Extraocular Muscle Biopsy in Chronic Progressive External Oph thalmoplegia Steven P. Ringel, MD, W. Bruce Wilson, MD, and Michael T. Barden

A quantitative assessment of t h e pathological changes in extraocular muscle is presented in 8 patients with chronic progressive external ophthalmoplegia (CPEO). Serial cross-sections of extraocular muscle were stained with a battery of histochemical and immunohistochemical techniques and compared with 36 normal extraocular muscles and 1 muscle from a patient who had longstanding third nerve palsy with anomalous reinnervation. Several of the patients had a striking increase in the number of ragged-red fibers in extraocular muscle, particularly if frequent ragged-red fibers also were found on limb muscle biopsy. One patient demonstrated extrajunctional acetylcholine receptor (AChR) in a small percentage of fibers, although this finding was not present in the reinnervated muscle, Numerous darkly staining central regions were noted in the ocular muscle fibers of a patient with Stephens syndrome (CPEO, peripheral neuropathy, and cerebellar disease) and in the reinnervated muscle. A patient with myotubular myopathy had single central nuclei in both limb and ocular muscle. All patients demonstrated in their extraocular muscles variation i n both t h e size and distribution of each of the three histochemical fiber types. Extraocular muscle biopsy proved to be a safe, reliable technique. As a similar quantitative analysis is applied to the study of further patients, a better understanding of the pathogenesis of CPEO should be possible. Ringel SP, Wilson WB, Barden MT: Extraocular muscle biopsy in chronic progressive external ophthalmoplegia. Ann Neurol 6:326-339, 1979

Previous descriptions of t h e pathological changes seen in extraocular muscle in patients with chronic progressive external ophthalmoplegia (CPEO) have emphasized that extraocular muscles are capable of only a limited range of pathological response [2, 4 , 5 , 9, 10, 27, 281. Many reports have been incomplete, and none have assessed alterations in the size o r distribution of histochemical fiber types in these disorders. I n this study, a complete histochemical analysis of a cross-sectional biopsy of extraocular muscle was performed in 8 patients with CPEO along with simultaneous limb muscle biopsy. A quantitative assessment of the size and distribution of each of the three fiber types in extraocular muscle was compared with an assessment of 36 normal human extraocular muscles [26]and with an ocular muscle from a patient who had a longstanding third nerve palsy with anomalous reinnervation. As an additional indicator of altered innervation, the presence of extrajunctional acetylcholine receptor (AChR) was assessed in each patient [23-251.

Methods All patients unJenvent an extensive clinical evaluation including complete neurological and neuroophthalmologica1

From the Departments of Neurology and Ophthalmology, University of Colorado Health Science Center, Denver, CO. Accepted for publication Mar 27, 1979

examination. Special ophthalmological investigations included electoretinogram (ERG), fluorescein angiography, and visual evoked response (VER). Laboratory evaluation was performed to exclude diseases known to produce ophthalmoplegia: myasthenia gravis (Tendon test), Refsum disease (phytanic acid), abetalipoproteinemia (lipoprotein electrophoresis), and thyroid ophthalmopathy (triiodothyronine, thyroxine). In addition, skull roentgenogram, CT scan, electroencephalogram, lumbar puncture, and electrocardiogram were performed. In most cases complete electrodiagnostic studies including electromyography (EMG) and nerve conduction velocities were done on limb muscles. Extraocular muscle EMG was done but will be the subject of a separate report. Each patient had a limb muscle biopsy and extraocular muscle biopsy performed under general or local anesthesia. A 2 mm cross-sectional biopsy of extraocular muscle was removed and mounted in gum tragacanth on a wooden chuck. The specimen was quick frozen in isopentane and immersed in liquid nitrogen. Serial 10 p cryostat sections of both limb and extraocular muscle were stained with a battery of 16 histochemical reactions LIZ] and an immunoperoxidase reaction for AChR [ 23-2 51. Positive controls for extrajunctional AChR included experimentally denervated rat limb muscle and monkey extraocular muscle as well as limb muscle biopsies from patients with denervating disorders. I n extraocular muscle the percentage of coarse, fine, and

Address reprint requests to D r Ringel, B-185 Department of Neurology, University of Colorado Health Science Center, 4200 E Ninth Ave, Denver, CO 80262.

326 O364-5134/7~/1OO326-14$01.25@ 1978 by Steven P. Ringel

granular fibers was determined [26]. Two hundred consecutive fibers were counted by two independent examiners using the reduced nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR) reaction. The fiber distribution was compared with the mean and standard deviation of 36 normal human extraocular muscles. Similar counts were made using the modified trichrome and the p H 4.6 preincubated myofibrillar adenosine triphosphatose (ATPase) reactions. N o significant discrepancy was noted except in Patient 5. The minimum diameter of 100 consecutive coarse, fine, and granular fibers was recorded; a histogram was constructed and compared with a similar histogram from normal individuals. For fine fibers, because of the limited number present, only 5 0 were measured. In normal human extraocular muscle a consistent, concentric arrangement of fibers has been noted [26]. A narrow peripheral zone consists almost entirely of coarse fibers. In several of the biopsies the peripheral zone was hard to identify. For this reason, fiber counts were performed only on the center of the biopsy and were compared with the large central zone from normal human extraocular muscle. It is not clear if the peripheral zone was destroyed during the biopsy o r if the primary patholog~cal process included alteration of this zone so that it was no longer recognizable. In 3 of the biopsies a normal peripheral zone was seen, which suggests that mechanical disruption may have caused the absence of this zone in the other cases.

