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

Abstract

Current tissue-clearing protocols for imaging in three dimensions (3D) are typically applied to optimally fixed, small-volume rodent brain tissue - which is not representative of the tissue found in diagnostic neuropathology laboratories. We present a method to visualise the cerebral cortical vasculature in 3D in human post-mortem brain tissue which had been preserved in formalin for many years. Tissue blocks of cerebral cortex from two control cases, two Alzheimer's brains and two cases from Alzheimer's patients immunised against Aβ were stained with fluorescent Lycopersicon esculentum agglutinin (Tomato lectin), dehydrated and cleared using an adapted three-dimensional imaging of solvent cleared organs (3DISCO) protocol to visualise the vascular endothelium. Tissue was imaged using light sheet and confocal microscopy and reconstructed in 3D using amira software. The method permits visualisation of the arrangement of the parallel penetrating cortical vasculature in the human brain. The presence of four vascular features including anastomosis, U-shaped vessels, spiralling and loops were revealed. In summary, we present a low cost and simple method to visualise the human cerebral vasculature in 3D compatible with prolonged fixation times (years), allowing study of vascular involvement in a range of normative and pathological states.

Advanced high resolution three-dimensional imaging to visualize the cerebral neurovascular network in stroke

“…The vasculature of the brain can also take full advantage of LSM through dye filling strategies, such as the special shape of cortical microvessels [99], and changes in blood vessels after cerebral ischemia [100]. The introduction of tissue removal provides three-dimensional imaging of the vasculature.…”

Optical Tissue Clearing: Illuminating Brain Function and Dysfunction

“…In the future, more innovative research based on the combination of optical clearing methods and in vivo imaging techniques are expected, and the depth of in vivo imaging is expected to increase further. As for human samples, Harrison et al reported a novel method based on 3DISCO clearing and fluorescent Lycopersicon esculentum agglutinin labeling to visualize the cerebral cortical vasculature from the long-fixed human brain 65 . This method permits the visualization of the parallel arrangement of the vasculature in the cortex and further assesses the physiological or pathological brain states 65 .…”

“…As for human samples, Harrison et al reported a novel method based on 3DISCO clearing and fluorescent Lycopersicon esculentum agglutinin labeling to visualize the cerebral cortical vasculature from the long-fixed human brain 65 . This method permits the visualization of the parallel arrangement of the vasculature in the cortex and further assesses the physiological or pathological brain states 65 . Additionally, optical clearance of the human dura mater is a potential method to aid the post-mortem investigation of human head injury since dissection of dura mater after skull removal may introduce other damage of brain samples 66 .…”

Optimizing tissue clearing and imaging methods for human brain tissue

“…In recent years, there have been substantial improvements in the techniques used to visualize rodent brains in three dimensions (3D) without the use of tissue sectioning. 1 – 7 These techniques include various tissue-clearing technologies as well as advances in confocal microscopy that allow images to be taken from deep inside the brain. 2 , 8 – 10 For example, iDISCO+ (immunolabeling-enabled three-dimensional imaging of solvent-cleared organs) has been used to image the vasculature of the adult mouse brain, 11 FDISCO (three-dimensional imaging of solvent-cleared organs with superior fluorescence-preserving capability) has been used to visualize neurons with weak fluorescence labeling in the whole mouse brain, 12 and MACS (m‐xylylenediamine-based aqueous clearing system) has enabled the clearing of whole mouse brains with robust lipophilic dye compatibility.…”