A Semi-Automated and Unbiased Microglia Morphology Analysis Following Mild Traumatic Brain Injury in Rats.

Mild traumatic brain injury (mTBI) affects over 40 million people every year. One of its features includes the activation of microglia, the resident immune cells of the brain. Microglia assume different morphological states depending on their level of activation, such as surveilling ramified and activated hypertrophic, ameboid, and rod-like microglia. These states can be distinguished by multiple features, including the shape, span, and branching of microglia. Male Sprague-Dawley rats sustained mTBI using the Closed-Head Impact Model of Engineered Rotational Acceleration (CHIMERA) (3 times, 1.5 J per impact) or sham treatment. Four days after the injury, brains were collected and stained for microglia using the ionized calcium-binding adapter molecule-1 (Iba-1) antibody. Cortical injury sites were identified in a subset of CHIMERA animals. Using the MicrogliaMorphology ImageJ plugin and the MicrogliaMorphologyR package, 27 morphological features were quantified from individual microglia, and k-means clustering was used to classify microglia as ramified, rod-like, ameboid, and hypertrophic states. The CHIMERA injury altered microglia morphology features, which contributed to increased hypertrophic (activated) and decreased ramified (inactive) microglia compared to the sham controls. Combined with the clinically relevant mTBI paradigm and semi-automated/unbiased approach, the current findings may contribute to microglia morphology classification.