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1.  Independent Passive Mechanical Behavior of Bovine Extraocular Muscle Compartments 
Purpose.
Intramuscular innervation of horizontal rectus extraocular muscles (EOMs) is segregated into superior and inferior (transverse) compartments, while all EOMs are also divided into global (GL) and orbital (OL) layers with scleral and pulley insertions, respectively. We sought evidence of potential independent action by examining passive mechanical coupling between EOM compartments.
Methods.
Putative compartments of each of the six whole bovine anatomical EOMs were separately clamped to a physiologically controlled, dual channel microtensile load cell (5-mN force resolution) driven by independent, high-speed, linear motors having 20-nm position resolution. One channel at a time was extended or retracted by 3 to 5 mm, with the other channel stationary. Fiducials distributed on the EOM global surface enabled optical tracking of local deformation. Loading rates of 5 to 100 mm/sec were applied to explore speeds from slow vergence to saccades. Control loadings employed transversely loaded EOM and isotropic latex.
Results.
All EOM bellies and tendons exhibited substantial compartmental independence when loaded in the physiologic direction, both between OL and GL, and for arbitrary transverse parsings of EOM width ranging from 60%:40% to 80%:20%. Intercompartmental force coupling in the physiologic direction was less than or equal to 10% in all six EOMS even for saccadic loading rates. Coupling was much higher for nonphysiologic transverse EOM loading and isotropic latex. Optical tracking demonstrated independent strain distribution between EOM compartments.
Conclusions.
Substantial mechanical independence exists among physiologically loaded fiber bundles in bovine EOMs and tendons, providing biomechanical support for the proposal that differential compartmental function in horizontal rectus EOMs contributes to novel torsional and vertical actions.
Dual-channel tensile loading demonstrates that adjacent extraocular muscle (EOMs) regions have marked mechanical independence. This finding supports the active pulley hypothesis and the proposal that topographic innervation within horizontal rectus EOMs could command torsional and vertical actions.
doi:10.1167/iovs.12-10318
PMCID: PMC4113332  PMID: 23188730
2.  Differential Lateral Rectus Compartmental Contraction during Ocular Counter-Rolling 
Purpose.
The lateral rectus (LR) and medial rectus (MR) extraocular muscles (EOMs) have largely nonoverlapping superior and inferior innervation territories, suggesting functional compartmental specialization. We used magnetic resonance imaging (MRI) in humans to investigate differential compartmental activity in the rectus EOMs during head tilt, which evokes ocular counter-rolling, a torsional vestibulo-ocular reflex (VOR).
Methods.
MRI in quasi-coronal planes was analyzed during target-controlled central gaze in 90° right and left head tilts in 12 normal adults. Cross sections and posterior partial volumes of the transverse portions of the four rectus EOMs were compared in contiguous image planes 2 mm thick spanning the orbit from origins to globe equator, and used as indicators of contractility.
Results.
Horizontal rectus EOMs had significantly greater posterior volumes and maximum cross sections in their inferior compartments (P < 10−8). In orbit tilt up (extorted) compared with orbit tilt down (intorted) head tilts, contractile changes in LR maximum cross section (P < 0.0001) and posterior partial volume (P < 0.05) were significantly greater in the inferior but not in the superior compartment. These changes were not explainable by horizontal or vertical eye position changes. A weaker compartmental effect was suggested for MR. The vertical rectus EOMs did not exhibit significant compartmental contractile changes during head tilt. Mechanical modeling suggests that differential LR contraction may contribute to physiological cyclovertical effects.
Conclusions.
Selective activation of the two LR, and possibly MR, compartments correlates with newly recognized segregation of intramuscular innervation into distinct compartments, and probably contributes to noncommutative torsion during the VOR.
Magnetic resonance imaging of extraocular muscles during ocular counter-rolling demonstrates selective activation of the lateral rectus inferior but not superior compartment. This is novel functional evidence that differential rectus compartmental activation contributes to a vestibulo-ocular reflex.
doi:10.1167/iovs.11-7929
PMCID: PMC3367472  PMID: 22427572
3.  Magnetic Resonance Imaging of the Effects of Horizontal Rectus Extraocular Muscle Surgery on Pulley and Globe Positions and Stability 
Purpose
Magnetic resonance imaging (MRI) was used to determine the effect of recessions and resections on horizontal extraocular muscle (EOM) paths and globe position.
Methods
Four adults with horizontal strabismus underwent contrast-enhanced, surface-coil MRI in central, secondary, and tertiary gazes, before and after horizontal EOM recessions and/or resections. EOM paths were determined from 2-mm thickness, quasicoronal MRI by analysis of cross-sectional area centroids in a normalized, oculocentric coordinate system. Globe displacement was determined by measuring the apparent shift of the bony orbit in eccentric gaze.
Results
In all subjects, the anteroposterior positions of the horizontal rectus pulleys shifted by less than 2 mm after surgery, indistinguishable from zero within measurement precision. In three subjects who underwent medial rectus (MR) recession or resection, postoperative globe position was similar in central gaze, but globe translation during vertical gaze shift changed markedly. There was no effect on globe translation in the subject who underwent only lateral rectus (LR) resection.
Conclusions
Recessions and resections of horizontal EOMs have minimal effect on anteroposterior EOM pulley positions. Because the pulley does not shift appreciably despite large alterations in the EOM insertion, the proximity of a recessed EOM to its pulley would be expected to introduce torsional and vertical actions in tertiary gazes. Connective tissue dissection during MR surgery may destabilize the globe’s vertical translational stability within the orbit, potentially changing the effective pulling directions of the rectus EOMs in vertical gazes. These changes may mimic oblique muscle dysfunction. LR surgery may avoid globe destabilization.
doi:10.1167/iovs.05-0498
PMCID: PMC1850672  PMID: 16384961
4.  Microanatomy of Adult Zebrafish Extraocular Muscles 
PLoS ONE  2011;6(11):e27095.
