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PurposeWe examined inferior oblique muscles from subjects with over-elevation in adduction for characteristics that might shed light on the potential mechanisms for their abnormal eye position.MethodsThe inferior oblique muscles were obtained at the time of surgery in subjects diagnosed with either primary inferior oblique overaction or Apert syndrome. The muscles were frozen and processed for morphometric analysis of myofiber size, central nucleation, myosin heavy chain (MyHC) isoform expression, nerve density, and numbers of neuromuscular junctions per muscle section.ResultsThe inferior oblique muscles from subjects with Apert Syndrome were smaller, and had a much more heterogeneous profile relative to myofiber cross-sectional area compared to controls. Increased central nucleation in the Apert syndrome muscles suggested on-going myofiber regeneration or reinnervation over time. Complex changes were seen in the MyHC isoform patterns that would predict slower and more sustained contractions than in the control muscles. Nerve fiber densities were significantly increased compared to controls for the muscles with primary inferior oblique overaction and Apert syndrome that had no prior surgery. The muscles from Apert syndrome subjects as well as those with primary inferior oblique overaction with no prior surgery had significantly elevated numbers of neuromuscular junctions relative to the whole muscle area.ConclusionsThe muscles from both sets of subjects were significantly different from control muscles in a number of properties examined. These data support the view that despite similar manifestations of eye misalignment, the potential mechanism behind the strabismus in these subjects is significantly different.

PurposeWe examined inferior oblique muscles from subjects with over-elevation in adduction for characteristics that might shed light on the potential mechanisms for their abnormal eye position.MethodsThe inferior oblique muscles were obtained at the time of surgery in subjects diagnosed with either primary inferior oblique overaction or Apert syndrome. The muscles were frozen and processed for morphometric analysis of myofiber size, central nucleation, myosin heavy chain (MyHC) isoform expression, nerve density, and numbers of neuromuscular junctions per muscle section.ResultsThe inferior oblique muscles from subjects with Apert Syndrome were smaller, and had a much more heterogeneous profile relative to myofiber cross-sectional area compared to controls. Increased central nucleation in the Apert syndrome muscles suggested on-going myofiber regeneration or reinnervation over time. Complex changes were seen in the MyHC isoform patterns that would predict slower and more sustained contractions than in the control muscles. Nerve fiber densities were significantly increased compared to controls for the muscles with primary inferior oblique overaction and Apert syndrome that had no prior surgery. The muscles from Apert syndrome subjects as well as those with primary inferior oblique overaction with no prior surgery had significantly elevated numbers of neuromuscular junctions relative to the whole muscle area.ConclusionsThe muscles from both sets of subjects were significantly different from control muscles in a number of properties examined. These data support the view that despite similar manifestations of eye misalignment, the potential mechanism behind the strabismus in these subjects is significantly different.

The extraocular muscles differ from other skeletal muscles in many respects but most strikingly in their response to neuromuscular diseases expected to affect the whole body. Oculomotor disturbances are not typical features of ALS. Recent data ascribe the muscle tissue an important role in the pathophysiology of ALS, with early involvement of the neuromuscular junctions and loss of axonal contact. We show that the extraocular muscles of terminal ALS donors and also of mice models of ALS maintain their morphology and well-preserved neuromuscular junctions until the end stages of the disease, whereas the limb muscles are severely affected and their neuromuscular junctions start losing contact with the supplying axons early in the course of ALS. There are intrinsic differences between the extraocular and limb muscles with respect to neurotrophic factors and Wnt isoforms and fundamental differences in their response to ALS that cannot be explained by the aging process. We propose that these differences may be instrumental in the selective sparing of the extraocular muscles in ALS.

Current models of ocular mechanics do not fully account for potentially large globe translations associated with eye rotation. Such combined motion can be measured using magnetic resonance imaging in axial planes. We imaged orbits of normal volunteers fixating horizontally eccentric targets. These data indicate that the human eye acts as if it rotates eccentrically about a varying point typically anterior to the geometric globe center, but significantly lateral in abduction and medial in adduction. Assumed eccentricity of the ocular rotational center would vary the torque lever arms for the horizontal rectus muscles, with an appreciably smaller relative lever arm for the medial rectus muscle in adduction than would be the case for oculocentric rotation. Such variation in ocular rotational center might alter muscle torque without commensurate change in muscle tension, as appears to happen in convergence.

