Change in the antioxidative capacity of extraocular muscles in patients with exotropia.

BACKGROUND: We compared the oxidative stress and antioxidant capacities of the medial rectus muscles (MRMs) between the patients with constant exotropia and control subjects. METHODS: A total of 40 MRMs from patients with constant exotropia and 40 MRMs from normal donor eyes (controls) were assessed. Neuronal nitric oxide synthase (nNOS), superoxide dismutase (SOD), and catalase levels were compared between the two groups using western blot analysis. In addition, the lipofuscin accumulation level was compared between the exotropic and control groups. RESULTS: According to western blot analysis, the nNOS level was significantly higher in the exotropic group than in the control group (P < 0.05). On the other hand, a catalase expression level was higher in the control group than in the exotropic group (P < 0.05). The SOD1 level did not show a significant difference between the two groups. The relative lipofuscin fluorescence intensity was higher in the exotropic group than in the control group (P < 0.001). CONCLUSIONS: Based on these findings, the MRMs of patients with exotropia had a redox imbalance status. Further study is needed to investigate whether this imbalance in antioxidant capacity is present in the extraocular muscles of patients with other strabismus.

Molecular analysis of the muscle pathology associated with mitochondrial DNA deletions.

Large-scale deletions of mitochondrial DNA (mtDNA) are associated with a subgroup of mitochondrial encephalomyopathies. We studied seven patients with Kearns-Sayre syndrome or isolated ocular myopathy who harboured a sub-population of partially-deleted mitochondrial genomes in skeletal muscle. Variable cytochrome c oxidase (COX) deficiencies and reduction of mitochondrially-encoded polypeptides were found in affected muscle fibres, but while many COX-deficient fibres had increased levels of mutant mtDNA, they almost invariably had reduced levels of normal mtDNA. Our results suggest that a specific ratio between mutant and wild-type mitochondrial genomes is the most important determinant of a focal respiratory chain deficiency, even though absolute copy numbers may vary widely.

Orbicularis oculi: morphological changes mimicking mitochondrial cytopathy in a series of control normal muscles.

INTRODUCTION: Orbicularis oculi (OO) muscle has recently proposed as a suitable muscle for biopsy to diagnose mitochondrial cyopathy: METHODS: Enzyme histochemical and immunohistochemical studies were performed on OO muscle obtained from 18 patients aged 37-87 years (median 64 years), 6 males, 12 females, who were undergoing routine upper blepharoplasty surgery. RESULTS: We confirmed the marked type II fibre (fast myosin heavy chain) predominance (89%) but also noted a different proportion and distribution of mitochondria in these fibres with occasional pseudo-'ragged-red' fibres with prominent subsarcolemmal and cytoplasmic aggregation of mitochondria. Cytochrome oxidase-negative fibres and true 'ragged-red' fibres were found at all ages over 40 years at levels that approach those used for diagnosis of mitochondrial cytopathy in peripheral or limb skeletal muscles. CONCLUSION: We would therefore urge caution in the use of OO as muscle biopsy for diagnosis of mitochondrial cytopathy and advise concomitant biopsy of limb skeletal muscle and/or supplementary genetic studies.

Palisade Endings Have an Exocytotic Machinery But Lack Acetylcholine Receptors and Distinct Acetylcholinesterase Activity.

