Multiple-particle tracking (MPT) is a microscopy technique capable of simultaneously tracking hundreds to thousands of nanoparticles in a biological sample and has been used extensively to characterize biological microenvironments, including the brain extracellular space (ECS). Machine learning techniques have been applied to MPT data sets to predict the diffusion mode of nanoparticle trajectories as well as more complex biological variables, such as biological age. In this study, we develop a machine learning pipeline to predict and investigate changes to the brain ECS due to injury using supervised classification and feature importance calculations. We first validate the pipeline on three related but distinct MPT data sets from the living brain ECSâage differences, region differences, and enzymatic degradation of ECS structure. We predict three ages with 86% accuracy, three regions with 90% accuracy, and healthy versus enzyme-treated tissue with 69% accuracy. Since injury across groups is normally compared with traditional statistical approaches, we first used linear mixed effects models to compare features between healthy control conditions and injury induced by two different oxygen glucose deprivation (1) exposure times. We then used machine learning to predict injury state using MPT features. We show that the pipeline predicts between the healthy control, 0.5 h OGD treatment, and 1.5 h OGD treatment with 59% accuracy in the cortex and 66% in the striatum, and identifies nonlinear relationships between trajectory features that were not evident from traditional linear models. Our work demonstrates that machine learning applied to MPT data is effective across multiple experimental conditions and can find unique biologically relevant features of nanoparticle diffusion.