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      Stereological Quantification of Extraocular Muscles in Humans Translated title: Cuantificación Estereológica de Músculos Extraoculares en Humanos

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          Abstract

          SUMMARY: The aim of this study is to quantify muscular and connective tissue volumes of extraocular muscles (EOM) in humans with no ophthalmological disease using stereology. EOM from five cadaveric non-strabismic humans were obtained. The number of muscle fibers in 5,000 µm2 and volume density (Vv) of muscle and collagen were measured using stereology. Comparisons between antagonist EOM were conducted using Wilcoxon signed rank test for paired samples. A secondary analysis examining differences between pairs of EOM was also conducted. Bilateral tests were performed, and significance was set at 0.05. The horizontal rectus muscles (medial and lateral rectus) had the highest Vv of muscle and the lowest Vv of collagen. The inferior rectus muscle tended to have a fewer number of fibers per 5,000 µm2 than the rest of the EOM. However, these differences did not reach statistical significance. This is the first published study describing the normal histology of human EOM using stereology. Our investigation, through the quantification of the proportion of muscle and collagen tissue, as well as the number of muscle fibers in 5,000 µ2, establishes normal stereological parameters for EOM of humans without ophthalmological disease.

          Translated abstract

          RESUMEN: El objetivo de este estudio es cuantificar el volumen de tejido muscular y conectivo de los músculos extraoculares en humanos sin enfermedad oftalmológica conocida utilizando estereología. Los músculos extraoculares fueron obtenidos de cinco cadáveres humanos sin estrabismo. El número de fibras musculares en 5.000 µm2 y la densidad de volumen (Vv) de músculo y colágeno fueron medidas usando estereología. Las comparaciones entre músculos extraoculares antagonistas se realizaron a través de la prueba de los rangos con signo de Wilcoxon para muestras pareadas. Un análisis secundario examinando diferencias entre pares de músculos extraoculares también fue llevado a cabo. Se realizaron pruebas bilaterales y la significancia fue fijada en 0,05. Los músculos rectos horizontales (recto medial y lateral) tuvieron el mayor Vv de músculo y el menor Vv de colágeno. El músculo recto inferior tuvo la tendencia a poseer menos número de fibras por 5.000 µm2 que el resto de los músculos extraoculares. No obstante, estas diferencias no llegaron a ser estadísticamente significativas. Este es el primer estudio publicado describiendo la histología normal de los músculos extraoculares usando estereología. Nuestra investigación, a través de la cuantificación de la proporción de tejido de músculo y colágeno, así como el número de fibras musculares en µm2, establece parámetros estereológicos normales para músculos extraoculares en humanos sin enfermedad oftalmológica.

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          Most cited references23

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          Mitochondrial morphology, topology, and membrane interactions in skeletal muscle: a quantitative three-dimensional electron microscopy study.

          Dynamic remodeling of mitochondrial morphology through membrane dynamics are linked to changes in mitochondrial and cellular function. Although mitochondrial membrane fusion/fission events are frequent in cell culture models, whether mitochondrial membranes dynamically interact in postmitotic muscle fibers in vivo remains unclear. Furthermore, a quantitative assessment of mitochondrial morphology in intact muscle is lacking. Here, using electron microscopy (EM), we provide evidence of interacting membranes from adjacent mitochondria in intact mouse skeletal muscle. Electron-dense mitochondrial contact sites consistent with events of outer mitochondrial membrane tethering are also described. These data suggest that mitochondrial membranes interact in vivo among mitochondria, possibly to induce morphology transitions, for kiss-and-run behavior, or other processes involving contact between mitochondrial membranes. Furthermore, a combination of freeze-fracture scanning EM and transmission EM in orthogonal planes was used to characterize and quantify mitochondrial morphology. Two subpopulations of mitochondria were studied: subsarcolemmal (SS) and intermyofibrillar (IMF), which exhibited significant differences in morphological descriptors, including form factor (means ± SD for SS: 1.41 ± 0.45 vs. IMF: 2.89 ± 1.76, P < 0.01) and aspect ratio (1.97 ± 0.83 vs. 3.63 ± 2.13, P < 0.01) and circularity (0.75 ± 0.16 vs. 0.45 ± 0.22, P < 0.01) but not size (0.28 ± 0.31 vs. 0.27 ± 0.20 μm(2)). Frequency distributions for mitochondrial size and morphological parameters were highly skewed, suggesting the presence of mechanisms to influence mitochondrial size and shape. In addition, physical continuities between SS and IMF mitochondria indicated mixing of both subpopulations. These data provide evidence that mitochondrial membranes interact in vivo in mouse skeletal muscle and that factors may be involved in regulating skeletal muscle mitochondrial morphology.
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            Tips for Studies with Quantitative Morphology (Morphometry and Stereology)

