Understanding biological sex differences in the glial response to neuropathology after a traumatic brain injury

Traumatic brain injury (TBI) triggers neuroinflammatory cascades primarily mediated by glia, including microglia and astrocytes. Despite intense investigations, the role of neuroinflammation in TBI pathophysiology remains unclear. It is postulated that the glial response promotes tissue repair in the short-term and may exacerbate neuropathology and symptoms in the months to years post-injury. Glia have been reported to exhibit sexually dimorphic roles in the developing, adult, and aged brain. Further, these dimorphic roles are begging to be reported in neurological conditions such as TBI. Therefore, the aim of this research was to examine the glial response across time following diffuse TBI in both males and females and whether this is dependent upon neuropathology.
The glial response in the early time period post-TBI, is theorised to be triggered by neuropathology. Therefore, this study examined microglia and astrocytes post-injury in a model of altered neuronal cytoskeleton, where mice were genetically engineered to be neurofilament knock outs (NFL-KO). Breakdown of the axonal cytoskeleton is a key component in TBI-induced neuropathology; therefore, we hypothesise NFL-KO mice would exhibit altered neuropathology and gliosis after TBI. Male and female NFL-KO and wild-type (WT) mice were subjected to midline fluid percussion injury (mFPI). Brains were collected at 3 hours, 1 day or 3 days post-injury and compared with naïve controls. All brain tissue was prepared for immunohistochemical analysis. The number of APP-positive (APP+) profiles (an indicator of axon pathology) was significantly increased across time post-injury throughout the brain of male and female WT and NFL-KO mice. Microglial morphological changes were evident as early as 3 hours post-injury where cells appeared hypertrophic (increased cell volume) with hyper-ramified processes (increased branch points). This was followed by an increase in the density of microglia and colocalisation with a surrogate marker of phagocytosis (CD68) at 3 days post-injury in WT and NFL-KO mice compared to naïve. Furthermore, astrocyte (GFAP) immunoreactivity was significantly increased across time post-injury in male and female WT and NFL-KO mice that was accompanied by astrocyte morphological changes including retracted processes (reduced branch length). In injured NFL-KO mice, the extent of APP+ neuropathology and microgliosis was exacerbated at all timepoints post-injury compared with WT, however, the astrocyte response was reduced. Biological sex differences were also evident in this study where APP+ neuropathology and gliosis were more robust in male mice, particularly at acute timepoints post-injury (3 hours at 1 day), compared with females. These results show that in the early stages following TBI, neuropathology and gliosis was evident to a greater extent in males compared females and that the structure of the axonal cytoskeleton and glial response are important in the sequelae of TBI pathophysiology in both biological sexes.
Next, how microglia and astrocytes respond to TBI in a model of altered neurodegeneration was examined. The sterile alpha and TIR motif containing 1 (SARM1) protein has been identified as a key regulator of delayed neurodegeneration following TBI. Therefore, we hypothesised that the inhibition of SARM1 would prevent glial reactivity and neurodegeneration in the delayed time period post-TBI and could be targeted for therapeutic intervention. TBI was modelled in WT and SARM1 knock-out (SARM1-KO) mice of both biological sexes by mFPI with the aim of investigating neuropathology and glial reactivity post-injury. At 7- or 28-days post-injury, blood and brains were collected and compared to naïve controls. Neurodegeneration and glial reactivity were examined via SIMOA and immunohistochemical analysis, respectively. The density of microglia and GFAP immunoreactivity was significantly increased in male and female WT and SARM1-KO mice across time post-injury compared with naïve. Furthermore, microglial morphological changes and increased colocalisation with CD68 were evident at 7 days post-injury in WT and SARM1- KO mice compared with naïve. Microglial and astrocyte reactivity corresponded with a blood biomarker of neurodegeneration (neurofilament light) where blood biomarker levels were significantly increased across time post-injury in WT and SARM1-KO mice compared with naïve. Microgliosis was altered in SARM1-KO mice, where microglial density and colocalisation with CD68 was greater in SARM1-KO animals compared with WT, regardless of TBI. However, the genetic deletion of SARM1 did not impact astrogliosis or blood biomarker levels. When investigating biological sexes, the TBI-induced increase in microglial density and cell volume was greater in male mice at 7 days post-injury. However, microglia were more deramified in female mice at 7 days post-injury that was accompanied by a greater extent of blood biomarker levels. There were no significant differences in astrogliosis between male and female mice. These results indicate that the genetic deletion of SARM1 is not sufficient to alter astrogliosis or delayed neurodegeneration, however, SARM1 appears to play a role in microglial functionality in the naïve and injured brain. Furthermore, females experience greater neurodegeneration in the delayed stages after TBI compared with males that corresponded with microglial morphological change.
Microglia are theorised to be the primary mediators of neuroinflammation following TBI. However, the progression of microglial function across time post-injury and whether this differs between biological sexes is not well understood. In this study, we examined the microglial proteome at 3-, 7-, or 28-days after a mFPI in male and female mice using label-free quantitative proteomics. We identified a reduction in microglial proteins involved with clearance of neuronal debris via phagocytosis at 3- and 7-days post-injury. At 28 days post?injury, pro-inflammatory proteins were decreased, and anti-inflammatory proteins were increased in microglia. These results indicate a reduction in microglial clearance of neuronal debris in the days post-injury with a shift to anti-inflammatory function by 28 days following TBI. The changes in the microglial proteome that occurred across time post-injury did not differ between biological sexes. However, we did identify an increase in microglial proteins related to pro-inflammation and phagocytosis as well as insulin and estrogen signalling in males compared with female mice that occurred with or without a brain injury. Although the microglial response was similar between males and females up to 28 days following TBI, biological sex differences in the microglial proteome, regardless of TBI, has implications for the efficacy of treatment strategies targeting the microglial response post-injury.
Overall, the outcomes from this work show that males exhibit greater neuropathology and gliosis in the short-term after TBI, whilst females display more extensive alterations in delayed neurodegeneration processes and glial reactivity. Although behavioural analysis was not conducted (COVID19 intervened), these results may have implications for differences in recovery time following TBI between males and females. Therefore, this research may have vast impacts on potential TBI therapeutic interventions in both biological sexes.