Purpose: The purpose of this work was to test whether palisade endings express structural and molecular features of exocytotic machinery, and are associated with acetylcholine receptors, and enzymes for neurotransmitter breakdown. Methods: Extraocular rectus muscles from six cats were studied. Whole-mount preparations of extraocular muscles (EOMs) were immunolabeled with markers for exocytotic proteins, including synaptosomal-associated protein of 25 kDa (SNAP25), syntaxin, synaptobrevin, synaptotagmin, and complexin. Acetylcholine receptors (AChRs) were visualized with α-bungarotoxin and with an antibody against AChRs, and acetylcholinesterase (AChE) was tagged with anti-AChE. Molecular features of multicolor labeled palisade endings were analyzed in the confocal scanning microscope, and their ultrastructural features were revealed in the transmission electron microscope. Results: All palisade endings expressed the exocytotic proteins SNAP25, syntaxin, synaptobrevin, synaptotagmin, and complexin. At the ultrastructural level, vesicles docked at the plasma membrane of terminal varicosities of palisade endings. No AChRs were associated with palisade endings as demonstrated by the absence of α-bungarotoxin and anti-AChR binding. AChE, the degradative enzyme for acetylcholine exhibited low, if any, activity in palisade endings. Axonal tracking showed that axons with multiple en grappe motor terminals were in continuity with palisade endings. Conclusions: This study demonstrates that palisade endings exhibit structural and molecular characteristics of exocytotic machinery, suggesting neurotransmitter release. However, AChRs were not associated with palisade endings, so there is no binding site for acetylcholine, and, due to low/absent AChE activity, insufficient neurotransmitter removal. Thus, the present findings indicate that palisade endings belong to an effector system that is very different from that found in other skeletal muscles.

Extraocular Muscle Reveals Selective Vulnerability of Type IIB Fibers to Respiratory Chain Defects Induced by Mitochondrial DNA Alterations.

Purpose: The purpose of this study was to gain insights on the pathogenesis of chronic progressive external ophthalmoplegia, thus we investigated the vulnerability of five extra ocular muscles (EOMs) fiber types to pathogenic mitochondrial DNA deletions in a mouse model expressing a mutated mitochondrial helicase TWINKLE. Methods: Consecutive pairs of EOM sections were analyzed by cytochrome C oxidase (COX)/succinate dehydrogenase (SDH) assay and fiber type specific immunohistochemistry (type I, IIA, IIB, embryonic, and EOM-specific staining). Results: The mean average of COX deficient fibers (COX-) in the recti muscles of mutant mice was 1.04 ± 0.52% at 12 months and increased with age (7.01 ± 1.53% at 24 months). A significant proportion of these COX- fibers were of the fast-twitch, glycolytic type IIB (> 50% and > 35% total COX- fibers at 12 and 24 months, respectively), whereas embryonic myosin heavy chain-expressing fibers were almost completely spared. Furthermore, the proportion of COX- fibers in the type IIB-rich retractor bulbi muscle was > 2-fold higher compared to the M. recti at both 12 (2.6 ± 0.78%) and 24 months (20.85 ± 2.69%). Collectively, these results demonstrate a selective vulnerability of type IIB fibers to mitochondrial DNA (mtDNA) deletions in EOMs and retractor bulbi muscle. We also show that EOMs of mutant mice display histopathological abnormalities, including altered fiber type composition, increased fibrosis, ragged red fibers, and infiltration of mononucleated nonmuscle cells. Conclusions: Our results point to the existence of fiber type IIB-intrinsic factors and/or molecular mechanisms that predispose them to increased generation, clonal expansion, and detrimental effects of mtDNA deletions.

Fatigue resistance of rat extraocular muscles does not depend on creatine kinase activity.

BACKGROUND: Creatine kinase (CK) links phosphocreatine, an energy storage system, to cellular ATPases. CK activity serves as a temporal and spatial buffer for ATP content, particularly in fast-twitch skeletal muscles. The extraocular muscles are notoriously fast and active, suggesting the need for efficient ATP buffering. This study tested the hypotheses that (1) CK isoform expression and activity in rat extraocular muscles would be higher, and (2) the resistance of these muscles to fatigue would depend on CK activity. RESULTS: We found that mRNA and protein levels for cytosolic and mitochondrial CK isoforms were lower in the extraocular muscles than in extensor digitorum longus (EDL). Total CK activity was correspondingly decreased in the extraocular muscles. Moreover, cytoskeletal components of the sarcomeric M line, where a fraction of CK activity is found, were downregulated in the extraocular muscles as was shown by immunocytochemistry and western blotting. CK inhibition significantly accelerated the development of fatigue in EDL muscle bundles, but had no major effect on the extraocular muscles. Searching for alternative ATP buffers that could compensate for the relative lack of CK in extraocular muscles, we determined that mRNAs for two adenylate kinase (AK) isoforms were expressed at higher levels in these muscles. Total AK activity was similar in EDL and extraocular muscles. CONCLUSION: These data indicate that the characteristic fatigue resistance of the extraocular muscles does not depend on CK activity.

