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S. Bose, C. Nien, C. Kenney, A. Wahlert, D. Wallace, J. V. Jester; Confocal Laser Scanning Microscopy: A Novel Technique for Quantitative Assessment of Normal and Hyperthyroid Extraocular Muscles in C57BL/6 Mice. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5626.
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The study aims to determine if confocal laser scanning microscopy (CLSM) is able to distinguish structural and functional changes in extraocular muscles (EOMs) of hyperthyroid mice known to differ from normal controls. We employed CLSM on EOMs using two-photon excited fluorescence (TPEF), second-harmonic image microscopy (SHIM), mitotracker, JC-1 and FITC-phalloidin fluorescence staining to quantify and localize myofibrils, mitochondria, and actin-myosin complex abnormalities of EOMs.
Hyperthyroidism was induced in C57BL/6 mice using daily injections of 3, 3’, 5 Triiodo-L-Thyronine (T3), n=20. After 4 weeks, animals were humanely euthanized and freshly obtained EOMs underwent light/electron microscopy, and Zeiss LSM confocal microscopy to determine myofibril size, mitochondrial density, oxidative potential, and the actin-myosin complex alterations using TPEF, SHIM, JC-1 and mitotracker staining. Results were compared with controls, n=24.
In contrast to controls, the EOMs of hyperthyroid mice demonstrated a significant increase in myofibril thickness (50.1± 9.1 vs. 23.8 ± 1.9 µm, p=0.001) greater mitochondrial density (157.9 ± 39.5 vs. 101.4 ± 25.1 µm2, p=0.02) and presence of numerous mitochondria in the A-band. EOM abnormalities in the hyperthyroid mice using CLSM suggest a role of mitochondria in the pathogenesis of hyperthyroid eye disease. The thickness of the control myofibrils obtained using 2 µm thin sections using light microscopy (24.7 ± 5.7 µm) correlated well with those obtained by confocal microscopy (p=0.2).
Confocal laser scanning microscopy is an important new tool that offers a detailed, accurate and rapid quantitative assessment of EOM abnormalities with several advantages over conventional techniques including controllable depth of field from thick EOM specimens, multi-photon, second harmonic imaging, simultaneous multicolor fluorescence/ antibody staining of fresh EOMs and analysis of actin-myosin complex.
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