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.

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 visualizing 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 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.

Conclusions: The effective use of CLARITY on large samples of human tissue opens up many potential avenues for detailed pathological and morphological studies.

Keywords: CLARITY; Lewy body pathology; human post mortem brain; three-dimensional visualization; tissue clearing.

Figure 1

Human cerebellar cortex stained with anti‐neurofilament primary antibody and subsequently AlexaFluor‐568‐conjugated donkey anti‐mouse secondary antibody and imaged using Leica SP5 confocal microscope with a 10× objective, Z‐projections of Z‐stacks 500‐μm thick (z‐stack step size 5 μm), each imaged using the same settings.

Figure 2

Immunofluorescence with neurofilament (NF) staining on human cortical tissue. a: A two‐dimensional image of NF staining showing fine axonal processes and neuronal somas. Scale bar = 50 μm. b: Z‐stack image of NF immunostaining on a 3‐mm block of human cortex with an imaging depth to 771.67 μm (z‐stack step size 5.3 μm).

Figure 3

Z‐stack image of Immunofluorescence with tyrosine hydroxylase (TH) staining on rat coronal block showing TH‐positive neuronal processes at the cortex and dense, homogenous staining within the striatum (z‐stack step size 3.3 μm) (a). Staining in the human midbrain block showed dense TH‐positive axonal processes (z‐stack step size 1.5 μm) (b).

Figure 4

Double immunofluorescence with anti‐neurofilament (a, green) and anti‐Iba‐1 (b, red) antibodies on human cortical block. c: Combined figure showing surveillance network of Iba‐1‐positive microglia around neuronal processes. Scale bar = 50 μm.

Figure 5

An orthogonal projection of a Lewy body‐like inclusion in a block of cleared tissue containing the nucleus basalis of Meynert in human stained with anti‐α SN antibody (green), showing a near‐spherical α SN shell of the inclusion in the XY, XZ and YZ planes (Scale bar = 50 μm) (a). A confocal Image of a Lewy body‐like inclusion in a standard 7‐μm‐thick section containing the nucleus basalis of Meynert stained with single immunofluorescence with anti‐α SN antibody (red) and a nuclear counterstain (DAPI, blue) (Scale bar = 20 μm) (b).

Figure 6

Z‐stack image of double immunofluorescence with anti‐α SN (green) and anti‐TH (red) antibodies on human midbrain block (z‐stack step size 1.5 μm).