Sparing of mdx extraocular muscles from dystrophic pathology is not attributable to normalized concentration or distribution of neuronal nitric oxide synthase.

Previous findings have led to speculations that decreased concentration of nNOS (neuronal nitric oxide synthase) may underlie some aspects of the pathophysiology of dystrophic muscle. We have tested whether the sparing of extraocular muscles (EOM) in muscular dystrophy is attributable to the presence of normal nNOS concentration and distribution in these muscles. Measurements of total nNOS concentration in control muscle showed that total nNOS comprises approximately 0.05% of total muscle protein, indicating a molar stoichiometry of approximately 60 and 20 to total dystrophin and syntrophin, respectively. Thus, most muscle nNOS is either not associated with the dystrophin complex, or binds to yet unidentified sites in the complex. nNOS concentration was at least two-fold greater in C57 EOM and tibialis anterior (TA) compared with mdx samples. No significant differences in nNOS concentration in EOM versus TA in either mdx or C57 mice were observed, nNOS was concentrated at the sarcolemma of all C57 samples, while mdx nNOS displayed a cytosolic distribution, except in fibers that reverted to express dystrophin. These data show that mdx EOM are spared by a mechanism other than normalized concentration and location of nNOS.

Quantification of muscle loss in the doxorubicin-treated orbicularis oculi of the monkey. Effect of local injection of doxorubicin into the eyelid.

Many neuromuscular diseases are characterized by involuntary and forceful muscle contraction. Doxorubicin has been shown to chemically remove the orbicularis oculi muscle after injection into the eyelid. The chemomyectomy effect of doxorubicin was quantified after injection of a 2-mg dose into the right lower eyelid. A significant loss of muscle was seen in the injected eyelids, with a mean loss of 87% of the summed cross-sectional area of all muscle fibers throughout representative cross-sections of the treated eyelids. This muscle loss was selective, with the greatest loss in the preseptal region of the orbicularis oculi. The authors found no differential loss of different muscle fiber types after doxorubicin treatment. This muscle loss was not accompanied by an increase in connective tissue, and surrounding eyelid structures were normal. The authors find that doxorubicin is a highly effective myotoxin and represents a novel permanent treatment for blepharospasm and related diseases because it is highly specific for muscle cells and is easy to localize within a given muscle.

Immunohistochemical identification of slow-tonic fibers in human extrinsic eye muscles.

A fluorescent antiserum against myosin from chicken anterior latissimus dorsi muscle, which stains specifically the multiply innervated slow fibers of birds and amphibians, was applied to frozen sections of human extraocular muscles. A proportion of fibers in oculorotatory muscles were labeled by the antiserum. In contrast, no labeled extrafusal fiber was present in the levator palpebrae or in other body muscles. Serial study of sections stained for acetylcholinesterase and myosin ATPase showed that the labeled fibers in human oculorotatory muscles are multiply innervated and display acid-stable myosin ATPase activity.

Sarco(endo)plasmic reticulum Ca2+ ATPases (SERCA1 and -2) in human extraocular muscles.

PURPOSE: To investigate the composition of the fibers in human extraocular muscles (EOMs) with respect to the sarco(endo)plasmic reticulum Ca(2+)ATPases (SERCA)-1 and -2 and to investigate possible correlations between SERCA and myosin heavy chain (MyHC) composition. METHODS: EOM samples were processed for immunocytochemistry with monoclonal antibodies specific against SERCA1 (fast isoform), SERCA2 (slow isoform), or different MyHCs. A total of 1571 fibers were analyzed. Microsomal EOM fractions were analyzed with SDS-PAGE and immunoblots. RESULTS: The fast fibers, containing MyHCIIa, accounted for 79% of the fibers in the orbital layer (OL) and 74% in the global layer (GL). More than 99% of these fibers contained SERCA1, and 86% of them coexpressed SERCA1 and -2. Almost all slow fibers stained with SERCA2; 54% of those in the GL and all in the OL coexpressed SERCA1 and -2. Fifteen percent of the fibers in the GL and less than 1% in the OL were MyHCeom(pos)/MyHCIIa(neg) fibers. All these contained SERCA1 and in the OL also stained strongly with anti-SERCA2. Biochemically SERCA2 was more abundant than SERCA1. CONCLUSIONS: The human EOMs had a very complex pattern of expression of the major protein regulating fiber relaxation rate. The coexistence of SERCA1 and -2, together with complex mixtures of MyHCs in most of the fibers provide the human EOMs with a unique molecular portfolio that allows a highly specific fine-tuning regimen of contraction and relaxation.