Binocular vision requires intricate control of eye movement to align overlapping visual fields for fusion in the visual cortex, and each eye is controlled by 6 extraocular muscles (EOMs). Disorders of EOMs are an important cause of symptomatic vision loss. Importantly, EOMs represent specialized skeletal muscles with distinct gene expression profile and susceptibility to neuromuscular disorders. We aim to investigate and describe the anatomy of adult zebrafish extraocular muscles (EOMs) to enable comparison with human EOM anatomy and facilitate the use of zebrafish as a model for EOM research. Using differential interference contrast (DIC), epifluorescence microscopy, and precise sectioning techniques, we evaluate the anatomy of zebrafish EOM origin, muscle course, and insertion on the eye. Immunofluorescence is used to identify components of tendons, basement membrane and neuromuscular junctions (NMJs), and to analyze myofiber characteristics. We find that adult zebrafish EOM insertions on the globe parallel the organization of human EOMs, including the close proximity of specific EOM insertions to one another. However, analysis of EOM origins reveals important differences between human and zebrafish, such as the common rostral origin of both oblique muscles and the caudal origin of the lateral rectus muscles. Thrombospondin 4 marks the EOM tendons in regions that are highly innervated, and laminin marks the basement membrane, enabling evaluation of myofiber size and distribution. The NMJs appear to include both en plaque and en grappe synapses, while NMJ density is much higher in EOMs than in somatic muscles. In conclusion, zebrafish and human EOM anatomy are generally homologous, supporting the use of zebrafish for studying EOM biology. However, anatomic differences exist, revealing divergent evolutionary pressures.
doi:10.1371/journal.pone.0027095
PMCID: PMC3223174  PMID: 22132088
5.  The Viscoelastic Properties of Passive Eye Muscle in Primates. III: Force Elicited by Natural Elongations 
PLoS ONE  2010;5(3):e9595.
We have recently shown that in monkey passive extraocular muscles the force induced by a stretch does not depend on the entire length history, but to a great extent is only a function of the last elongation applied. This led us to conclude that Fung's quasi-linear viscoelastic (QLV) model, and more general nonlinear models based on a single convolution integral, cannot faithfully mimic passive eye muscles. Here we present additional data about the mechanical properties of passive eye muscles in deeply anesthetized monkeys. We show that, in addition to the aforementioned failures, previous models also grossly overestimate the force exerted by passive eye muscles during smooth elongations similar to those experienced during normal eye movements. Importantly, we also show that the force exerted by a muscle following an elongation is largely independent of the elongation itself, and it is mostly determined by the final muscle length. These additional findings conclusively rule out the use of classical viscoelastic models to mimic the mechanical properties of passive eye muscles. We describe here a new model that extends previous ones using principles derived from research on thixotropic materials. This model is able to account reasonably well for our data, and could thus be incorporated into models of the eye plant.
doi:10.1371/journal.pone.0009595
PMCID: PMC2833209  PMID: 20221406
6.  Quasilinear Viscoelastic Behavior of Bovine Extraocular Muscle Tissue 
Purpose
Until now, there has been no comprehensive mathematical model of the nonlinear viscoelastic stress-strain behavior of extraocular muscles (EOMs). The present study describes, with the use of a quasilinear viscoelastic (QLV) model, the nonlinear, history-dependent viscoelastic properties and elastic stress-strain relationship of EOMs.
Methods
Six oculorotary EOMs were obtained fresh from a local abattoir. Longitudinally oriented specimens were taken from different regions of the EOMs and subjected to uniaxial tensile, relaxation, and cyclic loading testing with the use of an automated load cell under temperature and humidity control. Twelve samples were subjected to uniaxial tensile loading with 1.7%/s strain rate until failure. Sixteen specimens were subjected to relaxation studies over 1500 seconds. Cyclic loading was performed to validate predictions of the QLV model characterized from uniaxial tensile loading and relaxation data.
Results
Uniform and highly repeatable stress-strain behavior was observed for 12 specimens extracted from various regions of all EOMs. Results from 16 different relaxation trials illustrated that most stress relaxation occurred during the first 30 to 60 seconds for 30% extension. Elastic and reduced relaxation functions were fit to the data, from which a QLV model was assembled and compared with cyclic loading data. Predictions of the QLV model agreed with observed peak cyclic loading stress values to within 8% for all specimens and conditions.
Conclusions
Close agreement between the QLV model and the relaxation and cyclic loading data validates model quantification of EOM mechanical properties and will permit the development of accurate overall models of mechanics of ocular motility and strabismus.
doi:10.1167/iovs.08-3245
PMCID: PMC2742171  PMID: 19357357
7.  The Effect of Resection on Satellite Cell Activity in Rabbit Extraocular Muscle 
Purpose
A common treatment for motility disorders of the extraocular muscles (EOMs) is a resection procedure in which there is surgical shortening of the muscle. This procedure results in rotation of the globe toward the resected muscle, increased resting tension across the agonist–antagonist pair, and stretching of the elastic components of the muscles. In the rabbit, due to orbital constraints and limited rotation, resection results in more significant stretch of the surgically treated muscle than the antagonist. This surgical preparation allows for the examination of the effects of surgical shortening of one rectus muscle and passive stretch of its ipsilateral antagonist.
