Next generation histology pipeline for three-dimensional visualisation of archival chronic traumatic encephalopathy brain tissues

Abstract

Aims: There are currently many techniques available for the three-dimensional visualisation of cleared tissues but none have been optimised for use on human brain tissues. In particular, there have been great difficulties in the processing and visualisation of tissues with prolonged formalin fixation, such as the historic chronic traumatic encephalopathies (CTE) brain tissues from the Corsellis Collection. We aimed to develop a three-dimensional histology protocol that was optimised for human brain materials. We also aimed to explore different labelling strategies for the human brain, including chemical staining, immunostaining and retrograde tracers to visualise vascular and glial pathology in three-dimensions. Method: Formalin-fixed tissue (including archival CTE tissues with over 50 years in fixative) were processed and immunostained for glial fibrillary acidic protein (GFAP) using FASTClear. In addition, chemical staining with lectin and Nissl, as well as retrograde tracer staining with DiI were performed. Results: We have successfully simplified the process of tissue clearing for archival material and achieved immunostaining of glial cells down to a depth of 300 µm. This has enabled us to visualise the blood-brain-barrier of CTE tissue in three-dimensions. Combining with a novel refractive index-matching reagent, OPTIClear, we were also able to perform chemical staining for neurons and vasculature in formalin-fixed tissues. Finally, we have visualised dendritic spines and mossy fibre rosettes in the human cerebellum using DiI retrograde labelling. Conclusion: We have succeeded in developing a next generation three-dimensional histology pipeline for human formalin-fixed tissue, which will hopefully free up archival materials for future tissue clearing research.