Optimisation of specimen handling and mitochondrial analysis in patient skeletal muscle biopsies for nutritional and ageing research

Introduction: Skeletal muscle biopsies are valuable in research for understanding effects of nutritional interventions on muscle ageing. Traditional freezing methods cause artefacts, leading to misinterpretation of research findings. Moreover, existing mitochondrial analytical workflows focus on cells instead of tissue samples.
Aim: This study aimed to improve cryopreservation techniques for muscle biopsies and create a reliable method for mitochondrial analysis, essential for research on muscle health, nutrition and ageing.
Materials & Methods: Human samples preserved with varying formaldehyde concentrations were evaluated for freeze-thaw effects. Edge and belly of muscles were compared to investigate how Optimal Cutting Temperature compound (OCT) affects ice crystal formation. Six cryopreservation techniques were tested on rat samples using liquid nitrogen and precooled isopentane as freezing media. Each medium involved direct immersion, OCT dip before immersion, and placement in histocassettes before immersion. Effectiveness was evaluated using histological and immunohistochemical staining. Mitochondrial analysis in both myofibre types was attempted using Trainable Weka Segmentation plugin in Fiji.
Results: Histological staining revealed freeze-thaw and freezing artefacts. Quantitative differences in ice crystal artefacts between the edge and belly of rat whole muscle samples demonstrated effects of OCT on crystal formation. Histological staining of rat muscle biopsies revealed that isopentane/histocassette combination best preserved tissue integrity. The optimized Fiji workflow enabled accurate quantification and mapping of mitochondrial networks.
Discussion: The isopentane/histocassette combination ensured artefact-free preservation of entire tissue sections. Our Fiji workflow provided a reliable method for mitochondrial analysis in skeletal muscle tissues, facilitating future studies in muscle health, nutrition and ageing.
Lay Summary: Research on effects of dietary interventions on muscle ageing warrants meticulous handling and analysis to ensure reliability of research findings. We optimised cryopreservation techniques and developed a mitochondrial analysis workflow to achieve this.
References:
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