Methods
The insertional 6 mm of the superior rectus muscles of adult rabbits were resected and reattached to the original insertion site. After 7 and 14 days, the animals were injected intraperitoneally with bromodeoxyuridine (BrdU) every 2 hours for 12 hours, followed by a 24-hour BrdU-free period. All superior and inferior rectus muscles from both globes were examined for BrdU incorporation, MyoD expression, neonatal and developmental myosin heavy chain (MyHC) isoform expression, and myofiber cross-sectional area alterations.
Results
In the resected muscle and in the passively stretched antagonist muscle, there was a dramatic increase in the number of myofibers positive for neonatal MyHC and in the number of BrdU- and MyoD-positive satellite cells. The addition of BrdU-positive myonuclei increased from 1 per 1000 myofibers in cross sections of control muscles to 2 to 3 per 100 myofibers in the resected muscles. Single myofiber reconstructions showed that multiple BrdU-positive myonuclei were added to individual myofibers. Addition of new myonuclei occurred in random locations along the myofiber length of single fibers. There was no correlation between myofibers with BrdU-positive myonuclei and neonatal MyHC isoform expression.
Conclusions
Both active and passive stretch of the rectus muscles produced by strabismus surgery dramatically upregulated the processes of satellite cell activation, integration of new myonuclei into existing myofibers, and concomitant up-regulation of immature myosin heavy chain isoforms. Understanding the effects of strabismus surgery on muscle cell biological reactions and myofiber remodeling may suggest new approaches for improving surgical outcomes.
doi:10.1167/iovs.05-1069
PMCID: PMC1780261  PMID: 16431957
8.  RNA Expression Analysis of Passive Transfer Myasthenia Supports Extraocular Muscle as a Unique Immunological Environment 
Purpose.
Myasthenia gravis demonstrates a distinct predilection for involvement of the extraocular muscles (EOM), and we have hypothesized that this may be due to a unique immunological environment. To assess this hypothesis, we took an unbiased approach to analyze RNA expression profiles in EOM, diaphragm, and extensor digitorum longus (EDL) in rats with experimentally acquired myasthenia gravis (EAMG).
Methods.
Experimentally acquired myasthenia gravis was induced in rats by intraperitoneal injection of antibody directed against the acetylcholine receptor (AChR), whereas control rats received antibody known to bind the AChR but not induce disease. After 48 hours, animals were killed and muscles analyzed by RNA expression profiling. Profiling results were validated using qPCR and immunohistochemical analysis.
Results.
Sixty-two genes common among all muscle groups were increased in expression. These fell into four major categories: 12.8% stress response, 10.5% immune response, 10.5% metabolism, and 9.0% transcription factors. EOM expressed 212 genes at higher levels, not shared by the other two muscles, and a preponderance of EOM gene changes fell into the immune response category. EOM had the most uniquely reduced genes (126) compared with diaphragm (26) and EDL (50). Only 18 downregulated genes were shared by the three muscles. Histological evaluation and disease load index (sum of fold changes for all genes) demonstrated that EOM had the greatest degree of pathology.
Conclusions.
Our studies demonstrated that consistent with human myasthenia gravis, EOM demonstrates a distinct RNA expression signature from EDL and diaphragm, which is based on differences in the degree of muscle injury and inflammatory response.
The preferential involvement of extraocular muscle by myasthenia gravis is clinically well known. Here we provide evidence that the muscle is a unique immunological environment that contributes to its targeting by myasthenia gravis.
doi:10.1167/iovs.14-14422
PMCID: PMC4102389  PMID: 24917137
myasthenia gravis; acetylcholine receptor; autoimmunity; complement; skeletal muscle
9.  Functional Morphometry of Horizontal Rectus Extraocular Muscles during Horizontal Ocular Duction 
Purpose.
We explored multiple quantitative measures of horizontal rectus extraocular muscle (EOM) morphology to determine the magnetic resonance imaging (MRI) measure best correlating with duction and thus contractility.
Methods.
Surface coil coronal MRI was obtained in target-controlled central gaze and multiple positions of adduction and abduction in 26 orbits of 15 normal volunteers. Duction angles were determined by position changes of the globe-optic nerve junction. Cross-sectional areas, partial volumes, and location of peak cross-sections of the horizontal rectus EOMs were computed in contiguous image planes 2-mm thick spanning the EOM origins to the globe equator.
Results.
All measures correlated significantly with duction angle (P < 0.0001). The best measures obtainable in single image planes were the maximum change in the cross-sectional area between equivalent image planes, with coefficients of determination R2 = 0.92 for medial rectus (MR) and 0.91 for lateral rectus (LR), and percentage change in maximum cross-section with R2 = 0.79 for MR and 0.78 for LR. The best partial volume measure of contractility was the change in partial volumes in four contiguous posterior planes (R2 = 0.86 MR and for 0.89 LR), particularly when combined with the corresponding change in partial volume for the antagonist EOM (R2 = 0.95 for MR and LR).
Conclusions.
EOM morphologic changes are highly correlated with degrees of duction and thus contractility. Both changes in single-plane maximum cross-sectional areas and posterior partial volumes provide accurate, quantitative measures of EOM contractility.
Magnetic resonance morphometry of horizontal rectus extraocular muscles is highly correlated with duction angle in normal subjects, suggesting that local volume and cross-section muscle features reflect contractile state.
doi:10.1167/iovs.12-9730
PMCID: PMC3481603  PMID: 22997285
10.  Dystrophic Changes in Extraocular Muscles after Gamma Irradiation in mdx:utrophin+/− Mice 
PLoS ONE  2014;9(1):e86424.
