Bringing CLARITY to the human brain: visualization of Lewy pathology in three dimensions

Abstract

Aims: CLARITY is a novel technique which enables three-dimensional visualisation of immunostained tissue for the study of circuitry and spatial interactions between cells and molecules in the brain. In this study we aimed to compare methodological differences in the application of CLARITY between rodent and large human post-mortem brain samples. In addition, we aimed to investigate if this technique could be used to visualise Lewy pathology in a post-mortem Parkinson’s brain. Methods: Rodent and human brain samples were clarified and immunostained using the passive version of the CLARITY technique. Samples were then immersed in different refractive index matching media before mounting and visualising under a confocal microscope. Results: We found that tissue clearing speed using passive CLARITY differs according to species (human vs rodents), brain region and degree of fixation (fresh vs formalin-fixed tissues). Furthermore, there were advantages to using specific refractive index matching media. We have applied this technique and have successfully visualised Lewy body inclusions in three dimensions within the nucleus basalis of Meynert, and the spatial relationship between monoaminergic fibres and Lewy pathologies among nigrostriatal fibres in the midbrain without the need for physical serial sectioning of brain tissue. Conclusions: The effective use of CLARITY on large samples of human tissue opens up many potential avenues for detailed pathological and morphological studies.