Neuroinflammation in Immunodeficient Mice Following Traumatic Brain Injury

Abstract

Background: Traumatic Brain Injury (TBI) is a cause of disability and death worldwide. It is characterized by primary mechanical damage followed by secondary neuroinflammatory damage, that involves oxidative stress and immune activation, which exacerbates the brain tissue injury. The immune system plays an immense role in TBI. Both innate immune responses (mediated by microglia, neutrophils, and kallikrein-kinin system) and adaptive immune responses (mediated by T and B lymphocytes) play crucial roles in neuroinflammation. The role of adaptive immunity in modulating inflammatory responses after TBI has gained attention, with lymphocytes implicated in amplifying inflammatory cascades. The recombination activating gene 1 (RAG1) is essential for developing mature T and B lymphocytes, and knocking out RAG1 results in mice lacking adaptive immune responses. However, the precise role of adaptive immunity in TBI remains unclear. Hypothesis & Aim: We hypothesized that the absence of the RAG1 gene and adaptive immune responses lead to a reduction in neuroinflammatory responses following TBI. To investigate this hypothesis, we aimed to compare key inflammatory responses in RAG1 knockout mice versus wildtype (WT) mice subjected to TBI. Methods: For the purpose of this study, moderate TBI was induced in 48 male/female WT and RAG1-/- mice (n=3/group) using controlled cortical impact (CCI) (4 m/s, 1.5 mm depth, 1s dwell time) targeting the right parietal cortex. Sham controls received craniotomy only. Mice were sacrificed on days 3 or 7 post-injury. Cortical RNA was extracted for RT-qPCR analysis of inflammatory genes and normalized to GAPDH. All procedures were approved by AUB's IACUC and complied with ethical guidelines for animal research. Results: Our study revealed that RAG1 significantly modulates neuroinflammatory responses following TBI in a time- and sex-dependent manner. Both genotypes and sexes showed astrocyte activation (GFAP↑ at D3 and D7). RAG1+/+ mice exhibited sustained neuroinflammation, with prolonged elevation of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, IFN-γ), microglial markers (Galectin-3, CD68), and oxidative enzymes (NOX1&4) through D7, particularly in females. However, RAG1-/- mice demonstrated resolution of these responses. This suggests that adaptive immunity perpetuates secondary injury. Notably, RAG1 was required for anti-inflammatory signaling (IL-10, TGF-β1), since their levels didn’t increase in RAG1-/- females, unlike RAG1+/+ females. Therefore, it has a dual role in both exacerbating early inflammation and facilitating later repair. In addition, sex differences were prominent, with RAG1+/+ females showing delayed but amplified cytokine/KKS activation whereas males had a persistent oxidative stress post-TBI. Conclusion: Our findings demonstrate that adaptive immunity plays a critical and complex role in TBI since it is responsible for both detrimental and reparative processes. Knocking out the RAG1 gene attenuated pathological inflammatory processes and reduced sustained microglial activation, pro-inflammatory cytokine production, and oxidative stress, particularly in females. However, RAG1 deficiency also impaired beneficial adaptive immune functions due to the blunted anti-inflammatory responses, especially in RAG1-/- females. These findings reveal that while RAG1 drives neuroinflammation, it is still essential for anti-inflammatory effects and repair. Therefore, this highlights the dual nature of adaptive immunity in TBI pathophysiology.