Extraocular muscles (EOM) have a strikingly different disease profile than limb skeletal muscles. It has long been known that they are spared in Duchenne (DMD) and other forms of muscular dystrophy. Despite many studies, the cause for this sparing is not understood. We have proposed that differences in myogenic precursor cell properties in EOM maintain normal morphology over the lifetime of individuals with DMD due to either greater proliferative potential or greater resistance to injury. This hypothesis was tested by exposing wild type and mdx:utrophin+/− (het) mouse EOM and limb skeletal muscles to 18 Gy gamma irradiation, a dose known to inhibit satellite cell proliferation in limb muscles. As expected, over time het limb skeletal muscles displayed reduced central nucleation mirrored by a reduction in Pax7-positive cells, demonstrating a significant loss in regenerative potential. In contrast, in the first month post-irradiation in the het EOM, myofiber cross-sectional areas first decreased, then increased, but ultimately returned to normal compared to non-irradiated het EOM. Central nucleation significantly increased in the first post-irradiation month, resembling the dystrophic limb phenotype. This correlated with decreased EECD34 stem cells and a concomitant increase and subsequent return to normalcy of both Pax7 and Pitx2-positive cell density. By two months, normal het EOM morphology returned. It appears that irradiation disrupts the normal method of EOM remodeling, which react paradoxically to produce increased numbers of myogenic precursor cells. This suggests that the EOM contain myogenic precursor cell types resistant to 18 Gy gamma irradiation, allowing return to normal morphology 2 months post-irradiation. This supports our hypothesis that ongoing proliferation of specialized regenerative populations in the het EOM actively maintains normal EOM morphology in DMD. Ongoing studies are working to define the differences in the myogenic precursor cells in EOM as well as the cellular milieu in which they reside.
doi:10.1371/journal.pone.0086424
PMCID: PMC3897728  PMID: 24466085
11.  Perimysial Fibroblasts of Extraocular Muscle, as Unique as the Muscle Fibers 
Fibroblasts derived from the extraocular muscle demonstrate distinct properties that differentiate them from fibroblasts of the hindlimb skeletal muscles. The results of this study indicate that extraocular muscles interact with fibroblasts in a fundamentally different manner and appear to benefit from both cell–cell contact and soluble trophic interactions.
Purpose.
Extraocular muscle (EOM) has a distinct skeletal muscle phenotype. The hypothesis for the study was that fibroblasts support the unique EOM phenotype and that perimysial fibroblasts derived from EOM have properties that distinguish them from fibroblasts derived from other skeletal muscle.
Methods.
Perimysial fibroblasts from leg muscle (LM-Fibro) and EOM (EOM-Fibro) of mice were derived and maintained in culture. EOM- and LM-Fibro were assessed morphologically and for vimentin, smooth muscle actin, and Thy-1 immunoreactivity. DNA microarray analysis was performed on LM- and EOM-Fibro grown in conditions that support myoblast differentiation. To assess trophic interactions, co-cultures of myoblasts from established cell lines, CL-EOM and CL-LM with, EOM- or LM-Fibro were performed in direct contact and in a permeable filter support culture. The degree of myotube maturation was assessed by the percentage of myotubes with more than three myonuclei per myotube.
Results.
EOM- and LM-Fibro cells exhibited distinct morphologies. Both cell types proliferated as a monolayer and expressed vimentin. Fifty-five percent (SD 4.4%) of EOM-Fibro were Thy-1 positive compared with only 24% (SD 4.4%) of LM-Fibro. DNA microarray analysis demonstrated differential expression of structural, immune response, and metabolism-related genes between EOM- and LM-Fibro. Co-cultures demonstrated that mature myotube formation in EOM-derived cell lines was supported to a greater extent by EOM-Fibro than by LM-Fibro, compared with CL-EOM grown with LM-Fibro.
Conclusions.
Fibroblasts from EOM demonstrate distinct properties that distinguish them from leg muscle–derived fibroblasts. The distinct properties of EOM-Fibro may support the unique EOM phenotype and contribute to their differential involvement in disease.
doi:10.1167/iovs.08-2857
PMCID: PMC2869047  PMID: 19661226
12.  The Viscoelastic Properties of Passive Eye Muscle in Primates. II: Testing the Quasi-Linear Theory 
PLoS ONE  2009;4(8):e6480.
We have extensively investigated the mechanical properties of passive eye muscles, in vivo, in anesthetized and paralyzed monkeys. The complexity inherent in rheological measurements makes it desirable to present the results in terms of a mathematical model. Because Fung's quasi-linear viscoelastic (QLV) model has been particularly successful in capturing the viscoelastic properties of passive biological tissues, here we analyze this dataset within the framework of Fung's theory.
We found that the basic properties assumed under the QLV theory (separability and superposition) are not typical of passive eye muscles. We show that some recent extensions of Fung's model can deal successfully with the lack of separability, but fail to reproduce the deviation from superposition.
While appealing for their elegance, the QLV model and its descendants are not able to capture the complex mechanical properties of passive eye muscles. In particular, our measurements suggest that in a passive extraocular muscle the force does not depend on the entire length history, but to a great extent is only a function of the last elongation to which it has been subjected. It is currently unknown whether other passive biological tissues behave similarly.
