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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 labeled for immunoglobulin G (IgG; plasma protein) and laminin-α4 (capillary basement membrane). A Machine Learning Image Analysis program (Zeiss ZEN Intellisis) was trained to recognize and segment laminin-α4 to equivocally identify blood vessels in large sets of images. An IgG subclass based on a threshold intensity 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 analyzed 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 visualizing 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.

Automated Quantitative Analysis of ex vivo Blood-Brain Barrier Permeability Using Intellesis Machine-Learning