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S. Bose, C. J. Nien Shy, L. T. Lam, J. V. Jester; Orbital and Global Layer Differences of Normal and Hyperthyroid Extraocular Muscles in C57BL/6 Mice. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4120. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Extraocular muscles (EOMs) have a unique laminar organization consisting of a thin outer orbital layer (OL) and a thicker inner global layer (GL) that presumably serve distinctly different functions. These muscle layers have been well characterized in rats, humans and rabbits, but little is known regarding the mouse EOM. The purpose of this study was to characterize the EOM of the mouse and to determine the effects of hyperthyroidism on the OL and GL of the EOM.
Two month old, female C57BL/6 mice were used. Hyperthyroidism was induced in five mice by daily IP injection with 3, 3’, 5 Triiodo-L-Thyronine for 4 weeks. Control mice (n = 5) received daily IP injections of vehicle. At 4 weeks mice were sacrificed, orbit exenterated and fixed in 4% paraformaldehyde in PBS. Tissue was processed for frozen sectioning following equilibration in serial sucrose from 15% up to 30%, embedding in OCT and freezing liquid nitrogen. Coronal sections (7µm) were taken through EOM starting at the muscle insertion and extending to the common ring tendon. Sections were stained with anti-rabbit Troponin T (Abcam), mitotracker green FM (Molecular Probes) and DAPI and then viewed on a Nikon Ellipse E600 fluorescent microscope. Images were analyzed using Metamorph 7.0 and Graphpad prism 3.0.
The OL of the EOM from control mice contained small diameter muscle fibers averaging 113.2 ± 3.66 µm2 with a high density of fibers staining for Troponin T (90%). The GL of the normal EOM had significantly larger fibers (p = 0.001) averaging 411 ± 13.84 µm2 with fewer Troponin positive fibers (10%). The GL muscle fibers stained by Mitotracker were significantly more intense (p < 0.001) relative to OL muscle fibers (0.68 ± 0.02 compared to 0.26 ± 0.02 normalized intensity, respectively). In hyperthyroid mice fiber size increased in the OL to 205.9 ± 5.3 µm2 and decreased in the GL to 271.7 ± 7.5 µm2 but remained significantly different (p < 0.001) with a general loss of anti-Troponin staining in the OL. Mitotracker staining of hyperthyroid EOM showed a significant switch in the intensity of staining (p < 0.002) with OL muscle fibers having a normalized intensity of 0.49 ± 0.04 compared to 0.22 ± 0.03 for GL fibers.
Similar to other species, the OL of the EOM from C57BL/6 mice contain smaller diameter muscle fibers that stain more predominantly for Troponin T and appear to contain less active mitochondria compared to GL muscle fibers. These differences are significantly altered in hyperthyroid EOMs. Understanding the mechanism underlying these changes may help elucidate the pathophysiology of thyroid eye disease.
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