doi:10.1371/journal.pone.0006480
PMCID: PMC2715107  PMID: 19649257
13.  Extraocular Muscle Susceptibility to Myasthenia Gravis Unique Immunological Environment? 
Extraocular muscle (EOM) is susceptible to neuromuscular junction disorders, in particular, myasthenia gravis (MG). While EOM physiological characteristics and the ocular motor system requirements contribute to the propensity of ocular motor deficits observed among patients with MG, the authors propose that EOM have immunological features that place the muscles at risk for immune attack. Genomic profiling studies have demonstrated that genes associated with the immune response are differentially expressed in EOM, with particular differences in both classical and alternative complement-mediated immune response pathways. Intrinsic complement regulators are expressed at lower levels at rodent EOM neuromuscular junctions, which would put them at risk for the complement-mediated injury that occurs in MG. In fact, systemic C inhibition in experimental autoimmune MG (EAMG) induced by administration of acetylcholine receptor (AChR) antibodies or immunization with AChR will eliminate complement deposition at junctions of other skeletal muscle, but not EOM. Also, EOM junctions have greater injury in active and passive EAMG by several measures, suggesting that the lack of complement inhibition puts the EOM at risk. Among ocular myasthenia patients, serum AChR antibody levels are low, which would support the concept that EOM junctions are more susceptible to antibody injury than are other junctions. These observations suggest that complement inhibitory therapies may prove to be particularly effective in treatment of ocular myasthenia.
doi:10.1196/annals.1405.037
PMCID: PMC2527818  PMID: 18567871
complement; myasthenia gravis; ocular muscles
14.  The Role of Pitx2 in Maintaining the Phenotype of Myogenic Precursor Cells in the Extraocular Muscles 
PLoS ONE  2013;8(3):e58405.
Many differences exist between extraocular muscles (EOM) and non-cranial skeletal muscles. One striking difference is the sparing of EOM in various muscular dystrophies compared to non-cranial skeletal muscles. EOM undergo continuous myonuclear remodeling in normal, uninjured adults, and distinct transcription factors are required for the early determination, development, and maintenance of EOM compared to limb skeletal muscle. Pitx2, a bicoid-like homeobox transcription factor, is required for the development of EOM and the maintenance of characteristic properties of the adult EOM phenotype, but is not required for the development of limb muscle. We hypothesize that these unique properties of EOM contribute to the constitutive differences between EOM and non-craniofacial skeletal muscles. Using flow cytometry, CD34+/Sca1−/CD45−/CD31− cells (EECD34 cells) were isolated from extraocular and limb skeletal muscle and in vitro, EOM EECD34 cells proliferated faster than limb muscle EECD34 cells. To further define these myogenic precursor cells from EOM and limb skeletal muscle, they were analyzed for their expression of Pitx2. Western blotting and immunohistochemical data demonstrated that EOM express higher levels of Pitx2 than limb muscle, and 80% of the EECD34 cells expressed Pitx2. siRNA knockdown of Pitx2 expression in EECD34 cells in vitro decreased proliferation rates and impaired the ability of EECD34 cells to fuse into multinucleated myotubes. High levels of Pitx2 were retained in dystrophic and aging mouse EOM and the EOM EECD34 cells compared to limb muscle. The differential expression of Pitx2 between EOM and limb skeletal muscle along with the functional changes in response to lower levels of Pitx2 expression in the myogenic precursor cells suggest a role for Pitx2 in the maintenance of constitutive differences between EOM and limb skeletal muscle that may contribute to the sparing of EOM in muscular dystrophies.
doi:10.1371/journal.pone.0058405
PMCID: PMC3591328  PMID: 23505501
15.  A stretch reflex in extraocular muscles of species purportedly lacking muscle spindles 
It is generally assumed that proprioceptive feedback plays a crucial role in limb posture and movement. However, the role of afferent signals from extraocular muscles (EOM) in the control of eye movement has been a matter of continuous debate. These muscles have atypical sensory receptors in several species and it has been proposed that they are not supported by stretch reXexes. We recorded electromyographic activity of EOM during passive rotations of the eye in sedated rats and squirrel monkeys and observed typical stretch reXexes in these muscles. Results suggest that there is a similarity in the reXexive control of limb and eye movement, despite substantial differences in their biomechanics and sensory receptors. Like in some limb skeletal muscles, the stretch reflex in EOM in the investigated species might be mediated by other length-sensitive receptors, rather than muscle spindles.
doi:10.1007/s00221-006-0833-8
PMCID: PMC3230225  PMID: 17216145
Motor control; Sensorimotor integration; Eye movement; Proprioception; Electromyogram
16.  Myosin Heavy Chain Expression in Mouse Extraocular Muscle: More Complex Than Expected 
The expression of myosin heavy chains in mouse extraocular muscle is complex, with developmental isoforms retained and coexpression of multiple isoforms in single muscle fibers. The myosin expression may reflect the need of the ocular motor system for fine gradation of force generation.
Purpose.
To characterize the expression patterns of myosin heavy chain (MyHC) isoforms in mouse extraocular muscles (EOMs) during postnatal development.
Methods.
MyHC isoform expression in mouse EOMs from postnatal day (P)0 to 3 months was evaluated by quantitative polymerase chair reaction (qPCR) and immunohistochemistry. The longitudinal and cross-sectional distribution of each MyHC isoform and coexpression of certain isoforms in single muscle fibers was determined by single, double, and triple immunohistochemistry.
Results.
MyHC isoform expression in postnatal EOMs followed the developmental rules observed in other skeletal muscles; however, important exceptions were found. First, developmental isoforms were retained in the orbital layer of the adult EOMs. Second, expression of emb-MyHC, neo-MyHC, and 2A-MyHC was restricted to the orbital layer and that of 2B-MyHC to the global layer. Third, although slow-MyHC and 2B-MyHC did not exhibit obvious longitudinal variations, emb-MyHC, neo-MyHC, and 2A-MyHC were more abundant distally and were excluded from the innervational zone, whereas eom-MyHC complemented their expression and was more abundant in the mid-belly region in both the orbital and global layers. Fourth, coexpression of MyHC isoforms in single global layer fibers was rare, but it was common among the orbital layer fibers.
Conclusions.
