Extraocular Muscle Fibers Research Articles

Succinylcholine-induced intraocular hypertension reflects muscular contracture. An experimental study in pigs

The increase in intraocular pressure (IOP) following administration of succinylcholine (SCH) was believed to result from an increase in extraocular muscle tone, stimulation of orbital smooth muscle and choroidal vasodilation [1]. Recently, a different mechanism, based on SCH induced cycloplegia and increased resistance to aqueous humour outflow was postulated, and any significant contribution from extraocular muscle tone was denied [2]. We have assessed the muscular contribution to ΔIOP in an experimental setting. With approval from the regional ethics committee, we have investigated the mechanism of SCH induced IOP increase in six acutely instrumented, anaesthetized pigs (9±1 weeks, 25-28 kg piritramide + N2O), using a previously described model [3]. IOP was measured continuously manometrically in the anterior chambers of both eyes. All extraocular muscles of one eye were removed surgically, the other eye served as control. After haemodynamic variables had stabilised, 1.5 mg kg−1 SCH were given. When IOP had peaked and reached a plateau, dantrolene was administered (5 mg kg−1 by rapid infusion). After IOP had returned to baseline, a second dose of SCH was given. Pressure in the intact eyes increased significantly with the first dose of SCH; this effect was not significant in the eyes that had had the extraocular muscles removed nor in either eye after the second dose, following dantrolene. SCH produces a contracture of multiple innervated extraocular muscle fibres, which is abolished by dantrolene [4]. We believe that, in our study, the transient, pronounced increase in IOP in the intact eyes may be ascribed to such contracture; the minor increase in IOP observed in the operated eyes and also after (intracellular) muscle relaxation with dantrolene possibly reflects choroidal vascular engorgement or other phenomena. An increase in resistance to aqueous humour outflow resulting from SCH induced cycloplegia [2] is unlikely to account for an IOP increase as rapid as that seen with the administration of SCH. Although the origin of SCH induced intraocular pressure rise is probably multifactorial, the largest proportion of this effect is due to extraocular muscle tone.

Muscles of a different 'color'

The rules that govern many aspects of skeletal muscle structure and function are very different for the extraocular muscle allotype. The myoblast lineages present in the extraocular muscle primordia are permissive for generation of an unusually wide range of fiber types. The balance that is struck between genetic specification and activity dependent factors in shaping fiber phenotype to suit the demands of complex visuomotor systems is not yet well defined. Because skeletal muscle has high energy demands, diversity in fiber types is needed to maximize efficiency; greater diversity in fiber composition then indicates a more diverse functional repertoire. Together, the characteristics of small motor unit size, precise dependence of muscle force upon motor neuron discharge rate, high contractile speed but low tension development, and contractile protein heterogeneity contribute toward the high precision and diversity that is required for eye movements. Finally, the structural and functional characteristics and plasticity of the individual extraocular muscle fiber types play an important role in determining their response to disease or manipulation. The lack of uniform responses across the muscle allotypes in disease, or in response to pharmaceutical or surgical interventions, requires that we obtain a better understanding of the fundamental differences that exist between muscle groups.

The gamma-Subunit of the Acetylcholine Receptor Is Not Expressed in the Levator Palpebrae Superioris

In extraocular muscle (EOM), expression of the gamma-subunit, which is associated with the fetal-type acetylcholine receptor (AChR), may offer a differential target for immune-mediated damage and could explain the preponderance of ocular manifestations caused by myasthenia gravis (MG). Using Poly(A)+ RNA hybridization, we investigated expression of the gamma-subunit in bovine levator palpebrae superioris (LP), a muscle also differentially involved by MG. There were no transcripts of the gamma-subunit of the AChR, but the epsilon-subunit, associated with the adult-type AChR, was present. The results indicate that the susceptibility of LP to MG is not mediated by gamma-subunit expression and suggest that multiterminal fibers in EOM may be the site of gamma-subunit expression.

Characterization of the sarcoplasmic reticulum proteins in the thermogenic muscles of fish.

