Purpose of Review The goal of this review is to summarize the unique regenerative milieu within mature mammalian extraocular muscles (EOMs). This will aid in understanding disease propensity for and sparing of EOMs in skeletal muscle diseases as well as the recalcitrance of the EOM to injury. Recent Findings The EOMs continually remodel throughout life and contain an extremely enriched number of myogenic precursor cells that differ in number and functional characteristics from those in limb skeletal muscle. The EOMs also contain a large population of Pitx2-positive myogenic precursor cells that provide the EOMs with many of their unusual biological characteristics, such as myofiber remodeling and skeletal muscle disease sparing. This environment provides for rapid and efficient remodeling and regeneration after various types of injury. In addition, the EOMs show a remarkable ability to respond to perturbations of single muscles with coordinated changes in the other EOMs that move in the same plane. Summary These data will inform Ophthalmologists as they work toward developing new treatments for eye movement disorders, new approaches for repair after nerve or direct EOMs injury, as well as suggest potential explanations for the unusual disease propensity and disease sparing characteristics of human EOM.

The hypothalamic-pituitary-thyroid (HPT) axis is known to play a crucial role in the development of teleost fish. However, knowledge of endogenous transcription profiles of thyroid-related genes in developing teleosts remains fragmented. We selected two model teleost species, the fathead minnow (Pimephales promelas) and the zebrafish (Danio rerio), to compare the gene transcription ontogeny of the HPT axis. Control organisms were sampled at several time points during embryonic and larval development until 33 days post-fertilization. Total RNA was extracted from pooled, whole fish, and thyroid-related mRNA expression was evaluated using quantitative polymerase chain reaction. Gene transcripts examined included: thyrotropin-releasing hormone receptor (trhr), thyroid-stimulating hormone receptor (tshr), sodium-iodide symporter (nis), thyroid peroxidase (tpo), thyroglobulin (tg), transthyretin (ttr), deiodinases 1, 2, 3a, and 3b (dio1, dio2, dio3a and 3b), and thyroid hormone receptors alpha and beta (thrα and β). A loess regression method was successful in identifying maxima and minima of transcriptional expression during early development of both species. Overall, we observed great similarities between the species, including maternal transfer, at least to some extent, of almost all transcripts (confirmed in unfertilized eggs), increasing expression of most transcripts during hatching and embryo-larval transition, and indications of a fully functional HPT axis in larvae. These data will aid in the development of hypotheses on the role of certain genes and pathways during development. Furthermore, this provides a background reference dataset for designing and interpreting targeted transcriptional expression studies both for fundamental research and for applications such as toxicology.

PurposeWe examined the pattern and extent of connective tissue distribution in the extraocular muscles (EOMs) and determined the ability of the interconnected connective tissues to disseminate force laterally.MethodsHuman EOMs were examined for collagens I, III, IV, and VI; fibronectin; laminin; and elastin using immunohistochemistry. Connective tissue distribution was examined with scanning electron microscopy. Rabbit EOMs were examined for levels of force transmission longitudinally and transversely using in vitro force assessment.ResultsCollagens I, III, and VI localized to the endomysium, perimysium, and epimysium. Collagen IV, fibronectin, and laminin localized to the basal lamina surrounding all myofibers. All collagens localized similarly in the orbital and global layers throughout the muscle length. Elastin had the most irregular pattern and ran longitudinally and circumferentially throughout the length of all EOMs. Scanning electron microscopy showed these elements to be extensively interconnected, from endomysium through the perimysium to the epimysium surrounding the whole muscle. In vitro physiology demonstrated force generation in the lateral dimension, presumably through myofascial transmission, which was always proportional to the force generated in the longitudinally oriented muscles.ConclusionsA striking connective tissue matrix interconnects all the myofibers and extends, via perimysial connections, to the epimysium. These interconnections are significant and allow measurable force transmission laterally as well as longitudinally, suggesting that they may contribute to the nonlinear force summation seen in motor unit recording studies. This provides strong evidence that separate compartmental movements are unlikely as no region is independent of the rest of the muscle.

Recent microarray and RNAseq experiments provided evidence that glial derived neurotrophic factor (GDNF) levels were decreased in extraocular muscles from human strabismic subjects compared to age-matched controls. We assessed the effect of sustained GDNF treatment of the superior rectus muscles of rabbits on their physiological and morphological characteristics, and these were compared to naïve control muscles. Superior rectus muscles of rabbits were implanted with a sustained release pellet of GDNF to deliver 2μg/day, with the contralateral side receiving a placebo pellet. After one month, the muscles were assessed using in vitro physiological methods. The muscles were examined histologically for alteration in fiber size, myosin expression patterns, neuromuscular junction size, and stem cell numbers and compared to age-matched naïve control muscles. GDNF resulted in decreased force generation, which was also seen on the untreated contralateral superior rectus muscles. Muscle relaxation times were increased in the GDNF treated muscles. Myofiber mean cross-sectional areas were increased after the GDNF treatment, but there was a compensatory increase in expression of developmental, neonatal, and slow tonic myosin heavy chain isoforms. In addition, in the GDNF treated muscles there was a large increase in Pitx2-positive myogenic precursor cells. One month of GDNF resulted in significant extraocular muscle adaptation. These changes are interesting relative to the decreased levels of GDNF in the muscles from subjects with strabismus and preliminary data in infant non-human primates where sustained GDNF treatment produced a strabismus. These data support the view that GDNF has the potential for improving eye alignment in subjects with strabismus.

One major difference between limb and extraocular muscles (EOM) is the presence of an enriched population of Pitx2-positive myogenic precursor cells in EOM compared to limb muscle. We hypothesize that retinoic acid regulates Pitx2 expression in EOM myogenic precursor cells and that its effects would differ in leg muscle. The two muscle groups expressed differential retinoic acid receptor (RAR) and retinoid X receptor (RXR) levels. RXR co-localized with the Pitx2-positive cells but not with those expressing Pax7. EOM-derived and LEG-derived EECD34 cells were treated with vehicle, retinoic acid, the RXR agonist bexarotene, the RAR inverse agonist BMS493, or the RXR antagonist UVI 3003. In vitro, fewer EOM-derived EECD34 cells expressed desmin and fused, while more LEG-derived cells expressed desmin and fused when treated with retinoic acid compared to vehicle. Both EOM and LEG-derived EECD34 cells exposed to retinoic acid showed a higher percentage of cells expressing Pitx2 compared to vehicle, supporting the hypothesis that retinoic acid plays a role in maintaining Pitx2 expression. We hypothesize that retinoic acid signaling aids in the maintenance of large numbers of undifferentiated myogenic precursor cells in the EOM, which would be required to maintain EOM normalcy throughout a lifetime of myonuclear turnover.