MyHC isoforms have complex expression patterns, exhibiting not only longitudinal and cross-sectional variation of each isoform, but also of coexpression in single fibers. The highly heterogeneous MyHC expression reflects the complex contractile profiles of EOMs, which in turn are a function of the requirements of eye movements, which range from extremely fast saccades to sustained position, each with a need for precise coordination of each eye.
doi:10.1167/iovs.10-5937
PMCID: PMC3055759  PMID: 20610840
17.  Extraocular Muscles in Patients With Infantile Nystagmus 
Archives of ophthalmology  2012;130(3):343-349.
Objective
To test the hypothesis that the extraocular muscles (EOMs) of patients with infantile nystagmus have muscular and innervational adaptations that may have a role in the involuntary oscillations of the eyes.
Methods
Specimens of EOMs from 10 patients with infantile nystagmus and postmortem specimens from 10 control subjects were prepared for histologic examination. The following variables were quantified: mean myofiber cross-sectional area, myofiber central nucleation, myelinated nerve density, nerve fiber density, and neuromuscular junction density.
Results
In contrast to control EOMs, infantile nystagmus EOMs had significantly more centrally nucleated myofibers, consistent with cycles of degeneration and regeneration. The EOMs of patients with nystagmus also had a greater degree of heterogeneity in myofiber size than did those of controls, with no difference in mean myofiber cross-sectional area. Mean myelinated nerve density, nerve fiber density, and neuromuscular junction density were also significantly decreased in infantile nystagmus EOMs.
Conclusions
The EOMs of patients with infantile nystagmus displayed a distinct hypoinnervated phenotype. This represents the first quantification of changes in central nucleation and myofiber size heterogeneity, as well as decreased myelinated nerve, nerve fiber, and neuromuscular junction density. These results suggest that deficits in motor innervation are a potential basis for the primary loss of motor control.
Clinical Relevance
Improved understanding of the etiology of nystagmus may direct future diagnostic and treatment strategies.
doi:10.1001/archophthalmol.2011.381
PMCID: PMC3759680  PMID: 22411664
18.  Analysis of Neurotrophic Factors in Limb and Extraocular Muscles of Mouse Model of Amyotrophic Lateral Sclerosis 
PLoS ONE  2014;9(10):e109833.
Amyotrophic lateral sclerosis (ALS) is currently an incurable fatal motor neuron syndrome characterized by progressive weakness, muscle wasting and death ensuing 3–5 years after diagnosis. Neurotrophic factors (NTFs) are known to be important in both nervous system development and maintenance. However, the attempt to translate the potential of NTFs into the therapeutic options remains limited despite substantial number of approaches, which have been tested clinically. Using quantitative RT-PCR (qRT-PCR) technique, the present study investigated mRNA expression of four different NTFs: brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4) and glial cell line-derived neurotrophic factor (GDNF) in limb muscles and extraocular muscles (EOMs) from SOD1G93A transgenic mice at early and terminal stages of ALS. General morphological examination revealed that muscle fibres were well preserved in both limb muscles and EOMs in early stage ALS mice. However, in terminal ALS mice, most muscle fibres were either atrophied or hypertrophied in limb muscles but unaffected in EOMs. qRT-PCR analysis showed that in early stage ALS mice, NT-4 was significantly down-regulated in limb muscles whereas NT-3 and GDNF were markedly up-regulated in EOMs. In terminal ALS mice, only GDNF was significantly up-regulated in limb muscles. We concluded that the early down-regulation of NT-4 in limb muscles is closely associated with muscle dystrophy and dysfunction at late stage, whereas the early up-regulations of GDNF and NT-3 in EOMs are closely associated with the relatively well-preserved muscle morphology at late stage. Collectively, the data suggested that comparing NTFs expression between limb muscles and EOMs from different stages of ALS animal models is a useful method in revealing the patho-physiology and progression of ALS, and eventually rescuing motor neuron in ALS patients.
doi:10.1371/journal.pone.0109833
PMCID: PMC4198138  PMID: 25334047
19.  Physically-based Modeling and Simulation of Extraocular Muscles 
Dynamic simulation of human eye movements, with realistic physical models of extraocular muscles (EOMs), may greatly advance our understanding of the complexities of the oculomotor system and aid in treatment of visuomotor disorders. In this paper we describe the first three dimensional (3D) biomechanical model which can simulate the dynamics of ocular motility at interactive rates. We represent EOMs using “strands”, which are physical primitives that can model an EOM's complex nonlinear anatomical and physiological properties. Contact between the EOMs, the globe, and orbital structures can be explicitly modeled.
Several studies were performed to assess the validity and utility of the model. EOM deformation during smooth pursuit was simulated and compared with published experimental data; the model reproduces qualitative features of the observed non-uniformity. The model is able to reproduce realistic saccadic trajectories when the lateral rectus muscle was driven by published measurements of abducens neuron discharge. Finally, acute superior oblique palsy, a pathological condition, was simulated to further evaluate the system behavior; the predicted deviation patterns agree qualitatively with experimental observations. This example also demonstrates potential clinical applications of such a model.
doi:10.1016/j.pbiomolbio.2010.09.002
PMCID: PMC3003910  PMID: 20868704
physically-based modeling; extraocular muscle; interactive simulation; soft tissue motion; eye movement; biomechanics
20.  Effects of Recession versus Tenotomy Surgery without Recession in Adult Rabbit Extraocular Muscle 
The EOMs are particularly adaptive to changes induced by recession and tenotomy surgery, responding with modulations in fiber remodeling and myosin expression and also with changes in antagonist and contralateral muscles. These results suggest the possibility that these processes are manipulated immediately after surgery to improve surgical success rates.