Marlins, sailfish, spearfishes, and swordfish have extraocular muscles that are modified into thermogenic organs beneath the brain. The modified muscle cells, called heater cells, lack organized myofibrils and are densely packed with sarcoplasmic reticulum (SR), transverse (T) tubules, and mitochondria. Thermogenesis in the modified extraocular muscle fibers is hypothesized to be associated with increased energy turnover due to Ca2+ cycling at the SR. In this study, the proteins associated with sequestering and releasing Ca2+ from the SR (ryanodine receptor, Ca2+ ATPase, calsequestrin) of striated muscle cells were characterized in the heater SR using immunoblot and immunofluorescent techniques. Immunoblot analysis with a monoclonal antibody that recognizes both isoforms of nonmammalian RYRs indicates that the fish heater cells express only the alpha RYR isoform. The calcium dependency of [3H]ryanodine binding to the RYR isoform expressed in heater indicates functional identity with the non-mammalian alpha RYR isoform. Fluorescent labeling demonstrates that the RYR is localized in an anastomosing network throughout the heater cell cytoplasm. Measurements of oxalate supported 45Ca2+ uptake, Ca2+ ATPase activity, and [32P]phosphoenzyme formation demonstrate that the SR contains a high capacity for Ca2+ uptake via an ATP dependent enzyme. Immunoblot analysis of calsequestrin revealed a significant amount of the Ca2+ binding protein in the heater cell SR. The present study provides the first direct evidence that the heater SR system contains the proteins necessary for Ca2+ release, re-uptake and sequestration, thus supporting the hypothesis that thermogenesis in the modified muscle cells is achieved via an ATP-dependent cycling of Ca2+ between the SR and cytosolic compartments.

Different in situ hybridization patterns of mitochondrial DNA in cytochrome c oxidase-deficient extraocular muscle fibres in the elderly.

Previous studies have revealed an increase of cytochrome c oxidase-deficient fibres/cells in the skeletal and heart muscle of humans during ageing. The enzyme defect is due to a lack of both mitochondrial and nuclear coded enzyme subunits. In the present investigation in situ hybridization of mitochondrial DNA (mtDNA) has been performed on extraocular muscles of humans over 70 years of age to show whether mutated mtDNA with the so called common deletion of 4,977 basepairs at position 8,482-13,460 of mtDNA accumulates in the cytochrome c oxidase-deficient fibres. The cytochrome c oxidase-deficient fibres revealed different hybridization patterns: a normal hybridization signal with three different mtDNA probes, a reduced or lacking signal with all three probes indicating depletion of mtDNA and a selective hybridization defect with the probe recognizing the "common deletion" region of mtDNA as evidence of mtDNA deletion. The results suggest that during ageing defects of cytochrome c oxidase are associated with different molecular alterations of mtDNA. Deletion and depletion of mtDNA are not the only nor probably the leading mechanisms responsible for the loss of respiratory chain capacity during ageing. The normal hybridization signal in most of the cytochrome c oxidase-deficient fibres and the loss of mitochondrial and nuclear protein subunits indicate the involvement of other, especially nuclear factors.

Extraocular muscles are spared in advanced Duchenne dystrophy.

Fast-twitch extremity muscle fibers are preferentially affected in Duchenne's and Becker's muscular dystrophy. Since saccades are thought to be mediated by fast-twitch fibers, saccadic velocities would be expected to be decreased in these patients. Using infrared oculography, we found that the peak velocities of saccades in 3 patients with advanced Duchenne's or Becker's muscular dystrophy were normal. Clinical findings in 7 other patients with these forms of dystrophy were normal. This investigation is the first study of ocular motility in Duchenne's and Becker's muscular dystrophy. It demonstrates that extraocular muscle function is preserved and suggests that fast-twitch fibers in extraocular muscles possess properties that protect against degeneration.

Expression of alpha-cardiac myosin heavy chain in mammalian skeletal muscle.

We have investigated the reactivity of different human, rat and cat muscles to a monoclonal antibody directed against human alpha-cardiac myosin heavy chain. We have found that special fiber subpopulations of human masseter and extraocular muscles, as well as the bag fibers of human, rat and cat muscle spindles, were reactive to this antibody, indicating that these fibers expressed alpha-cardiac myosin heavy chain or a closely related isoform. This isomyosin was present in the spindle bag fibers at early fetal stages, whereas its expression in masseter and extraocular muscle fibers was not detected during the first 22 weeks of gestation. Our results add to the list of muscle proteins which are expressed in locations or at developmental stages other than those initially described, suggesting that a revision of the present nomenclature of the subgroups of myosin heavy chains might be considered in the future.

