Poster K12 - The Impact of Injury Severity and Subconcussive Preconditioning on Microglia Morphology

Saturday, October 26, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Kyulee Kim

Undergraduate Researcher
University of Pennsylvania
Philadelphia, Pennsylvania, United States

Co-Author(s)

EA

Erin Anderson

PhD Candidate
University of Pennsylvania, United States

Primary Investigator(s)

DM

David Meaney, Dr.

Principal Investigator
University of Pennsylvania, United States

Introduction: Microglia, the resident immune cells of the central nervous system, are critically important in the brain’s response to head trauma. Microglia serve a variety of roles, including surveillance, coagulation, and phagocytosis, and change their morphology and localization to accommodate these roles. For example, they may become amoeboid for phagocytosis or hyper-ramified for increased surveillance. Furthermore, these morphological changes scale with injury severity. Detailed microglial morphological analysis enhances our understanding of injury progression and brain recovery.

Repeated mild microglial stimulation has been shown to either potentiate or ameliorate the subsequent immune response to a larger microglia stimulus. Subconcussive impacts have been shown to activate microglia in the absence of cognitive deficits, potentially serving as a form of mild microglial stimulation. We asked if repeated exposure to subconcussive impacts could stimulate microglia and modify their response to concussion.This study aims to 1) investigate how injury severity affects microglia and 2) whether the combination of repetitive subconcussive and concussive impacts produces different microglial responses compared to either impact alone.

Materials and

Methods: To deliver a precisely-controlled diffuse injury, we subjected mice to closed-head controlled cortical impacts (CCI). We used a velocity of 3.5m/s for a concussive impact and a velocity of 1m/s for a subconcussive impact. We divided the mice into four experimental conditions: 1) Sham, 2) Single Subconcussion, 3) Single Concussion, and 4) Preconditioned Concussion - two subconcussive impacts followed by one concussive impact, spaced 1 minute apart.

We perfused mice for immunohistochemistry at 9 days post-injury (DPI). We prepared 20um thick coronal brain slices under the injury site and analyzed three sections per brain from three brains per condition (9 total). We identified microglia using anti-Iba1 and acquired images using the Keyence BZ-X800 microscope at 20X magnification and processed images using ImageJ.

We evaluated microglia in 2mm² areas of the ipsilateral and contralateral cortices. We used custom ImageJ macros to assess microglia density and perform Sholl analysis on 30 randomly selected microglia per section. For Sholl analysis, we manually outlined cell somas and traced the branches to evaluate the branching patterns and degree of ramification in the microglia.

Results, Conclusions, and Discussions: We found no significant differences in microglia density (count/mm²) across the conditions in either the ipsilateral or contralateral cortex (Figure 2). However, there were significant differences in standard error of means between sham vs. subconcussive group (p = 0.0494) and sham vs. preconditioned (p= 0.0549), suggesting variable microglial number and/or localization following head injury regardless of severity.

The Sholl analysis of microglia revealed no significant differences between the sham and injury conditions in either the ipsilateral or contralateral cortex (Figure 3). In Figure 3(a) and 3(b), the sham group is noticeably less ramified than injured groups for both ipsilateral and contralateral cortex.

Uninjured brains showed some differences compared to the injured groups. The three injury groups (subconcussive, concussive, and preconditioned) had more variable microglia density than the sham group, indicating that injury, regardless of scale, induces microglial differences, possibly due to varying recovery speeds.

Sholl analysis revealed no significant effect of injury intensity (subconcussive vs. concussive) on microglial morphological changes 9 days post-injury, suggesting that injury scale does not solely determine microglial changes. This highlights the need for additional time points before 9 days to fully understand the microglial response.

Despite low subconcussive preconditioning not showing significant microglial morphological differences at 9 days post-injury, all injury groups had similar results. This raises the question of whether microglial morphology recovers and converges over time across injury groups, warranting investigation at an acute time point of 1 DPI.

Although these results point to similar microglial responses to disparate injury severity, it is possible that we would observe different pathology in other regions at a more acute time point. To that end, we are examining microglia in the hippocampus at 9dpi and microglia in the cortex and hippocampus at 1 dpi to understand whether the acute response mirrors the chronic response and whether the hippocampus responds similarly to the cortex. Understanding the time course and localization of microglial pathology will give us greater insight into how the brain’s immune system responds to varying severities and combinations of head injury to allow us to better tailor treatments and monitor recovery.

Acknowledgements (Optional):