PurposeWe used magnetic resonance imaging (MRI) to ascertain effects of optic nerve (ON) traction in adduction, a phenomenon proposed as neuropathic in primary open-angle glaucoma (POAG).MethodsSeventeen patients with POAG and maximal IOP ≤ 20 mm Hg, and 31 controls underwent MRI in central gaze and 20° to 30° abduction and adduction. Optic nerve and sheath area centroids permitted computation of midorbital lengths versus minimum paths.ResultsAverage mean deviation (±SEM) was -8.2 ± 1.2 dB in the 15 patients with POAG having interpretable perimetry. In central gaze, ON path length in POAG was significantly more redundant (104.5 ± 0.4% of geometric minimum) than in controls (102.9 ± 0.4%, P = 2.96 × 10-4). In both groups the ON became significantly straighter in adduction (28.6 ± 0.8° in POAG, 26.8 ± 1.1° in controls) than central gaze and abduction. In adduction, the ON in POAG straightened to 102.0% ± 0.2% of minimum path length versus 104.5% ± 0.4% in central gaze (P = 5.7 × 10-7), compared with controls who straightened to 101.6% ± 0.1% from 102.9% ± 0.3% in central gaze (P = 8.7 × 10-6); and globes retracted 0.73 ± 0.09 mm in POAG, but only 0.07 ± 0.08 mm in controls (P = 8.8 × 10-7). Both effects were confirmed in age-matched controls, and remained significant after correction for significant effects of age and axial globe length (P = 0.005).ConclusionsAlthough tethering and elongation of ON and sheath are normal in adduction, adduction is associated with abnormally great globe retraction in POAG without elevated IOP. Traction in adduction may cause mechanical overloading of the ON head and peripapillary sclera, thus contributing to or resulting from the optic neuropathy of glaucoma independent of IOP.

PurposeWe used magnetic resonance imaging (MRI) to ascertain effects of optic nerve (ON) traction in adduction, a phenomenon proposed as neuropathic in primary open-angle glaucoma (POAG).MethodsSeventeen patients with POAG and maximal IOP ≤ 20 mm Hg, and 31 controls underwent MRI in central gaze and 20° to 30° abduction and adduction. Optic nerve and sheath area centroids permitted computation of midorbital lengths versus minimum paths.ResultsAverage mean deviation (±SEM) was -8.2 ± 1.2 dB in the 15 patients with POAG having interpretable perimetry. In central gaze, ON path length in POAG was significantly more redundant (104.5 ± 0.4% of geometric minimum) than in controls (102.9 ± 0.4%, P = 2.96 × 10-4). In both groups the ON became significantly straighter in adduction (28.6 ± 0.8° in POAG, 26.8 ± 1.1° in controls) than central gaze and abduction. In adduction, the ON in POAG straightened to 102.0% ± 0.2% of minimum path length versus 104.5% ± 0.4% in central gaze (P = 5.7 × 10-7), compared with controls who straightened to 101.6% ± 0.1% from 102.9% ± 0.3% in central gaze (P = 8.7 × 10-6); and globes retracted 0.73 ± 0.09 mm in POAG, but only 0.07 ± 0.08 mm in controls (P = 8.8 × 10-7). Both effects were confirmed in age-matched controls, and remained significant after correction for significant effects of age and axial globe length (P = 0.005).ConclusionsAlthough tethering and elongation of ON and sheath are normal in adduction, adduction is associated with abnormally great globe retraction in POAG without elevated IOP. Traction in adduction may cause mechanical overloading of the ON head and peripapillary sclera, thus contributing to or resulting from the optic neuropathy of glaucoma independent of IOP.

PURPOSE. To investigate the distribution of the intermediate filament (IF) proteins desmin, vimentin, and nestin in human extraocular muscles (EOMs). METHODS. Healthy adult EOM samples were serially sectioned (5 and 1 mu m) and processed for immunohistochemistry, with specific antibodies (Abs) against desmin, vimentin, and nestin and different myosin heavy chains (MyHCs), including the newly characterized Ab MYH7b against MyHC slow tonic. The distribution of desmin was also studied in EOMs at 16 to 18 weeks of gestation. RESULTS. Desmin was present in the vast majority of muscle fibers. Notably, muscle fibers that contained MyHC slow tonic were either unlabeled or very weakly labeled with three different Abs against desmin. These muscle fibers had normal cytoarchitecture and intact basement membrane. In fetal muscle, desmin was also absent or weak in myotubes containing MyHC slow tonic. Nestin was detected in a large proportion of muscle fibers in the orbital layer and to some extent also in the global layer, whereas no muscle fibers contained vimentin. Desmin and nestin were enriched at neuromuscular junctions, as in limb muscle. In contrast, some myotendinous junctions lacked desmin or nestin. CONCLUSIONS. The human EOMs differed significantly from the other muscles in the body with respect to their IF composition. Desmin, hitherto regarded as a ubiquitous muscle cytoskeletal protein, was absent or only present in trace amounts in a subset of normal muscle fibers in adult and fetal EOMs. Nestin, normally downregulated early in the postnatal period, was present in a high proportion of adult muscle fibers.