Diltiazem reduces the contractility of extraocular muscles in vitro and in vivo.

Diltiazem, a Ca-channel blocker that is used clinically for the treatment of hypertension and cardiac arrhythmias, reduces the contractility of extraocular muscles. Exposure of rabbit extraocular muscle to diltiazem in vitro reduces the sustained tension that is generated by the tonic, multiply innervated fibers, and decreases the baseline, or resting, tension of the muscle. When diltiazem is injected into a selected extraocular muscle in the rabbit in vivo, it causes a temporary weakening of the muscle, which is indicated by a deviation of eye position. These in vivo effects are of short duration, are easily reproducible, and vary with dosage. The results of this study raise the possibility that diltiazem may be used as an alternative to the surgical treatment of strabismus and other oculomotor dysfunctions including blepharospasm.

The development of longitudinal variation of Myosin isoforms in the orbital fibers of extraocular muscles of rats.

PURPOSE: To examine the appearance of longitudinal variation of extraocular and embryonic myosin heavy chain (MyHC) isoforms during the development of orbital singly innervated fibers of rat extraocular muscles (EOMs). METHODS: EOMs were dissected from rat pups of various ages and stained with isoform-specific monoclonal antibodies to the embryonic and extraocular MyHC isoforms and to neurofilaments, as well as with labeled alpha-bungarotoxin. The orbital layers of whole muscles were examined by confocal microscopy. RNase protection assays for the embryonic (Myh3) and extraocular (Myh13) MyHC isoform mRNAs were also performed. RESULTS: At 10 days postpartum, the EOM MyHC RNA was first detected by RNase protection assay. At 11 days postpartum, the extraocular isoform was detected in the orbital fibers as two thin stripes just proximal and distal to the neuromuscular junction (NMJ). Over the next few weeks, the area occupied by the extraocular isoform increased to include the entire central region of the orbital fibers at and surrounding the NMJ. At the same time, the embryonic isoform became excluded from the region of the NMJ. CONCLUSIONS: The orbital layer fibers of rat EOMs contain a longitudinal variation in MyHC isoforms not seen in other skeletal muscles. Development of this longitudinal variation begins as a late event postpartum; and the first appearance of it may be closely linked to neural contact. This targeting of MyHC isoforms to distinct domains is unique to EOMs.

Doxorubicin chemomyectomy is enhanced when performed two days following bupivacaine injections: the effect coincides with the peak of muscle satellite cell division.

PURPOSE: Doxorubicin is effective in permanently removing muscle after direct injection into the eyelid for treatment of blepharospasm and hemifacial spasm. However, patients often require two or more injection series before full abatement of their spasms is achieved. Local anesthetics cause muscle necrosis, followed by regeneration, a process that requires activation and division of muscle satellite cells. This study examined whether the muscle toxicity of doxorubicin could be amplified by injection of doxorubicin into the eyelid of rabbits 2 days after a local anesthetic injury, perhaps exploiting the toxic effects of doxoribicin on satellite cells at the peak time of their division after injury. METHODS: Rabbit eyelids received two series of injections of bupivacaine and hyaluronidase spaced 18 hours apart. Two days later, the eyelids were injected with either 0.5 or 1 mg doxorubicin. Animals were monitored daily for onset and duration of skin injury. After 1 month, the eyelids were assessed for muscle loss using histologic and morphometric techniques. RESULTS: Injection of doxorubicin during the peak of satellite cell activation and division 2 days after injury significantly increased muscle loss over doxorubicin alone. This treatment did not result in increased skin injury compared with doxorubicin alone. CONCLUSIONS: Permanent muscle loss was increased when doxorubicin was injected at the peak of satellite cell division 2 days after injury of the muscle with bupivacaine in rabbit eyelid, taking advantage of the antimitotic effects of doxorubicin on satellite cell division during the period of active regeneration. When local anesthetic injection immediately preceded the doxorubicin injection, increased myotoxicity was not seen. The injection of doxorubicin into muscle 2 days after a previous injury maximizes muscle loss. The increased muscle loss provided by this double treatment may decrease the number of injection visits required by blepharospasm and hemifacial spasm patients during their course of treatment, thus reducing the number of patients with side effects, which increases with repeated exposures of the eyelid to doxorubicin.

