Measuring the Effects of Pharmacological Compounds on Mitochondrial Morphology in Mouse Aging Models

Aging-related molecular damage accumulation can contribute to changes in mitochondrial morphology and metabolic dysfunction, particularly in tissues with high energy expenditures. Pharmacological compounds that ameliorate metabolic dysfunction and restore healthy, 'young' mitochondrial morphologies may thus represent effective modalities to extend healthspan (i.e., the portion of one's lifespan free from the burden of aging-related chronic disease) in mammals. Herein, methods for performing subcutaneous osmotic pump implantations in aged mice for the stable delivery of hydrophobic pharmacological compounds are described. Then, tissues are fixed, cryosectioned, stained, and imaged using super-resolution confocal light microscopy. Finally, an automated pipeline for the three-dimensional segmentation, classification, and quantitative measurement of mitochondrial structures is presented. Thus, these methods may be useful for quantitatively assessing changes in mitochondrial three-dimensional structure in different tissues across the mouse lifespan. Moreover, these techniques may aid in the reproducible identification of small-molecule drugs that mitigate aging-related metabolic dysfunction.