A controversial issue in the eye movement field involves the compartmentalization of extraocular muscle (EOM). Conventionally, it has been believed that each individual EOM is innervated uniformly by its motor nerve, and behaves uniformly. However, recent studies have revealed that motor nerves innervating horizontal rectus EOMs are bifurcated into two divisions that control, respectively, the superior and inferior zones of muscle fibers. This finding has motivated the proposition that each of the two compartments of these EOMs might be controlled independently, so that each EOM could behave as it were two parallel but independent actuators. The compartmental independence postulate requires a mechanically low degree of transverse force coupling among parallel EOM and extraocular tendon (EOT) fibers. This project aimed to employ biomechanical characterization to EOM and EOT to test the plausibility of this hypothesis.The first study described the biomechanical characterization of EOT, in both at the level of individual fibers and whole tendon. Nano indentation of EOT fiber bundles analyzed within the Hertzian framework effectively characterized the transverse Young's modulus for bovine EOT fiber bundles using atomic force microscopy (AFM). A precise estimate of the Poisson ratio (PR) of bovine EOTs was calculated using optical coherence tomography (OCT) during tensile loading. The second study investigated mechanical potential for independent compartmental behavior in EOM and EOT. Extensive experimental study was performed to examine the passive mechanical interaction between sets compartments using a custom fabricated dual channel micro-tensile load cell. Independent active contractile behavior of EOM compartments was also examined using calcium-induced EOM contraction. Both passive loading and active contraction findings indicate that EOM and tendon have a high degree of biomechanical independence, sufficient to support the proposed functional diversity of actions

A controversial issue in the eye movement field involves the compartmentalization of extraocular muscle (EOM). Conventionally, it has been believed that each individual EOM is innervated uniformly by its motor nerve, and behaves uniformly. However, recent studies have revealed that motor nerves innervating horizontal rectus EOMs are bifurcated into two divisions that control, respectively, the superior and inferior zones of muscle fibers. This finding has motivated the proposition that each of the two compartments of these EOMs might be controlled independently, so that each EOM could behave as it were two parallel but independent actuators. The compartmental independence postulate requires a mechanically low degree of transverse force coupling among parallel EOM and extraocular tendon (EOT) fibers. This project aimed to employ biomechanical characterization to EOM and EOT to test the plausibility of this hypothesis.The first study described the biomechanical characterization of EOT, in both at the level of individual fibers and whole tendon. Nano indentation of EOT fiber bundles analyzed within the Hertzian framework effectively characterized the transverse Young's modulus for bovine EOT fiber bundles using atomic force microscopy (AFM). A precise estimate of the Poisson ratio (PR) of bovine EOTs was calculated using optical coherence tomography (OCT) during tensile loading. The second study investigated mechanical potential for independent compartmental behavior in EOM and EOT. Extensive experimental study was performed to examine the passive mechanical interaction between sets compartments using a custom fabricated dual channel micro-tensile load cell. Independent active contractile behavior of EOM compartments was also examined using calcium-induced EOM contraction. Both passive loading and active contraction findings indicate that EOM and tendon have a high degree of biomechanical independence, sufficient to support the proposed functional diversity of actions

A controversial issue in the eye movement field involves the compartmentalization of extraocular muscle (EOM). Conventionally, it has been believed that each individual EOM is innervated uniformly by its motor nerve, and behaves uniformly. However, recent studies have revealed that motor nerves innervating horizontal rectus EOMs are bifurcated into two divisions that control, respectively, the superior and inferior zones of muscle fibers. This finding has motivated the proposition that each of the two compartments of these EOMs might be controlled independently, so that each EOM could behave as it were two parallel but independent actuators. The compartmental independence postulate requires a mechanically low degree of transverse force coupling among parallel EOM and extraocular tendon (EOT) fibers. This project aimed to employ biomechanical characterization to EOM and EOT to test the plausibility of this hypothesis.The first study described the biomechanical characterization of EOT, in both at the level of individual fibers and whole tendon. Nano indentation of EOT fiber bundles analyzed within the Hertzian framework effectively characterized the transverse Young's modulus for bovine EOT fiber bundles using atomic force microscopy (AFM). A precise estimate of the Poisson ratio (PR) of bovine EOTs was calculated using optical coherence tomography (OCT) during tensile loading. The second study investigated mechanical potential for independent compartmental behavior in EOM and EOT. Extensive experimental study was performed to examine the passive mechanical interaction between sets compartments using a custom fabricated dual channel micro-tensile load cell. Independent active contractile behavior of EOM compartments was also examined using calcium-induced EOM contraction. Both passive loading and active contraction findings indicate that EOM and tendon have a high degree of biomechanical independence, sufficient to support the proposed functional diversity of actions