Ocular pathology in mitochondrial superoxide dismutase (Sod2)-deficient mice.

PURPOSE: To characterize the pathologic features in retina, optic nerve, and extraocular muscle of mitochondrial superoxide dismutase (Sod2)-deficient mice, a model of increased mitochondrial production of reactive oxygen species. METHODS: Morphometric and ultrastructural study of eyes of 43 homozygous sod2(tm1Cje-/-) mice and wild-type control animals. For retinal morphometric analysis, 32 manganese 5,10,15,20-tetrakis (4-benzoic acid) porphyrin (MnTBAP)-treated animals aged either 9 to 10 days or 20 to 21 days were studied. Ultrastructural examination was performed on tissue from the treated animals, and 11 additional untreated mutant and control animals. RESULTS: In treated Sod2-deficient animals, the photoreceptor layer was thinner centrally at 9 to 10 days than in control animals (mean 8.8 vs. 14.7 microm). By 20 to 21 days, all retinal layers apart from the outer nuclear layer and retinal pigment epithelium (RPE) were thinner centrally in mutant animals (total retinal thickness, 233.2 vs. 272.6 microm; combined nerve fiber layer, ganglion cell layer, and inner plexiform layer, 86.2 vs. 103.4 microm; inner nuclear layer, 51.8 vs. 60.3 microm; photoreceptors, 26.7 vs. 35.6 microm). Optic nerve cross-sectional area was less in 20- to 21-day-old treated Sod2-deficient animals than in control animals. Mitochondrial morphologic abnormalities (swelling, pale matrix, and disorganized cristae) were found predominantly in older mutant animals' (16 and 20 to 21 days) RPE and in extraocular muscle of a 16-day-old untreated mutant. CONCLUSIONS: In sod2(tm1Cje-/-) mice, there is relative progressive retinal thinning, with particular involvement of the inner retinal layers and an early effect on the photoreceptor layer, as well as mitochondrial morphologic abnormalities, all consistent with mitochondrial disease.

Definition, at the molecular level, of a thyroglobulin-acetylcholinesterase shared epitope: study of its pathophysiological significance in patients with Graves' ophthalmopathy.

The nature of the putative autoantigen in Graves' ophthalmopathy (Go) remains an enigma but the sequence similarity between thyroglobulin (Tg) and acetylcholinesterase (ACHE) provides a rationale for epitopes which are common to the thyroid gland and the eye orbit. In an attempt to define the shared epitope, we have screened a lambda gt 11 human thyroid cDNA library using a polyclonal antibody to Torpedo ACHE and isolated two clones, which upon sequencing, were shown to contain Tg segments, corresponding to portions of the C terminal part of the molecule which has a high similarity with ACHE. Having demonstrated the existence of an epitope common to Tg and ACHE, the clones have been further tested and found to be positive in lysis plaque assays with 1/10 sera from patients with Hashimoto's thyroiditis (HT), 8/8 from patients with Graves' ophthalmopathy and 0/8 normal sera. We have investigated the physiological significance of this common epitope by in situ immunolocalization studies in which the polyclonal antibody to Torpedo ACHE (which was used for screening the library) and immunoglobulins (Igs) from 6 Go patients tested were shown to bind to end plate regions of human foetal muscle fibres which were concurrently shown to be rich in cholinesterase activity: Igs from 3 normal individuals and 2 patients with Hashimoto's thyroiditis did not bind. The results demonstrate and characterize an epitope which is common to Tg and ACHE and show that Go patients Igs contain antibodies which bind to muscle end plates rich in cholinesterase. The significance of these findings to the pathogenesis of Go is discussed.