            SUMMARY: The quantitative morphology contributes to making the studies less subjective and reproducible. Quantitative results are analyzed with statistics and should be based on a well-defined sample. Quantitative variables can be ‘continuous’ or ‘discrete.' In this text, the term ‘morphometry’ is used to design a simpler procedure for measuring structures with a ‘ruler.' The term ‘stereology’ is used in estimating quantities in the 3-dimensional space analyzing 2-D cut sections. Correction factors may be necessary for the retraction and compression of the tissues. In histological sections, the ‘caps’ of the objects that have been sectioned tangentially are lost when chemical agents remove the paraffin distorting the analysis. Moreover, the analyses based on digital images should consider the same format and the same size (pixels) to all pictures allowing a comparison between groups. Stereology can be ‘model-based’: points within a frame are counted to estimate the ‘volume density’ (Vv) of a structure, and intercepts are counted to assess the ‘surface density’ (Sv). Counting structures within a frame allow estimating the ‘length density’ (Lv). Newer and more complex ‘design-based’ procedures are considered unbiased. The key point is that design-based inference does not require assumptions about the material and uses the ‘random sampling’ approach. The estimation of the number of objects requires a 3-D (volume) probe and therefore the ‘disector’ technique. This review aimed to contribute to the execution of the project, the correct sampling and the data obtained with morphometry and stereology.
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              Altered Protein Composition and Gene Expression in Strabismic Human Extraocular Muscles and Tendons

              Purpose To determine whether structural protein composition and expression of key regulatory genes are altered in strabismic human extraocular muscles. Methods Samples from strabismic horizontal extraocular muscles were obtained during strabismus surgery and compared with normal muscles from organ donors. We used proteomics, standard and customized PCR arrays, and microarrays to identify changes in major structural proteins and changes in gene expression. We focused on muscle and connective tissue and its control by enzymes, growth factors, and cytokines. Results Strabismic muscles showed downregulation of myosins, tropomyosins, troponins, and titin. Expression of collagens and regulators of collagen synthesis and degradation, the collagenase matrix metalloproteinase (MMP)2 and its inhibitors, tissue inhibitor of metalloproteinase (TIMP)1 and TIMP2, was upregulated, along with tumor necrosis factor (TNF), TNF receptors, and connective tissue growth factor (CTGF), as well as proteoglycans. Growth factors controlling extracellular matrix (ECM) were also upregulated. Among 410 signaling genes examined by PCR arrays, molecules with downregulation in the strabismic phenotype included GDNF, NRG1, and PAX7; CTGF, CXCR4, NPY1R, TNF, NTRK1, and NTRK2 were upregulated. Signaling molecules known to control extraocular muscle plasticity were predominantly expressed in the tendon rather than the muscle component. The two horizontal muscles, medial and lateral rectus, displayed similar changes in protein and gene expression, and no obvious effect of age. Conclusions Quantification of proteins and gene expression showed significant differences in the composition of extraocular muscles of strabismic patients with respect to important motor proteins, elements of the ECM, and connective tissue. Therefore, our study supports the emerging view that the molecular composition of strabismic muscles is substantially altered.
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                Author and article information

                Journal
                ijmorphol
                International Journal of Morphology
                Int. J. Morphol.
                Sociedad Chilena de Anatomía (Temuco, , Chile )
                0717-9502
                April 2021
                : 39
                : 2
                : 506-511
                Affiliations
                [1] Santiago de Chile orgnamePontificia Universidad Católica de Chile orgdiv1Facultad de Medicina orgdiv2Departamento de Oftalmología Chile
                [2] Cataluña orgnameUniversitat Autónoma de Barcelona orgdiv1Escuela de Medicina orgdiv2Departamento de Ciencias Morfológicas Spain
                [3] Cataluña orgnameUniversitat Autónoma de Barcelona orgdiv1Escuela de Medicina orgdiv2Departamento de Ciencias Morfológicas Spain
                Article
                S0717-95022021000200506 S0717-9502(21)03900200506
                3d3952a1-2460-4cdd-a7e6-84b357652585

                This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

                History
                : 13 January 2021
                : 18 December 2020
                Page count
                Figures: 0, Tables: 0, Equations: 0, References: 23, Pages: 6
                Product

                SciELO Chile


                Humans,Histología,Anatomía,Oculomotor muscles,Histology,Anatomy,Músculos oculomotores,Humanos

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