Alzheimer's disease is a progressive neurodegenerative disease characterised by the accumulation of amyloid-beta plaques. Recent therapeutic strategies involve using monoclonal antibodies to target amyloid-beta for clearance and are considered the first disease-modifying treatments. However, their pharmaceutical efficacy is constrained by poor blood-brain barrier penetrance. Exploiting receptor-mediated transcytosis via the transferrin receptor found on brain endothelial cells offers a mechanism to actively shuttle targeted therapeutics from the blood into the brain parenchyma. This project aimed to establish a three-dimensional volumetric imaging pipeline to study the penetrance and localisation of these therapeutics as well as Alzheimer’s disease pathology. This involved combining tissue clearing with light-sheet fluorescence microscopy to quantify amyloid-beta pathology and attempting to evaluate the spatial distribution of transferrin-targeted therapeutics in an Alzheimer’s transgenic mouse model.

Tissue clearing is the process by which animal tissue is made optically transparent. This can be achieved using organic solvents to delipidate tissue but the process is slow and delicate epitopes may not survive. A shortened method which instead uses brain sections to validate epitope survival and antibody compatibility was developed and tested, allowing for faster screening of different labelling approaches. Subsequently, a humanised version of the murine bispecific antibody (rmAb158-scFv8D3) targeting both the transferrin receptor and amyloid-beta was injected in Alzheimer’s model mice and visualised using a labelled secondary antibody. Whole-hemisphere light-sheet volumetric analysis was able to resolve age-dependent expansion of amyloid-beta plaque volume in males between 6.5 and 10 months. Furthermore, the pipeline proved sensitive to sex-based differences, detecting accelerated pathological exacerbation in female animals, and successfully established the framework for visualisation of markers critical to the study of Alzheimer’s disease.

However, definitive ex vivo tracking of the therapeutic’s parenchymal penetrance was precluded by suspected secondary-antibody cross-reactivity. Furthermore, a significant bottleneck in 3D data analysis limited quantification to 1 mm3 cropped regions in the cortex. Ultimately, this light-sheet fluorescence microscopy pipeline establishes a robust, high-resolution framework for quantifying endogenous Alzheimer’s pathology, laying the critical methodological foundation required for future spatial evaluations of blood-brain barrier-crossing immunotherapies.