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

Abstract

Aims CLARITY is a novel technique which enables three‐dimensional visualization 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 visualize Lewy pathology in a post mortem Parkinson's brain.MethodsRodent 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 visualizing under a confocal microscope.ResultsWe 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 visualized 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.ConclusionsThe effective use of CLARITY on large samples of human tissue opens up many potential avenues for detailed pathological and morphological studies.

A novel method to visualise the three‐dimensional organisation of the human cerebral cortical vasculature

“…Use of antibodies in small volumes of fresh and fixed human brain tissue has been reported (Liu et al. ), although antibody penetration in large volumes of tissue appears to be challenging. Recent work has focused on the development of methods that allow for repeated rounds of tissue labelling in human and rodent tissue (Murray et al.…”

Free of acrylamide sodium dodecyl sulphate (SDS)‐based tissue clearing (FASTClear): a novel protocol of tissue clearing for three‐dimensional visualization of human brain tissues

“…Also we, and other groups, have reported tissue expansion after tissue clearing with CLARITY . Although it was often claimed that the tissue expansion is a transient effect which will be adjusted by subsequent refractive index matching , the effects in human brain tissues, especially after prolonged (>40 days) passive tissue clearing, appeared to be irreversible . In addition, immunolabelling with antibodies, particularly on larger samples, remains challenging because the depth of antibody penetration is still limited .…”

“…After that, the tissue was washed thoroughly in PBS‐Triton (5 × 1 hr; then overnight) and proceeded to refractive index matching. For tissues that have been rendered optically transparent at the delipidation step, immersion in 47% 2,2′‐thiodiethanol (vol/vol) in 0.01 M PBS without saline or 70% w/v sorbitol in 0.1 M phosphate buffer as previously described was done for refractive index matching. For tissues that did not achieve transparency at the delipidation step (or if microscope objectives are designed for high refractive index solution), they can be dehydrated and refractive index matched as per the 3DISCO protocol .…”

Mapping causal pathways from genetics to neuropsychiatric disorders using genome‐wide imaging genetics: Current status and future directions

“…Imaging many serial 2‐D sections to reconstruct 3‐D images is possible but inefficient, and may distort features within the images. Several tissue‐clearing techniques have recently been developed to address this problem . These techniques remove light‐scattering lipids while preserving cellular spatial arrangements of proteins to yield intact, transparent tissue amenable to light microscopy.…”