Histochemistry of human extraocular muscle.

A reliable method for evaluating biopsy specimens of human extraocular muscles is presented to better understand the pathological responses of these highly organized striated muscles. Three muscle fiber types and their distribution are described with morphological and histochemical measurements used commonly for limb muscle. The granular and fine fibers have single end plates and may be comparable to limb-twitch fibers (type 2 and type 1 fibers). The coarse fibers have multiple end plates and may correspond to multiple end plated tonic fibers found in avian and amphibian limb muscles. The fibers of extraocular muscles are arranged in three concentric zones. Because of the zonal arrangement, a complete cross section should be evaluated in diseases of the ocular muscles to estimate any changes in fiber type distribution.

Structural features of extraocular muscles of children with strabismus.

Ninety extraocular muscle (EOM) biopsy specimens from 80 children with strabismus and normal ocular alignment were studied by light microscopy, enzyme histochemistry, and electron microscopy. Light microscopy showed considerable variation in muscle fiber shape and size with sarcomere disruption, sharp increases in endomysial and perimysial collagen, numerous vacuoles and subsarcolemmal inclusions. Electron microscopy showed disruption of myofilaments, nemaline rods, abnormal mitochondria, leptomeric profiles, occasional "myelin figures," glycogen, and lipid-like droplets. Some intramuscular nerves contained long-spacing collagen ("Luse bodies"). Unlike somatic skeletal muscle, EOM showed a consistent lack of mosaic pattern and reciprocal stain activity between fiber types noted by enzyme histochemistry. Quantitative observations indicate that minimal but consistent morphologic differences exist between strabismic and nonstrabismic EOM. The role these changes play in the pathogenesis of strabismus and their significance remains unclear.

Effects of alpha-isoproterenol on denervation atrophy in orbicularis oculi muscle fibers.

The effect of the beta-adrenoceptor agonist isoproterenol on the denervated orbicularis oculi muscle was studied. We cut the facial nerve on the right side of eight rats and injected isoproterenol (5 mg/kg) into four rats every day. The animals were sacrificed after three weeks, and small pieces of the orbicularis oculi muscle were removed. Muscle fibers were separated into single fibers. Cytochrome oxidase enzyme staining was applied, and density of cytochrome oxidase enzyme product and diameter of individual muscle fibers were analyzed by computer. Some of the muscle fibers were observed under electron microscopy. The results showed that diameter and cytochrome oxidase enzyme activity of the orbicularis oculi muscle decreased by denervation, otherwise, it increased to near the normal level by administration of isoproterenol. Electron microscopic observation showed that the myosion filaments became thin and their arrangement were disordered by denervation and their findings recovered to be almost normal by isoproterenol administration. We concluded that isoproterenol could prevent the orbicularis oculi muscle from atrophying which caused by denervation.

Visual system maldevelopment disrupts extraocular muscle-specific myosin expression.

The genetic and epigenetic influences that are responsible for the establishment and maintenance of the unique phenotype of the extraocular muscles (EOMs) are poorly understood. A role for visual cues in shaping EOM maturation was assessed in rats by using two visual deprivation paradigms, dark rearing and monocular deprivation. Isoforms of the contractile protein myosin heavy chain (MHC) were used as an index of phenotypic change in developing and adult EOMs after these visual insults. In rats that were dark reared during the visual critical period, the proportion of EOM fibers expressing either fast or slow MHCs was decreased significantly. EOM-specific myosin was also sensitive to dark rearing during the critical period, as evidenced by a significant decrease in its mRNA in EOMs from these rats. EOM-specific MHC did not change in either dark-reared rats returned to normally illuminated conditions or in adult rats denied visual experience for a similar time period. These data suggest that there may be a critical period during development when alterations in visual activity have significant consequences for the eye muscle phenotype. In contrast to dark rearing, monocular deprivation had a minimal effect on expression of the typical myosin isoforms and no effect on EOM-specific myosin expression. Collectively, these data confirm the hypothesis that visual input to the oculomotor system during development modulates EOM-specific MHC expression.