Type-specific changes in fiber morphometry following denervation of canine extraocular muscle

To investigate the changes that occur in extraocular muscle morphometry following denervation, 12 beagles were subjected to intracranial section of the left oculomotor nerve. The inferior and medial rectus muscles were removed from both orbits of four dogs killed at each 4-week postoperative interval. Fiber diameters and fiber-type ratios of denervated muscles were compared with those from paired muscles in the normal orbit. We found significant, persistent atrophy of the singly innervated fibers in both the global and orbital layers of denervated extraocular muscle. The multiinnervated fibers were predominantly spared from denervation atrophy. We also found a significant increase in the proportion of multiinnervated fibers in the orbital layer only. These results suggest a relative neurotrophic independence of multiinnervated fibers in extraocular muscle.

Immunohistochemical study of carbonic anhydrase III in the extraocular muscles of human embryos.

The differentiation of extraocular muscles was studied immunohistochemically in externally normal human embryos (Carnegie stages 13-23), using antibodies to carbonic anhydrase (CA) III and beta-enolase as the markers of type 1 and type 2 muscle fibers, respectively. At stage 18, some myoblasts were immunoreactive to beta-enolase antibodies, however, CA-III immunoreactivity was not observed around the optic vesicle. At stage 20, CA-III immunoreactivity appeared in some muscle fibers of extraocular muscles. From stage 21 to stage 23, CA-III-immunoreactive fibers increased and almost equalled the number of beta-enolase-immunoreactive fibers. These findings suggest that CA-III-immunoreactive type 1 fibers appear in the late stage of myogenesis compared with beta-enolase-immunoreactive type 2 fibers.

Saccadic eye movements analysis in the early diagnosis of myasthenia gravis

Saccadic eye movements were examined by electro-oculography in 21 patients with suspected myasthenia gravis. The presence of dissociated nystagmus and quiver eye movements was also assessed. The aim of the study was to assess the diagnostic value of saccadic abnormalities in the early stage of the disease. Pathological oculographic findings consisted of intra and post saccadic disorders and intersaccadic variability. A fatigue test was useful in detecting latent disorders in most patients. A Tensilon test was positive in 11 out of 16 patients with oculographic abnormalities. Diagnostic problems, apparently paradoxical findings and differential involvement of extraocular muscle fibers are discussed.

Succinylcholine-stimulated muscle tensions following botulinum injection in the domestic cat

Succinylcholine (SCh) selectively stimulates, and can therefore selectively assay, the multiple innervated (MI) fiber system of the extraocular muscles. Since botulinum-A toxin has been observed to induce changes in eye position in humans. SCh was used to assess the effect of botulinum-A on the SCh-sensitive MI fibers of extraocular muscles. Intravenous SCh infusion (40 μg −1 kg −1 min −1) was performed in the anesthetized domestic cat. Thirty-eight infusions were performed in 19 normal controls, measuring the peak tensions generated in the four horizontal and four vertical rectus muscles. Succinylcholine-stimulated muscle tensions (SSMT) were then repeated in nine animals, 4 weeks and 10 weeks following injection of botulinum-A toxin into both medial rectus muscles. Mean peak SSMTs were unchanged at 4 and 10 weeks following botulinum injection when compared to controls. We propose that botulinum chemo-denervation has no acute or chronic effect on the MI SCh-sensitive muscle fibers of the medial recti of the domestic cat. This lack of effect on the postsynaptic MI fibers indirectly supports light and electron microscopic studies which show changes predominantly in the singly innervated (SI), rather than the MI fibers following botulinum injection. Mean peak SSMTs were also greater for medial and superior rectus muscles compared to lateral and inferior recti respectively, suggesting a greater number or proportion of MI fibers in medial and superior recti.

Age-Related Conversion of Dystrophin-Negative to -Positive Fiber Segments of Skeletal but not Cardiac Muscle Fibers in Heterozygote mdx Mice

Immunoreactive dystrophin was examined in muscle fibers of quadriceps, extraocular muscles and cardiac ventricular muscles of female heterozygote mdx mice at 10, 35 and 60 days of age, with microscopic immunoperoxidase method and by immunoblots. In quadriceps muscle fibers there was a marked gradual diminution of the dystrophin-negative fiber segments between age 10 and 60 days. We suggest that this was partly due to a spontaneous fusion of dystrophin-competent satellite cells into the dystrophin-negative fiber segments and partly to an expansion of the cytoplasmic domain of dystrophin expression related to the original myonuclei. In cardiac muscle that lacks satellite cells, there was persistence of a large number of dystrophin-negative fiber segments even at age 60 days and probably beyond. The findings of this study have implications for the detection of heterozygote female carriers of Duchenne muscular dystrophy (DMD) and for the possible therapy of DMD muscles by myoblast transfer.

