Determining regions of altered brain physiology after diffuse brain injury is challenging. Microglia, brain immune cells with ramified and dynamically moving processes, constantly surveil the parenchyma for dysfunction which, when present, results in a changed morphology. Our purpose was to define the spatiotemporal changes in microglia morphology over 28 days following rat midline fluid percussion injury (mFPI) as a first step in exploiting microglia morphology to reflect altered brain physiology. Microglia morphology was quantified from histological sections using Image J skeleton and fractal analysis procedures at three time points and in three regions post-mFPI: impact site, primary somatosensory cortex barrel field (S1BF), and a remote region. Microglia ramification (process length/cell and endpoints/cell) decreased in the impact and S1BF but not the remote region (p < 0.05). Microglia complexity was decreased in the S1BF (p = 0.003) and increased in the remote region (p < 0.02). Rod-shaped microglia were present in the S1BF and had a 1.8:1.0 length:width ratio. An in-depth quantitative morphologic analysis revealed diverse and widespread changes to microglia morphology in the cortex post-mFPI. Due to their close link to neuronal function, changes in microglia morphology, summarized in this study, likely reflect altered physiology with diverse and widespread impact on neuronal and circuit function.

Neuropathology after traumatic brain injury (TBI) is the result of both the immediate impact injury and secondary injury mechanisms. Unresolved post-traumatic glial activation is a secondary injury mechanism that contributes to a chronic state of neuroinflammation in both animal models of TBI and human head injury patients. We recently demonstrated, usingin vitromodels, that p38α MAPK signaling in microglia is a key event in promoting cytokine production in response to diverse disease-relevant stressors and subsequent inflammatory neuronal dysfunction. From these findings, we hypothesized that the p38α signaling pathway in microglia could be contributing to the secondary neuropathologic sequelae after a diffuse TBI. Mice where microglia were p38α-deficient (p38α KO) were protected against TBI-induced motor deficits and synaptic protein loss. In wild-type (WT) mice, diffuse TBI produced microglia morphological activation that lasted for at least 7 d; however, p38α KO mice failed to activate this response. Unexpectedly, we found that the peak of the early, acute phase cytokine and chemokine levels was increased in injured p38α KO mice compared with injured WT mice. The increased cytokine levels in the p38α KO mice could not be accounted for by more infiltration of macrophages or neutrophils, or increased astrogliosis. By 7 d after injury, the cytokine and chemokine levels remained elevated in injured WT mice but not in p38α KO mice. Together, these data suggest that p38α balances the inflammatory response by acutely attenuating the early proinflammatory cytokine surge while perpetuating the chronic microglia activation after TBI.

Microglia are specialized immune cells unique to the central nervous system (CNS). Microglia have a highly plastic morphology that changes rapidly in response to injury or infection. Qualitative and quantitative measurements of ever-changing microglial morphology are considered a cornerstone of many microglia-centric research studies. The distinctive morphological variations seen in microglia are a useful marker of inflammation and severity of tissue damage. Although a wide array of damage-associated microglial morphologies has been documented, the exact functions of these distinct morphologies are not fully understood. In this review, we discuss how microglia morphology is not synonymous with microglia function, however, morphological outcomes can be used to make inferences about microglial function. For a comprehensive examination of the reactive status of a microglial cell, both histological and genetic approaches should be combined. However, the importance of quality immunohistochemistry-based analyses should not be overlooked as they can succinctly answer many research questions.

Microglial morphology is used to measure neuroinflammation and pathology. For reliable inference, it is critical that microglial morphology is accurately quantified and that results can be easily interpreted and compared across studies and laboratories. The process through which microglial morphology is quantified is a key methodological choice and little is known about how this choice may bias conclusions. We applied five of the most commonly used ImageJ-based methods for quantifying the microglial morphological response to a stimulus to identical photomicrographs and individual microglial cells isolated from these photomicrographs, which allowed for direct comparisons of results generated using these approaches. We found a lack of comparability across methods that analyzed full photomicrographs, with significant discrepancies in results among the five methods. Quantitative methods to analyze microglial morphology should be selected based on several criteria, and combinations of these methods may give the most biologically accurate representation of microglial morphology.