. There were no ragged red fibers. Apart from the extensive collagenous and fatty connective tissue, most of the ultrastructural changes in the EOM tissue reported in our patient mirror those previously reported in limb skeletal muscle biopsies of MG patients irrespective of whether the patients had detectable AChR or MuSK antibodies. Limb muscles have demonstrated the presence of myofiber atrophy, subsarcolemmal mitochondrial aggregation (or rims), and myofibrillar disarray with Z-line streaming (8,9). These features did not seem to correlate with MG disease severity. Padua et al (10) found that the histology of limb muscles from AChR/MuSK antibody-negative patients was similar to AChR antibody-positive patients with mild myofiber atrophy, but ultrastructual analyses were not performed. Although some authors report slightly more evidence of mitochondrial dysfunction in MuSK antibody-positive MG (9), overall it seems that MG, regardless of the pathogenic antibody subtype, results in 416 ACKNOWLEDGMENTS The authors thank Dr. Maureen Duffield and Mr. Ebrahim Dollie for their assistance. REFERENCES 1. Benatar M, McDermott MP, Sanders DB, Wolfe GI, Barohn RJ, Nowak RJ, Hehir M, Juel V, Katzberg H, Tawil R; the Muscle Study Group. Efficacy of prednisone for the treatment of ocular myasthenia (EPITOME): a randomized, controlled trial. Muscle Nerve. 2016;53:363-369. 2. Binks S, Vincent A, Palace J. Myasthenia gravis: a clinicalimmunological update. J Neurol. 2016;263:826-834. 3. Soltys J, Gong B, Kaminski HJ, Zhou Y, Kusner LL. Extraocular muscle susceptibility to myasthenia gravis: unique immunological environment? Ann N Y Acad Sci. 2008;1132:220-224. 4. Huda S, Woodhall MR, Vincent A, Heckmann JM. 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