Systematic variation in myosin expression along extraocular muscle fibres of the adult rat

Monoclonal antibodies (McAB) specific for fast (C14) and slow (S58) myosin, and a myosin antigenically similar to neonatal/embryonic myosin in mammals (ALD180), were used to characterize the myosin distribution in orbital layer fibres of rat extraocular muscles (EOM) in relation to innervation patterns. The orbital layer is composed of both singly-innervated (SIF) and multiply-innervated (MIF) fibres. The SIFs have the characteristics of twitch fibres, while the MIFs, in addition to possessing many small endings characteristic of tonic fibres, also have an en-plaque-like innervation in the endplate band resembling that of the adjacent SIFs. Myosin expression in MIFs and SIFs is unusual and varies systematically along the length of the fibres. Both SIFs and MIFs label with ALD180, but this labelling is absent in both fibre types in the endplate band region, where all fibres label with C14. Distally and also proximally to the endplate band, SIFs label with both ALD180 and C14, while the MIFs, innervated by many small, superficial endings in these regions, label with ALD180 only. This pattern of myosin expression could also be demonstrated in isolated fibres. The results are discussed in relation to the hypothesis that both populations of orbital layer fibres express constitutively both fast and the neonatal-like myosin, and that superimposed on this constitutive expression twitch or tonic innervation acts locally to selectively suppress either neonatal-like or fast myosin, respectively.

New growth of extraocular muscle in peripheral nerve autografts: Ultrastructural relationships

We assessed the ultrastructural relationships of extraocular muscle (eom) fibers grown in a nonmuscle, extracellular matrix. Free sections of autogenous, extraorbital, peripheral nerve were implanted between the cut edge of a healthy eom and a denervated eom in four dogs. Light microscopy at 4 and 8 weeks demon-strated muscle fiber growth throughout the length of the implanted segments. Electron microscopy showed muscle fibers surrounded by collagen, frequently beside degenerating myelinated nerve fibers, but never growing within Schwann tubes or completely surrounded by the basal lamina of one nerve or Schwann cell. The muscle fibers were, however, bordered by abundant fibroblast processes (always separated by collagen), which often partially encircled the fiber. All muscle fiber growth occurred in parallel with the nerve axis. These findings indicate that peripheral nerve may contain factors conducive to the growth and orderly alignment of eom fibers. The identification of these factors may be helpful in the development of a synthetic matrix for eom graft growth.

Conduction velocities of extraocular muscle fibers

A new multi-electrode needle was applied to record the conduction velocities of cat and human extraocular muscle fibers. This needle contained 10 micro-electrodes which each had a diameter of 25 μm and were regularly arranged in intervals of 60 μm.In cat muscle fibers, the conduction velocities of 95 units were measured in situ under stimulus and non-stimulus conditions. Conduction velocities of lateral rectus muscles without nerve stimulation ranged from 0.9 to 4.8 m/s (mean 2.0 m/s) and those of retractor bulbi muscles ranged from 1.7 to 4.8 m/s (mean 3.6 m/s). The velocities of lateral rectus by the electrical stimulation of the abducens nerve in the pontine exit ranged from 1.2 to 5.3 m/s (mean 2.9 m/s).In human muscle fibers, the conduction velocities of inferior rectus muscles (29 units) during central fixation ranged from 1.0 to 5.5 m/s (mean 2.6 m/s) and those of lateral rectus muscles (44 units) ranged from 1.2 to 3.3 m/s (mean 1.9 m/s).These results reconfirmed electrophysiologically that the mu...

Mitochondrial abnormalities in extraocular muscles in myotonic dystrophy

This is the first description of ‘ragged red fibers’ in extraocular muscles of myotonic dystrophy. Muscle biopsies were obtained from the biceps brachii and superior rectus of a 53-year-old man who had suffered from chronic progressive myotonia and muscular weakness in limbs, blepharoptosis, external ophthalmoparesis, myotonic cataract, and foveal pigmentary dystrophy. The extraocular as well as skeletal muscle cells showed centrally placed multiple nuclei with type 1 fiber predominance and increased intermyofibrillary connective tissue. About 20% of extraocular muscle fibers were identified as ‘ragged red fibers’ with the modified Gomori trichrome stain, most of which showed reduced activity in the cytochrome c oxidase reaction. An increased number of enlarged mitochondria were clustered beneath the sarcolemma and between the myofibrils, some of which contained irregularly or circularly arranged cristae and paracrystalline inclusions. Biochemical assessments showed in both skeletal and extraocular muscle...

