AUTHOR=Nesbit Michael , Mamo John C. , Majimbi Maimuna , Lam Virginie , Takechi Ryusuke TITLE=Automated Quantitative Analysis of ex vivo Blood-Brain Barrier Permeability Using Intellesis Machine-Learning JOURNAL=Frontiers in Neuroscience VOLUME=Volume 15 - 2021 YEAR=2021 URL=https://www.frontiersin.org/journals/neuroscience/articles/10.3389/fnins.2021.617221 DOI=10.3389/fnins.2021.617221 ISSN=1662-453X ABSTRACT=Background: An increase in blood brain barrier permeability commonly precedes neuro-inflammation and cognitive impairment in models of dementia. Common methods to estimate capillary permeability have potential confounders, or require laborious and subjective semi-manual analysis New method: Here we used snap frozen mouse and rat brain sections that were double-immunofluorescent labelled for immunoglobulin G (IgG; plasma protein) and laminin-α4 (capillary basement membrane). A Machine Learning Image Analysis program (Zeiss ZEN Intellisis) was trained to recognise and segment laminin-α4 to equivocally identify blood vessels in large sets of images. An IgG subclass based on a thresholdintensity was segmented and quantitated only in extravascular regions. The residual parenchymal IgG fluorescence is indicative of blood-to-brain extravasation of IgG and was accurately quantitated. Results: Automated machine-learning and threshold based segmentation of only parenchymal IgG extravasation accentuates otherwise indistinct capillary permeability, particularly frequent in minor BBB leakage. Comparison with Existing Methods: Large datasets can be processed and analysed quickly and robustly to provide an overview of vascular permeability throughout the brain. All human bias or ambiguity involved in classifying and measuring leakage is removed. Conclusion: Here we describe a fast and precise method of visualising and quantitating BBB permeability in mouse and rat brain tissue, while avoiding the confounding influence of unphysiological conditions such as perfusion and eliminating any human related bias from analysis.