Optimisation of specimen handling & mitochondrial analysis in patient skeletal muscle biopsies

Introduction: Skeletal muscle biopsies are valuable in pharmacological research for identifying drug targets in muscle-related conditions. Traditional freezing methods cause artefacts that can lead to misinterpretation of research findings (1). Proper handling of muscle biopsies is critical for accurate histopathological and mitochondrial analysis. Preserving the entire tissue is essential, especially for small needle biopsies. While most research focuses on mitochondrial analysis in cells, there are few studies on tissue samples (2). This study aimed to improve cryopreservation techniques for muscle biopsies and create a reliable method for mitochondrial analysis in muscle tissues.
Methods: Human and rat muscle samples were preserved with different concentrations of formaldehyde after freezing with liquid nitrogen to study effects of freeze-thaw cycles. We compared the edge and belly of muscle samples embedded in Optimal Cutting Temperature compound (OCT) to see how OCT affects ice crystal formation. Rat muscle biopsies were frozen using direct liquid nitrogen immersion, liquid nitrogen with OCT dip, liquid nitrogen in a histocassette, pre-cooled isopentane immersion, pre-cooled isopentane with OCT dip, and pre-cooled isopentane in a histocassette. The effectiveness of these six methods was evaluated using histological and immunohistochemical staining. Mitochondrial analysis in type I and II myofibres was attempted by employing the Trainable Weka Segmentation plugin using Fiji.
Results: Picrosirius red-stained human tissue sections showed that freeze-thaw led to freezing artefacts, disrupted endomysium, and widely spaced cells. Quantitative differences in ice crystals between the edge and belly of rat whole muscle samples demonstrated effects of OCT in crystal formation. Picrosirius red and Haematoxylin and Eosin-stained tissue sections from rat muscle biopsies frozen in six different cryopreservation techniques revealed that only isopentane/histocassette combination preserved tissue integrity in both core and periphery of tissue sections. Moreover, an optimized Fiji workflow enabled quantification and mapping of mitochondrial networks.
Conclusion: The isopentane/histocassette combination was the most effective cryopreservation method, ensuring artefact-free preservation of both the core and periphery of the tissue sections. Our workflow utilising Trainable Weka Segmentation plugin provided a reliable method for mitochondrial analysis in skeletal muscle tissues, facilitating future studies in muscle research.

References
1.Meng H, Janssen PML, Grange RW, Yang L, Beggs AH, Swanson LC, Cossette SA, Frase A, Childers MK, Granzier H, Gussoni E, Lawlor MW. Tissue Triage and Freezing for Models of Skeletal Muscle Disease. J Vis Exp. 2014(89). https://dx.doi.org/10.3791/51586.
2.Hemel IMGM, Engelen BPH, Luber N, Gerards M. A Hitchhiker’s Guide to Mitochondrial Quantification. Mitochondrion. 2021;59:216-24. https://doi.org/10.1016/j.mito.2021.06.005.