Standardised TruAI automated quantification of intracellular neuromelanin granules in human brain tissue sections

Abstract

AimsTo standardise and automate the quantitation of human-unique neuromelanin granules in catecholamine neurons in post-mortem tissue sections from healthy individuals at different ages to understand any changes in these granules with age. Methods5-6 {micro}m-thick fixed and paraffin-embedded transverse midbrain tissue sections were supplied from 47 cases from three brain banks following ethical approvals. Sections were prepared and automated digital images acquired. Standardisation and automation of the quantification of neuromelanin granules was performed using the TruAI feature of the Olympus VS200 desktop platform. Comparisons between stained and unstained sections as well as correlations with age were performed. ResultsThe automated platform reliably identified both stained and unstained intracellular neuromelanin granules and extracellular pigments, showing high reproducibility in measurements across laboratories using different tissue processing methods. Extraneuronal pigments were significantly smaller than intracellular neuromelanin granules. Sections processed for haematoxylin and eosin staining impacted the size and colour of both neuromelanin and the neurons containing neuromelanin. Haematoxylin made neuromelanin bluer, and the increased tissue processing made the intracellular area occupied by neuromelanin smaller in younger people. There was an increase in neuromelanin optical density and colour change (browner) with age. ConclusionsThe TruAI automated platform reliably quantifies individual neuromelanin granules in catecholamine neurons. Extraneuronal pigments are considerably smaller in size than intracellular neuromelanin, and intracellular neuromelanin changes its properties with age. The darkening and colour change of intracellular neuromelanin suggests an increase in eumelanin over time in healthy individuals. These changes can be reliably identified using the automated platform. Key pointsO_LIStandardised, reliable TruAI automated quantitation of intracellular neuromelanin granules in human brain sections gives novel insights into their structure and function C_LIO_LIExtraneuronal pigments, including neuromelanin granules released from dying pigmented neurons, are significantly smaller than intracellular neuromelanin granules C_LIO_LIIntracellular neuromelanin granules change their properties with healthy ageing becoming darker, consistent with more antioxidant eumelanin C_LI