Patient Histories Clinical and laboratory data on the patients is summarized in Table 1.

Patient 1 A 34-year-old man had first noted progressive ptosis and ophthalmoparesis at age 17. There was no family history of neurological disease. General physical examination was normal. Ophthalmological examination revealed bilateral ptosis and mild exotropia. Severe symmetrical ophthalmoparesis was present in all directions, with only 5 degrees of movement. Mild orbicularis oculi weakness was seen, but the remainder of the facial muscles were normal. Funduscopic examination revealed a normal retina. Visual fields were normal, and visual acuity was 20/20 bilaterally. Pupils were normal. An ERG and VERs were normal. Neurological examination was otherwise normal. T h e only abnormal laboratory finding was a cerebrospinal fluid protein elevated to 59 mg per deciliter. A biopsy of the left biceps muscle revealed mild variation in fiber size with occasional angular fibers scattered throughout the biopsy. These angular fibers stained darkly with both the NADH-TR and nonspecific esterase reactions. One percent of the type 1 fibers demonstrated abnormal subsarcolemmal accumulations that stained red using the modified trichrome reaction (ragged-red fibers). Associated with this increase in red intermyofibrillar network was an occasional vacuole that stained positively with oil red 0 and Sudan black. The ragged-red fibers also stained darkly with oxidative reactions including the

NADH-TR and the succinic dehydrogenase (SDH) tests, suggesting that they were clusters o f mitochondria.

Patient 2 A 47-year-old man had first noted weakness in his lower extremities 15 years ago and weakness and incoordination in the right upper extremity several years later. Eight years before we saw him he was noted to have slowly progressive, bilateral ptosis and ophthalmoparesis, slurring of speech, and generalized incoordination. There was no family history of neurological disease. General physical and cardiovascular examinations were normal. Ophthalmological examination revealed bilateral ptosis, slightly greater on the left. Mild anisocoria was present with the left pupil measuring 6 mm and the right 5 mm, although both reacted briskly to light and accommodation. There was slight exotropia and moderately severe ophthalmoplegia, with 10 degrees of movement remaining on upward and downward gaze and 20 degrees on lareral gaze. Attempted saccadic eye movements elicited only slow pursuit speed movement in all directions. Funduscopic examination was normal. Visual acuity was 20/20 bilaterally and formal visual fields were normal. E R G and VERs were normal. Neurological examination revealed a stocking-glove distribution of sensory loss to pinprick stimulation in all four extremities, associated with absent deep tendon reflexes. Bilateral finger-nose and heel-knee-shin dysmetria was noted, and the patient walked with a broad-based, ataxic gait. Speech was halting and slurred. The EMG revealed fibrillations in all extremities associated with a reduction in the number of motor unit potentials under voluntary control. Several complex, polyphasic units were noted. Motor nerve conduction velocities were mildly decreased in all four extremities. A C T scan showed generalized cerebral cortical atrophy. An EEG revealed background beta activity and slowing of the alpha rhythm consistent with diffuse cerebral dysfunction. CSF protein was 5 3 mg per deciliter. An EKG revealed diffuse T-wave inversions in the anterolateral leads that were nonspecific but compatible with ischemia. A biopsy of the left biceps muscle revealed mild variation in fiber size. Two percent of the type 1 fibers were noted on modified trichrome stain to be typically ragged red with an increase in red intermyofibrillar network, particularly in the subsarcolemmal regions. In addition, occasional basophilic fibers with large vesicular nuclei were seen. No angular fibers, type grouping, o r group atrophy was noted.

Patient 3 A 16-year-old girl had first been noted to have progressive ophthalmoparesis at age 8 and progressive dysphagia and dysphonia beginning at age 12. There was no family history of neurological disease. l General physical examination revealed a thin g ~ r with marked facial diplegia but no other abnormalities. Ophthalmological examination revealed mild right esotropia with limitation of extraocular movements in all directions. Only a few degrees of elevation of each eye re-

Ringel et al: Extraocular Muscle in Ophthalmoplegia

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Table 1 . Summaq of Clinical and Laboratory FeatureJ Laboratory Studies Patient N o . and Age (yr)

Age at Onset iyr)

1, 34 2 , 47

17 32

3, 16

x

4 , 44

17

5 , 17

lnfancy

6 , 26

11

7 , 38

12

8, 5 5

‘t8

Clinical Features

EKG

CPEO CPEO; peripheral neuropath y; cerebellar degeneration CPEO; severe bulbar weakness; moderate extremity weak-

Normal T-wave inversions

CSF Protein (mgidl)

59 53

Normal

EMG

NCV

Normal Fibrillations; large polyphasic potentials

Normal Slow

Small polyphasic potentials

Normal

Inflammation in pharyngeal muscle biopsy

Other Cortical atrophy (CT); diffusely slow EEG

ness

CPEO; retinitis pigmentosa; hearing loss; mental retardation; mild bulbar and extremity weakness CPEO; retinitis pigmentosa; hearing loss; mental retardation; mild bulbar and extremity weakness; small stature; cerebellar degeneration; diabetes CPEO; cervical carcinoma CPEO; moderate facial and neck flexor weakness, distal > proximal; schizophrenia C P E O ; dysphagia; facial and neck flexor weakness

Right preterminal Q R S vectors and abnormal depolarization

140

Normal

Normal

Cortical atrophy (CT); theta bursts o n EEG; CPK 140 (normal,