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal neurodegenerative disorder characterised by selective loss of motor neurons with accompanying muscle paralysis and respiratory failure. Despite progressive paralysis in trunk and extremity muscles, disturbed eye motility is not a hallmark of ALS. Extraocular muscles (EOMs) of terminal ALS patients show far less morphological signs of disease than their limb muscles. One of the earliest signs of the disease in the transgenic G93A SOD1 mouse model of ALS is loss of motor neuron contact at the neuromuscular junctions (NMJ) in limb muscles. We used immunohistochemistry to identify NMJs and evaluate innervation in EOMs and limb muscles of G93A mice. In G93A limb muscles, loss of axonal contact was seen in 6-82 percent of the NMJs. On the contrary, the degree of endplate occupancy in the EOMs did not differ between transgenic mice and wild-type controls. We propose that EOM-specific properties make these muscles more resistant to the underlying pathophysiological process of ALS and that the EOMs are a useful model to advance our understanding of ALS.

We have recently shown that wild type mice are highly tolerant, whereas thyrotropin receptor (TSHR) knockout (KO) mice are susceptible to immunization with the mouse TSHR, the autoantigen in Graves' disease. However, because TSHR KO mice lack the endogenous TSHR, Graves-like hyperthyroidism cannot be expected to occur in these mice. We therefore performed adoptive transfer of splenocytes from TSHR KO mice into nude mice expressing the endogenous TSHR. Anti-TSHR autoantibodies were detected in approximately 50% recipient mice 4 wk after adoptive transfer of splenocytes (5 × 10 7/mouse) from TSHR KO mice immunized with adenovirus expressing mTSHR A subunit and persisted for 24 wk. Depletion of regulatory T cells by anti-CD25 antibody in the donor mice increased successful transfer rates without increasing antibody levels. Some recipient mice showed transient increases in thyroid-stimulating antibodies and T4 levels 4-8 wk after transfer, but many became thyroid-blocking antibody positive and hypothyroid 24 wk later. Adoptive transfer of splenocytes from naïve TSHR KO mice transiently induced very low antibody titers when the recipient mice were treated with anticytotoxic lymphocyte antigen 4 and antiprogrammed cell death 1 ligand 1 antibodies for 8 wk after transfer. Histologically, macrophages infiltrated the retrobulbar adipose tissues and extraocular muscles in a small fraction of the recipients. Our findings demonstrate successful adoptive transfer of anti-TSHR immune response from TSHR KO mice to nude mice. Although the recipient mice developed only transient and infrequent hyperthyroidism, followed by eventual hypothyroidism, induction of orbital inflammation suggests the possible role of anti-TSHR immune response for Graves' orbitopathy., Endocrinology, 153(4), pp.2034-2042; 2012

Skeletal muscles have the potential to regenerate by activation of quiescent satellite cells, however, the molecular signature that governs satellite cells during muscle regeneration is not well defined. Myosin light chains (Myls) are sarcomere-related proteins as traditional regulator of muscle contraction. In this report, we studied the possible role of Myl in the proliferation of skeletal muscle-derived myoblasts. Compared to diaphragm-derived myoblasts, the extraocular muscle-derived myoblasts with lower levels of Myl proliferated faster, maintained a longer proliferation phase, and formed more final myotubes. It was found that blockading Myl with anti-Myl antibody or knockdown of Myll by siRNA targeted against Myll could enhance the myoblast proliferation and delay the differentiation of myoblasts. Our results suggested that Myl, likely Myll, can negatively affect myoblast proliferation by facilitating myoblast withdrawal from cell cycle and differentiation.

The extraocular muscles (EOM) are anatomically and physiologically distinct from other skeletal muscles. EOM are preferentially affected in mitochondrial myopathies, but spared in Duchenne's muscular dystrophy. The anatomical and pathophysiological properties of EOM have been attributed to their unique molecular makeup: an allotype. We used expression profiling to define molecular features of the EOM allotype. We found 346 differentially expressed genes in rat EOM compared with tibialis anterior, based on a twofold difference cutoff. Genes required for efficient, fatigue-resistant, oxidative metabolism were increased in EOM, whereas genes for glycogen metabolism were decreased. EOM also showed increased expression of genes related to structural components of EOM such as vessels, nerves, mitochondria, and neuromuscular junctions. Additionally, genes related to specialized functional roles of EOM such as the embryonic and EOM-specific myosin heavy chains and genes for muscle growth, development, and/or regeneration were increased. The EOM expression profile was validated using biochemical, structural, and molecular methods. Characterization of the EOM expression profile begins to define gene transcription patterns associated with the unique anatomical, metabolic, and pathophysiological properties of EOM.

