Graded traumatic brain injury severity differentially modulates microglial and astrocytic polarization states and response to minocycline.

Abstract

Background: Traumatic brain injury (TBI) involves complex secondary injury cascades in which neuroinflammation is a prominent driver. The lack of standardized models capturing a spectrum of injury severities has hindered a systematic understanding of the associated cellular and molecular responses. This study aims to systematically characterize the dynamic responses and phenotypic shifts of neurons, microglia, and astrocytes during the acute and subacute phases following TBI of varying severities. By integrating macroscopic histopathological assessments with microscopic cellular analyses and correlating these with early peripheral biomarker changes, we seek to provide a solid experimental foundation for understanding TBI mechanisms and developing severity-stratified diagnostic and therapeutic strategies.

Methods: Male mice were randomly assigned using a computer-generated randomization sequence to the following experimental groups: Sham group, mice that underwent only craniotomy (n = 10 per group); Mild group, mice with a 0.5 mm depth impact on the right motor cortex (n = 10 per group); Moderate group, mice with a 1.0 mm depth impact on the right motor cortex (n = 10 per group); Severe group, mice with a 2.0 mm depth impact on the right motor cortex (n = 10 per group). On Days 1, 3, 7, and 14 after injury, tissue damage was assessed using Nissl staining; anxiety-like behavior was evaluated using the elevated plus maze; cognitive function was assessed using the Y-maze test; motor function was evaluated using the open field test, balance beam test, rotarod test, and gait analysis; neuronal apoptosis and glial cell polarization levels were assessed using immunofluorescence staining; and changes in peripheral serum markers were measured using enzyme-linked immunosorbent assay (Elisa). In addition, severely injured mice received minocycline treatment (45 mg/kg, n = 12 per group) from 30 min to Day 3 post-injury, while the control group received the same volume of saline. For comparisons between groups at a single time point, one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test was used. For comparisons between groups across multiple time points, two-way ANOVA followed by Tukey's multiple comparisons test was applied.

Results: Impact depth was directly correlated with histopathological lesion volume and dictated the trajectory of functional recovery. Motor deficits and neuronal apoptosis scaled with injury severity. The neuroimmune response was severity-dependent: mild TBI triggered a transient, reparative response dominated by M2 microglia and A2 astrocytes. In contrast, severe TBI provoked an early and sustained pro-inflammatory state, characterized by persistent M1 microglial and neurotoxic A1 astrocytic activation (n = 4 per group, p < 0.0001). Furthermore, severe injury led to significant acute elevations in serum interleukin-6 (IL-6) and ubiquitin carboxy-terminal hydrolase L1 (UCHL1) within 6 h post-injury (n = 3 per group, p < 0.0001). Minocycline treatment attenuated neuroinflammation, improved motor function, and promoted a shift in microglial polarization toward the protective M2 phenotype (n > 4 per group, all p < 0.05).

Conclusion: Our findings establish that TBI severity is a critical determinant of the post-injury neuroimmune microenvironment, with severe injuries driving a maladaptive, chronic inflammatory response. This graded model provides a robust framework for identifying severity-specific biomarkers and validates the rationale for developing precision immunomodulatory therapies stratified by injury severity.