Dependence of tonic tension on extracellular calcium in rat extraocular muscle.

Ca2+-free saline containing 3.0 mM Mg2+ virtually abolishes the tonic tension evoked by depolarization with a high K+ concentration of the tonic multiply innervated fibers of rat extraocular muscles. The tonic tension abolished by Ca2+ withdrawal is restored when Ca2+ is substituted by Sr2+ but not by Ni2+. The increase of Mg2+ reduces the tonic tension and displaces the tension-log K+ relationship to the right. Cd2+ significantly reduces the tension amplitude but does not shift the tension-log K+ relationship. The organic blocker of Ca2+ channels, nifedipine (1-10 microM), has no effect on the tonic tension. In contrast, diltiazem (20 microM) reduces the amplitude of the responses without changing the tension-log K+ relationship. Both foreign anions NO3- and SCN- potentiate tonic tension without changing the tension-log K+ relationship. SCN- increases the resting tension of the muscle; this effect depends on Ca2+. In conclusion, the disappearance of tonic tension after Ca2+ withdrawal is not due to depolarization of the fibers or inactivation of the contractile responses. It is suggested that entry of extracellular Ca2+, via a voltage-dependent Ca2+ conductance, or specific interactions of Ca2+ with membrane sites involved in the regulation of excitation-contraction coupling play a role in evoking tension in tonic fibers.

Fibre types in extraocular muscles: a new myosin isoform in the fast fibres.

We report on the existence of a myosin heavy chain (MHC) isoform with unique structural properties in extraocular (EO) muscles. Differences in MHC composition are apparent using a polyclonal antibody prepared against myosin isolated from bovine EO muscle myosin. In enzyme immunoassays and western blotting experiments, this anti-EO myosin antibody reacted specifically with the heavy chains of EO muscle myosin and not with the heavy chains of other myosins. The distribution of this new MHC isoform in the globe rotating muscles from different mammalian species was analysed using a panel of specific anti-myosin antibodies and comparing the histochemical myosin ATPase profile of muscle fibres with their isomyosin content. Most fibres which display a type 2 ATPase reaction pattern were selectively labelled by anti-EO antibodies. A few type 2 fibres were found to react with both anti-EO and anti-2A myosin antibodies and others, located almost exclusively in the orbital layers, reacted with anti-foetals as well as anti-EO antibodies. The presence of a distinct form of myosin in EO muscle fibres is probably related to the particular functional characteristics of these muscles, which are known to be exceptionally fast-contracting but display a very low tension output.

Effects of epinephrine on resting and tonic tensions of rat extraocular muscles.

"In vitro" epinephrine (10(-6) to 10(-4) M) stimulates the rat inferior rectus, one of the extraocular muscles (EOMs). When applied to resting muscles, it induces a slow rising tension in about one half of the preparations. The amplitude of this tension is equivalent to about 9% of the maximal tetanic response. This effect is blocked by propranolol (10(-7) to 10(-6) M). When epinephrine is applied prior to a depolarization of the muscle fibers with 30 mM K, the drug consistently induces a significant potentiation of the long lasting tonic tension normally evoked by that elevated K concentration. One possible explanation for these results is that epinephrine induces a slow rising tension by increasing a resting Ca conductance of the sarcolemma of the tonic or Felderstruktur fibers of EOM.

Motor innervation and acetylcholine receptor distribution of human extraocular muscle fibres

Motor innervation and acetylcholine receptor distribution of human extraocular muscle (EOM) fibres were investigated by using combined silver and acetylcholinesterase (AChE) staining method and [ 125I]α-bungarotoxin autoradiography. Three types of motor endplates were distinguished; (A) a large compact ending on a large-diameter fibre, (B) several endings regularly spaced on an intermediate-diameter fibre, each of which consisted of small stained particles and (C) numerous small endings scattered as a chain of beads on a small-diameter fibre. Type A fibre was singly innervated, while type B and C had multiterminal (possibly polyneuronal) innervation. These results indicate that the organization of human EOM endplates is quite different from that of limb muscle endplates and may provide some implications to understand the pathophysiology of the neuromuscular diseases, for example, myasthenia gravis.