Methods for In situ Quantification of Mitochondrial Morphology In Muscle and Terminal Schwann Cells of Mice

Abstract

Mitochondrial dysfunction is well described in many chronic illnesses including musculoskeletal, neurodegenerative, and cardiovascular diseases. Mitochondrial network morphology has been implicated as a biomarker of disease, correlating increased mitochondrial fragmentation to impaired cellular function. While advancements in imaging techniques further our understanding of mitochondrial dynamics in live cells, easily accessible approaches for accurate quantification of in situ mitochondrial networks in low abundance tissues are lacking. The purpose of this study was to validate a proof-of-concept method capable of quantifying 3D mitochondrial network morphology in whole mount skeletal muscle and then applying it to mitochondrial morphology analysis in cell types otherwise difficult to image within their native environment, terminal Schwann cells (tSCs). Herein, we report that mitochondrial networks were fragmented in dystrophic mouse muscle compared to healthy controls, as observed by others, and correlated with muscle pathology as expected. Using S100{beta} reporter mice to identify Schwann cells, we labeled tSC mitochondrial networks in vivo prior to rapid imaging in situ with high-resolution confocal microscopy. Moreover, these methods offer a comprehensive and novel approach enabling the quantification of mitochondria network morphology across multiple cell types (like muscle fibers and tSCs) using standard microscopy available in university core facilities. SummaryLocal injections of mitochondrial dye are used to label terminal Schwann cells for confocal microscopy imaging after proof of concept was demonstrated in skeletal muscle tissue from mice with healthy or diseased muscle.