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Novel sonomicrometry of ex vivo diaphragm after phrenic nerve injury: Role of matrix metalloproteinases

Authors

  • Huan Wang,

    1. Department of Molecular Oncology, Kyoto University Graduate School of Medicine, Kyoto, Japan
    2. Department of Trauma and Acute Critical Care Center, Osaka University Hospital, Osaka, Japan
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    • H.W. and Y.I. contributed equally to this work.

  • Yukio Imamura,

    Corresponding author
    1. Department of Trauma and Acute Critical Care Center, Osaka University Hospital, Osaka, Japan
    2. Unit for Livable Cities, Kyoto University Graduate School of Engineering and Medicine, Kyoto, Japan
    3. Human Health Science, Kyoto University Graduate School of Medicine, Kyoto, Japan
    • Department of Trauma and Acute Critical Care Center, Osaka University Hospital, Osaka, Japan
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    • H.W. and Y.I. contributed equally to this work.

  • Naoya Matsumoto,

    Corresponding author
    1. Department of Trauma and Acute Critical Care Center, Osaka University Hospital, Osaka, Japan
    • Department of Trauma and Acute Critical Care Center, Osaka University Hospital, Osaka, Japan
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  • Hong Wang,

    1. Auditory Section of 2nd Affiliated Hospital, Harbin Medical University, Harbin, China
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  • Hiroshi Ogura,

    1. Department of Trauma and Acute Critical Care Center, Osaka University Hospital, Osaka, Japan
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  • Takeshi Shimazu,

    1. Department of Trauma and Acute Critical Care Center, Osaka University Hospital, Osaka, Japan
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  • Akitoshi Seiyama

    1. Unit for Livable Cities, Kyoto University Graduate School of Engineering and Medicine, Kyoto, Japan
    2. Human Health Science, Kyoto University Graduate School of Medicine, Kyoto, Japan
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Abstract

Extracellular matrix (ECM) proteins and their proteolytic enzymes, matrix metalloproteinases (MMPs), implicate in neuromuscular junctions (NMJs) function during development. However, their pathophysiological mechanisms in the diaphragm remain obscure, because a well-characterized ex vivo experimental system has still been lacking. In the study, we aim to develop a novel ex vivo method of sonomicrometry and evaluate validity of the method with a mouse diaphragm twitch after phrenic nerve injury. In an ex vivo experiment using phrenic nerve-injured mice, diaphragm twitch during electrical pulse stimulation of phrenic nerve was transiently suppressed on day 1. Recombinant MMPs administered in recording solution exerted dose-responsive suppression on the diaphragm twitch in normal mice tissue. Furthermore, gelatinolytic and immunoblot experiments were performed to evaluate MMPs' involvement and NMJs' insults. After nerve injury, (1) in vivo levels of MMPs were transiently upregulated at day 1 and (2) expressions of ECM proteins, agrin (nicotinic acetylcholine receptor stabilizer) and laminin, were transiently reduced at day 1 in the diaphragm. These alterations were cancelled by preinjection of the MMP inhibitor. In conclusion, MMPs hamper NMJ synaptic function in association with the impairment of ECM milieu. Our novel experimental method using ex vivo sonomicrometry is necessary for examining the molecular pathophysiolgy for the dysfunction of NMJs in the diaphragm. Synapse, 2012. © 2012 Wiley Periodicals, Inc.

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