Purpose.
Surgical recession of an extraocular muscle (EOM) posterior to its original insertion is a common form of strabismus surgery, weakening the rotational force exerted by the muscle on the globe and improving eye alignment. The purpose of this study was to assess myosin heavy chain (MyHC) isoform expression and satellite cell activity as defined by Pax7 expression in recessed EOMs of adult rabbits compared with that in muscles tenotomized but not recessed and with that in normal control muscles.
Methods.
The scleral insertion of the superior rectus muscle was detached and sutured either 7 mm posterior to its original insertion site (recession surgery) or at the same site (tenotomy). One day before euthanatization, the rabbits received bromodeoxyuridine (BrdU) injections. After 7 and 14 days, selected EOMs from both orbits were examined for changes in fast, slow, neonatal, and developmental MyHC isoform expression, Pax7 expression, and BrdU incorporation.
Results.
Recession and tenotomy surgery resulted in similar changes in the surgical EOMs. These included a decreased proportion of fast MyHC myofibers, an increased proportion of slow MyHC myofibers, and increased BrdU-positive satellite cells. Similar changes were seen in the non-operated contralateral superior rectus muscles. The ipsilateral inferior rectus showed reciprocal changes to the surgical superior rectus muscles.
Conclusions.
The EOMs are extremely adaptive to changes induced by recession and tenotomy surgery, responding with modulations in fiber remodeling and myosin expression. These adaptive responses could be manipulated to improve surgical success rates.
doi:10.1167/iovs.10-5523
PMCID: PMC3061502  PMID: 20538996
21.  Magnetic Resonance Imaging Evidence for Widespread Orbital Dysinnervation in Dominant Duane’s Retraction Syndrome Linked to the DURS2 Locus 
Purpose
High-resolution, multipositional magnetic resonance imaging (MRI) was used to demonstrate extraocular muscles (EOMs) and associated motor nerves in Duane retraction syndrome (DRS) linked to the DURS2 locus on chromosome 2.
Methods
Five male and three female affected members of two autosomal dominant DURS2 pedigrees were enrolled in the study. Coronal T1-weighted MRI of the orbits was obtained in multiple gaze positions, as well as with heavy T2 weighting in the plane of the cranial nerves. MRI findings were correlated with motility.
Results
All subjects had unilateral or bilateral limitation of abduction, or of both abduction and adduction, with palpebral fissure narrowing and globe retraction in adduction. Orbital motor nerves were typically small, with the abducens nerve (cranial nerve [CN]6) often nondetectable. Lateral rectus (LR) muscles were structurally abnormal in seven subjects, with structural and motility evidence of oculomotor nerve (CN3) innervation from vertical rectus EOMs leading to A or V patterns of strabismus in three cases. Four cases had superior oblique, two cases superior rectus, and one case levator EOM hypoplasia. Only the medial and inferior rectus and inferior oblique EOMs were spared. Two cases had small CN3s.
Conclusions
DRS linked to the DURS2 locus is associated with bilateral abnormalities of many orbital motor nerves, and structural abnormalities of all EOMs except those innervated by the inferior division of CN3. The LR may be coinnervated by CN3 branches normally destined for any other rectus EOMs. Therefore, DURS2-linked DRS is a diffuse congenital cranial dysinnervation disorder involving but not limited to CN6.
doi:10.1167/iovs.06-0632
PMCID: PMC1850629  PMID: 17197533
22.  Sparing of Extraocular Muscle in Aging and Muscular Dystrophies: A Myogenic Precursor Cell Hypothesis 
Experimental cell research  2011;317(6):873-885.
The extraocular muscles (EOM) are spared from pathology in aging and many forms of muscular dystrophy. Despite many studies, this sparing remains an enigma. The EOM have a distinct embryonic lineage compared to somite-derived muscles, and we have shown that they continuously remodel throughout life, maintaining a population of activated satellite cells even in aging. This data suggested the hypothesis that there is a population of myogenic precursor cells (mpcs) in EOM that is different from those in limb, with either elevated numbers of stem cells and/or mpcs with superior proliferative capacity compared to mpcs in limb. Using flow cytometry, EOM and limb muscle mononuclear cells were compared, and a number of differences were seen. Using two different cell isolation methods, EOM have significantly more mpcs per mg muscle than limb skeletal muscle. One specific subpopulation significantly increased in EOM compared to limb was positive for CD34 and negative for Sca-1, M-cadherin, CD31, and CD45. We named these the EOMCD34 cells. Similar percentages of EOMCD34 cells were present in both newborn EOM and limb muscle. In addition, they were retained in aged EOM, whereas the population decreased significantly in adult limb muscle and were extremely scarce in aged limb muscle. Most importantly, the percentage of EOMCD34 cells were elevated in the EOM from both the mdx and the mdx/utrophin−/− (DKO) mouse models of DMD and extremely scarce in the limb muscles of these mice. In vitro, the EOMCD34 cells had myogenic potential, forming myotubes in differentiation media. After determining a media better able to induce proliferation in these cells, a fusion index was calculated. The cells isolated from EOM had a 40% higher fusion index compared to the same cells isolated from limb muscle. The EOMCD34 cells were resistant to both oxidative stress and mechanical injury. These data support our hypothesis that the EOM may be spared in aging and in muscular dystrophies due to a subpopulation of mpcs, the EOMCD34 cells, that are retained in significantly higher percentages in normal, mdx and DKO mice EOM, appear to be resistant to elevated levels of oxidative stress and toxins, and actively proliferate throughout life. Current studies are focused on further defining the EOMCD34 cell subtype molecularly, with the hopes that this may shed light on a cell type with potential therapeutic use in patients with sarcopenia, cachexia, or muscular dystrophy.
doi:10.1016/j.yexcr.2011.01.018
PMCID: PMC3072110  PMID: 21277300
extraocular muscles; muscular dystrophy; satellite cells; myogenic precursor cells; flow cytometry; CD34; aging
23.  High-Resolution Magnetic Resonance Imaging of the Extraocular Muscles and Nerves Demonstrates Various Etiologies of Third Nerve Palsy 
American journal of ophthalmology  2006;143(2):280-287.