The contractile properties of the rabbit inferior oblique muscle (IO) were studied in vitro with direct stimulation at temperatures between 20 and 35 degrees C. Isovelocity releases were used to determine the force/ velocity relation. Cooling the muscle from 35 degrees C to 20 degrees C increased contraction and half-relaxation times of single twitches with a temperature coefficient (Q(10)) of 0.4, but did not affect significantly the twitch tension. The tetanic tension increases with increasing temperature (Q(10) = 1.32). Cooling decreased the maximum shortening velocity of the IO with a Q(10) of 1.6 and the maximum mechanical power with a Q(10) of 2.3. At 35 degrees C, the maximum speed of shortening of the muscle (19 +/- 2 muscle lengths/s, mean+/-SEM) corresponded to a maximum shortening velocity of the sarcomeres of 57 +/- 6 mu m/s. This value is similar to data obtained for extraocular muscles (EOM) of smaller rodents (mice and rats). In comparison with mammalian limb muscles the isometric and force-velocity properties of mammalian EOM appear to be virtually independent of the size of the animal. Thus, IO is a fast-twitch muscle endowed with a maximum velocity of shortening higher than that of fast-twitch skeletal muscle, but using a tetanic mechanical power lower than that produced by slow-twitch muscle: the combination of these properties makes it ideally suited to move an ocular globe of low mass at high velocity.

We have measured the stiffness in cyclotorsion of the human eye using a scleral suction contact ring mounted on a shaft fitted with an eddy current motor to provide the torque to turn the eye and a shaft-position-encoder to register the torsion. The relation proved to be almost linear within the measuring range in four subjects with constant slope values of about 0.75, 0.60, 0.55 and 0.45 g·cm/deg. The average, 0.6 g·cm/deg, equals 0.5 g if applied at the radius of the globe. This value is rather low compared to the values obtained by other authors for horizontal eye movements. This may be due to different length-tension characteristics of oblique muscles compared to recti muscles.

We investigated the sideways displacement of recti muscles with the eye in various gaze-positions by making computed tomographic (CT) scans in a plane perpendicular to the muscle cone, posterior to the globe. We found no consistent sideways displacement of the horizontal recti in the up or down gaze or the vertical recti in the left or right gaze. We found a substantial displacement, however, of the contracting and relaxing muscles. Consequently, we made CT scans in a horizontal plane through medial and lateral recti and found that, with the eye in primary position, the recti muscles follow a curved path. The curvature of the path increased following muscle relaxation and decreased following muscle contraction.

The history of the elucidation of ocular counterrolling (OCR) is an interesting one. Although discovered two centuries ago, OCR has remained a contro- versial issue, even in our days. The main reason for this, is that rolling move- ments of the eyes about the line of sight are not easily detected or measured. OCR can be static, i.e. about 5 degrees counterrolling of the eyes which partly compensates for tilting of the head with respect to gravity, or it can be dynamic, that is to say a rotatory optokinetic and vestibular nystagmus. The rolling movement of the eyes about the line of sight can be seen when tilting the head of another person towards one of his shoulders. Close inspec- tion of the conjunctival vessels reveals the jerking movements of the quick phase of the rotatory nystagmus. This is what John Hunter must have seen some two hundred years ago, as he described dynamic OCR in 'The use of the oblique muscles', in a book called 'Observations on certain parts of the animal oeconomy' (1786).

Intraoperative continuous-registration length and tension curves of attached and detached eye muscles were made in 18 strabismic patients under general anesthesia. For relaxed eye muscles, we found an exponential relation between length and tension. An increased stiffness was quantified in Duane's syndrome, Graves' disease, orbital-floor fracture, and superior oblique palsy. The stiffnesses of agonist and antagonist were remarkably similar, not only in uncomplicated squint, but also when only one of the two had initially become stiffer. After intravenous administration of succinylcholine chloride, the eye muscles contracted, and the exponential length and tension curve changed into a set of straight, parallel lines. In addition, we measured stiffness in passive horizontal eye movement in awake volunteers and found 0.52 to 1.26 g/degrees (other eye in 5° of adduction), confirming other published results.