Simultaneous, real‐time assessment of mitochondrial function and archiving of multiple organs from a single mouse
Authors: Mar Curtis T. , Boddu Aayush , Howell Sarah A. , Mehta Aditya J. , Zemljic‐Harpf Alice E. , Schilling Jan M. 
Published: June 2021

Introduction

Many disease models utilizing mice involve multiple organs that, if analyzed and assayed in conjunction, could lead to a more complete and insightful understanding of disease. Many barriers exist to real‐time assays in multiple organs. Mitochondria are key to energy homeostasis and survival/death signaling and serve as end effectors of (dys)function in many organs. We attempted to develop a multi‐stage flow protocol for simultaneous assessment of mitochondrial respiration in six organs in conjunction with archiving parallel samples for end point analysis (i.e., RNA, protein, histology) from a single mouse.

Methods

Using the Oroboros O2k oxygraph for high‐resolution respirometry, the rate of oxygen consumption of tissues and cells can be measured. We initially optimized protocols for oxygen consumption in several murine organs individually (heart, skeletal muscle, hippocampus, liver, kidney, and lungs). We optimized tissue preparations to use mechanically and chemically isolated fibers (skeletal muscle), mechanically permeabilized fibers (heart), mechanically permeabilized tissue (liver, kidney, and lungs), and whole tissue homogenate (hippocampus) to assess oxygen flux. In parallel, multiple different organs were harvested for further biochemical and histological analysis. With optimized protocols for respiration and tissue preparation, we performed a substrate‐uncoupler‐inhibitor titration protocol utilizing ETS complex substrates (CI ‐ malate, glutamate; CII – succinate; CIV ‐ ascorbic acid/TMPD) and inhibitors (CI – rotenone; CIII ‐ antimycin A) on six organs simultaneously from a single mouse. Cytochrome c was used to test outer mitochondrial membrane integrity.

Results

Various organs utilized different amounts of oxygen normalized to tissue weight during combined complex I & II respiration [oxygen flux in pmol O2/(s*mg), N=3, Mean±SEM; heart (1260.5±284.6) > kidney (439.5±58.2) > skeletal muscle (211.3±17.8) > liver (112.0±13.8) > hippocampus (93.5±3.0) > lung (26.4±3.8)]. Cytochrome c response was highest in liver tissue indicating possible outer mitochondrial membrane damage as result of the preparation. Additional tissue collected was archived for further processing.

Conclusion

Assessing oxygen flux in parallel in primary preparations of several organs from one organism is possible within a short amount of time with good quality preparations utilizing our flow‐protocol for respiration and tissue preparation. Simultaneous preparations of multiple different organs from one organism could help to establish a metabolic organismal phenotype under various disease conditions, after drug treatment, and during toxicology studies leading to a more global understanding of how multiple organs may be impacted in certain models.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.