Abstract
Fragility fractures caused by osteoporosis affect millions of people worldwide every year with significant levels of associated morbidity, mortality and costs to the healthcare economy. The pathogenesis of declining bone mineral density is poorly understood but it is inherently related to increasing age. Growing evidence in recent years, especially that provided by mouse models, suggest that accumulating somatic mitochondrial DNA mutations may cause the phenotypic changes associated with the ageing process including osteoporosis. Methods to study mitochondrial abnormalities in individual osteoblasts, osteoclasts and osteocytes are limited and impair our ability to assess the changes seen with age and in animal models of ageing. To enable the assessment of mitochondrial protein levels, we have developed a quadruple immunofluorescence method to accurately quantify the presence of mitochondrial respiratory chain components within individual bone cells. We have applied this technique to a well-established mouse model of ageing and osteoporosis and show respiratory chain deficiency.
Highlights
Have documented whether respiratory chain deficiency associated with ageing occurs in bone cells, likely due to the absence of a reliable method of assessment
c oxidase (COX)/succinate dehydrogenase (SDH) histochemistry is applied to unfixed, frozen tissue sections and results in variable staining intensity depending on the degree of deficiency of the targeted respiratory chain components
In our endeavours to characterise respiratory chain dysfunction at a cellular level in bone tissue, we found the application of COX/SDH histochemistry to frozen mouse bone to be a challenging method due to loss of tissue morphology and difficulty in identifying individual cell types
Summary
Have documented whether respiratory chain deficiency associated with ageing occurs in bone cells, likely due to the absence of a reliable method of assessment. Assessment of mitochondrial respiratory chain dysfunction in individual cells in other tissues such as skeletal muscle, myocardium, brain and intestine is well documented. In the presence of COX, tissue stains brown and when completely deficient, only the blue SDH staining is visible Quantification using this method is challenging with notable inter- and intra-observer variability in classifying the fibres by colour intensity[27]. In our endeavours to characterise respiratory chain dysfunction at a cellular level in bone tissue, we found the application of COX/SDH histochemistry to frozen mouse bone to be a challenging method due to loss of tissue morphology and difficulty in identifying individual cell types. We have adapted a method developed for the assessment of mitochondrial respiratory chain components in muscle tissue sections[28] for use in bone tissue sections. This new method utilises a quadruple immunofluorescence technique that enables the simultaneous targeting of a cell marker and multiple mitochondrial epitopes, such that the respiratory chain protein expression of complexes I and IV can be quantified relative to mitochondrial mass in specific bone cell subtypes
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