Tissue Clearing and Expansion Methods for Imaging Brain Pathology in Neurodegeneration: From Circuits to Synapses and Beyond

Abstract

Studying the structural alterations occurring during diseases of the nervous system requires imaging heterogeneous cell populations at the circuit, cellular and subcellular levels. Recent advancements in brain tissue clearing and expansion methods allow unprecedented detailed imaging of the nervous system through its entire scale, from circuits to synapses, including neurovascular and brain lymphatics elements. Here, we review the state-of-the-art of brain tissue clearing and expansion methods, mentioning their main advantages and limitations, and suggest their parallel implementation for circuits-to-synapses brain imaging using conventional (diffraction-limited) light microscopy -such as confocal, two-photon and light-sheet microscopy- to interrogate the cellular and molecular basis of neurodegenerative diseases. We discuss recent studies in which clearing and expansion methods have been successfully applied to study neuropathological processes in mouse models and postmortem human brain tissue. Volumetric imaging of cleared intact mouse brains and large human brain samples has allowed unbiased assessment of neuropathological hallmarks. In contrast, nanoscale imaging of expanded cells and brain tissue has been used to study the effect of protein aggregates on specific subcellular structures. Therefore, these approaches can be readily applied to study a wide range of brain processes and pathological mechanisms with cellular and subcellular resolution in a time- and cost-efficient manner. We consider that a broader implementation of these technologies is necessary to reveal the full landscape of cellular and molecular mechanisms underlying neurodegenerative diseases.

Keywords: expansion microscopy; neurodegeneration; neuropathology; super-resolution microscopy; tissue clearing.

FIGURE 1

Brain tissue clearing and expansion approaches for neuropathological imaging. (A) Common steps required for whole-brain tissue clearing, imaging, and analysis of neuropathological features, allowing CNS-wide identification of vulnerable circuits and cell types. (B) Tissue expansion approaches can be used to study early pathological alterations caused by abnormal protein conformation, aggregation, and subsequent ultrastructural changes in specific subcellular compartments and organelles within affected cell types and circuits identified using tissue clearing. (C) Diffraction-limited light microscopy of cleared brain tissue allows imaging of large brain circuits at cellular resolution (a–c), including analysis of neuropathological markers (a,b), but it does not reach synaptic nanoscale resolution in non-expanded tissues (e). On the contrary, expansion microscopy of tissues and cells (d,f-i) allows nanoscale imaging using diffraction-limited microscopes, enabling detailed visualization of synapses (e) and other subcellular structures such as nuclear envelope invaginations (g–i) in healthy (h) and ALS pathological conditions (i). (a,b) LSFM images showing individual channels (a) and orthogonal optical planes (b) of a cleared transgenic AD mouse brain after staining of vasculature (yellow), Aβ plaques (Congo red; magenta) and microglia (Iba1; cyan) using the iDISCO protocol; adapted from Liebmann et al. (2016), with permission from Elsevier. (c-f) Epifluorescence images of a Thy1-YFP mouse brain stained with presynaptic (Bassoon, blue) and postsynaptic (Homer1, red) markers, before (c,e) and after (d-f) expansion microscopy; adapted from Chen et al. (2015), with permission from AAAS. (g-i) Confocal microscopy images of expanded human iPSC-derived motor neuron nuclei from healthy control (h) and ALS patient (i) [(g) inset: nuclear size before expansion], immunolabeled for LMNB1 (green) and DNA (blue); adapted from Ortega et al. (2020), with permission from Elsevier.