Abstract Body: Introduction: Major neurological disability from ischemic stroke is due to white matter injury (WMI) which varies by age and sex. In ischemic gray matter, the dual role of microglia in exacerbating injury or promoting neuroprotection has been well-reported. Previous reports also have identified a subset of dystrophic microglia found in human and rodent models with neurological diseases. However, the role of microglia in WMI remains to be investigated. We described the microglial dynamics in WMI by characterizing the morphological characteristics in response to ischemic injury as a function of age and sex.
Method: We used isolated mouse optic nerves, a pure white matter tract, from 3-,12-, and 24-month-old male and female Cx3Cr1-GFP mice to capture the microglia morphological changes during an ex vivo WM ischemic model. Live cell images (z-stack of 10µm total thickness) were captured for 5 minutes every 20 minutes during baseline, 1 hour of oxygen-glucose deprivation (OGD), and 1 hour of recovery. Images were processed and scored for 8 morphological characteristics using ImageJ (NIH) with the fractal analysis (FracLac) plugins. Microglial characteristics were separated by sex and age, analyzed using principal component analysis, and grouped by Euclidean dendrogram and cluster mapping in R. Additionally, the tissues were processed for immunohistochemical labeling for dystrophic microglia using plin2, h2a.x, and 4HNE antibodies. For dark microglia, tissues were processed for ultrastructure EM and immunohistochemical labeling with FTL antibody.
Result: We observed significant differences in microglial morphology at baseline between age groups. Furthermore, three distinct clusters were identified as activated, unresponsive, and dystrophic based on morphological changes during OGD. Compared to young male (YM) microglia, a higher percentage of aged male (AM) microglia are classified as activated or dystrophic after OGD. YM-activated and dystrophic microglia had greater structural variance between time points than AM microglia. Interestingly, we also observed sex differences in the distribution of microglia sub-types at baseline and after OGD.
Conclusion: We highlighted an unbiased method to define microglia behavioral subtypes during acute OGD in WM. Our findings indicate microglia vary in activation and resiliency during OGD as a function of age and sex. We proposed that dystrophic microglia play a crucial role in WMI and warrant further investigation.