Abstract
Background: Structural details of vertebrate extraocular muscles (EOMs) have shown an anatomically and functionally distinct laminar organization into an outer orbital (OL) and an inner global layer (GL). Since hyperthyroidism alters tissue oxidative metabolism through mitochondrial enzymes, it is expected that structural/mitochondrial changes may be seen in hyperthyroid EOMs. We investigated the alterations in the laminar organization and mitochondrial changes in hyperthyroid mouse EOMs. Methods: Hyperthyroidism was induced in C57BL/6 mice and fresh rectus muscles were obtained to identify functional mitochondria using MitoTracker® Green and confocal microscopy; frozen sections from rectus muscles were stained with anti-rabbit Troponin T (selectively present in the OL) to demonstrate changes in the OL and GL of the EOMs. Ultrastructural features of EOMs were studied using transmission electron microscopy (TEM). Results: Of all four rectus EOMs studied, the maximum change was seen in the inferior rectus muscle (IR) followed by medial rectus (MR). Myofiber cross-sectional area measurements and Troponin T staining in the control IR EOMs demonstrated a smaller OL (113.2 ± 3.66 μm2) and higher density staining with Troponin T (90%) and a larger GL (411 ± 13.84 μm2) with low intensity staining (10%), while hyperthyroidism resulted in an increased OL (205.9 ± 5.3 μm2) and decreased GL (271.7 ± 7.5 μm2) p = 0.001. Confocal microscopy demonstrated an intense staining especially in the outer rims in the hyperthyroid IR which was confirmed by TEM showing structural alterations in the mitochondria and a subsarcolemmal migration. Conclusions: The outer, thinner, OL of the mouse EOM contains smaller diameter myofibers and fewer mitochondria while the inner, larger GL contains larger diameter myofibers and larger density of mitochondria. Hyperthyroidism results in a significant alteration in the laminar organization and mitochondrial alterations of mouse EOMs.
Highlights
The extraocular muscles (EOMs) are unique from other skeletal muscles regarding their embryologic development, innervations, vascularization, metabolic profile, mitochondrial content, and gene expression (Budak et al, 2004)
Extraocular muscles changes Evaluating the intensity of MitoTracker® Green staining to illustrate the size and/or number of mitochondria along the myofibers, we observed a marked increase in the intensity of stain in the inferior rectus muscle when hyperthyroidism was induced (Figure 1)
OL and global layer (GL) changes in inferior rectus muscle Due to the differences noted in the inferior rectus, we further characterized these changes using immunohistochemistry
Summary
The extraocular muscles (EOMs) are unique from other skeletal muscles regarding their embryologic development, innervations, vascularization, metabolic profile, mitochondrial content, and gene expression (Budak et al, 2004). It has been demonstrated that both layers differ in thickness, fiber size, metabolic activity, and vascular density (Budak et al, 2004) These distinct muscle layers have been described in rats, monkeys, humans, rabbits, and dogs (Porter et al, 1995; Wasicky et al, 2000; Oh et al, 2001a,b; Lucas and Hoh, 2003; Budak et al, 2004; Wicke et al, 2007; Wiesen et al, 2007). Hyperthyroidism results in a significant alteration in the laminar organization and mitochondrial alterations of mouse EOMs
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