Purpose
The etiology of third nerve palsy is usually diagnosed by history, motility examination, and presence of lid and pupil involvement, as well as cranial and vascular imaging. We used high-resolution magnetic resonance imaging (hrMRI) of the oculomotor nerve and affected extraocular muscles (EOMs) to investigate oculomotor palsy.
Design
Prospective, noncomparative, observational case series in an academic referral setting.
Methods
Twelve patients with non-aneurysmal oculomotor palsy of duration 0.75 to 252 months were studied. In the orbit and along the intracranial oculomotor nerve, hrMRI at 1–2mm thickness was performed. Coronal plane images of each orbit were obtained in multiple, controlled gaze positions. Structural abnormalities of the oculomotor nerve and associated changes in EOM volume and contractility were evaluated.
Results
Cases were categorized as tumor-related, congenital, diabetic, traumatic, and idiopathic according to clinical characteristics and hrMRI findings. Reduction of volume and contractility of affected EOMs were noted in six patients; however, there was no significant EOMs atrophy in two cases with diabetic oculomotor palsy, and four cases with aberrant regeneration. hrMRI demonstrated the oculomotor nerve at the midbrain and at EOMs in all cases, and in two case with previous normal neuroimaging elsewhere demonstrated contrast-enhancing tumors on the oculomotor nerve. One patient with apparently unilateral congenital inferior division oculomotor palsy had no detectable ipsilateral and a hypoplastic contralateral oculomotor nerve exiting the midbrain.
Conclusions
hrMRI provides valuable information in patients with oculomotor palsy, such as structural abnormalities of the orbit and oculomotor nerve, and atrophy and diminished contractility of innervated EOMs. This information could be helpful in diagnosis and management of oculomotor palsy.
doi:10.1016/j.ajo.2006.10.035
PMCID: PMC1850712  PMID: 17173848
24.  Development of extraocular muscles require early signals from periocular neural crest and the developing eye 
Archives of ophthalmology  2011;129(8):1030-1041.
Purpose
Identify and explain morphologic changes of the extraocular muscles (EOMs) in anophthalmic patients.
Methods
Retrospective chart review of patients with congenital anophthalmia, using MRI and intraoperative findings to characterize EOM morphology. We then employ molecular biology techniques in zebrafish and chick embryos to determine the relationships among the developing eye, periocular neural crest, and EOMs.
Results
In three human patients with bilateral congenital anophthalmia and preoperative orbital imaging, we observed a spectrum of EOM morphologies ranging from indiscernible muscle tissue to well-formed, organized EOMs. Timing of eye loss in zebrafish and chick embryos correlated with the morphology of EOM organization in the orbit (“eye socket”). In congenitally eyeless Rx3 zebrafish mutants, or following genetic ablation of the cranial neural crest cells, EOMs failed to organize, which was independent of other craniofacial muscle development.
Conclusions
Orbital development is dependent on interactions between the eye, neural crest, and developing EOMs. Timing of the ocular insult, in relation to neural crest migration and EOM development, is a key determinant of aberrant EOM organization. Additional research will be required to study patients with unilateral and syndromic anophthalmia, and assess for possible differences in clinical outcomes among patients with varied EOM morphology.
Clinical relevance
The presence and organization of EOMs in anophthalmic sockets may serve as a marker for the timing of genetic or teratogenic insults, improving genetic counseling, and assisting with surgical reconstruction and family counseling efforts.
doi:10.1001/archophthalmol.2011.75
PMCID: PMC3248700  PMID: 21482859
25.  Underdeveloped extraocular muscles in the naked mole-rat (Heterocephalus glaber) 
The extraocular muscles (EOM), the effector arm of the ocular motor system, have a unique embryological origin and phenotype. The naked mole-rat (NMR) is a subterranean rodent with an underdeveloped visual system. It has not been established if their ocular motor system is also less developed. The NMR is an ideal model to examine the potential codependence of oculomotor and visual system development and evolution. Our goal was to compare the structural features of NMR EOMs to those of the mouse, a similar sized rodent with a fully developed visual system. Perfusion-fixed whole orbits and EOMs were dissected from adult NMR and C57BL mice and examined by light and electron microscopy. NMR orbital anatomy showed smaller EOMs in roughly the same distribution around the eye as in mouse and surrounded by a very small Harderian gland. The NMR EOMs did not appear to have the two-layer fiber distribution seen in mouse EOMs; fibers were also significantly smaller (112.3 ± 46.2 vs. 550.7 ± 226sq μm in mouse EOMs, *P<0.05). Myofibrillar density was less in NMR EOMs, and triad and other membranous structures were rudimentary. Finally, mitochondrial volume density was significantly less in NMR EOMs than in mouse EOM (4.5%± 1.9 vs. 21.2%± 11.6 respectively, *P<0.05). These results demonstrate that NMR EOMs are smaller and less organized than those in the mouse. The “simpler” EOM organization and structure in NMR may be explained by the poor visual ability of these rodents, initially demonstrated by their primitive visual system.
doi:10.1002/ar.21107
PMCID: PMC3086521  PMID: 20186962
extraocular muscle; Heterocephalus glaber; naked mole-rat; mitochondria

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