Sexually dimorphic extracellular vesicle responses after chronic spinal cord injury are associated with neuroinflammation and neurodegeneration in the brain
Introduction: Advancements in treatment and healthcare allow many patients suffering a spinal cord injury (SCI) to live for several decades after their traumatic event. Amongst the long-term consequences of SCI, cognitive decline has garnered high interest that is further complicated by the factor of biological sex. Emerging data suggest that male and female utilize different pathological mechanisms after injury, which may in turn lead to differences in functional outcome. Extracellular vesicles (EVs), containing microRNAs (miRs), proteins, and lipids from their originating cells, have emerged as potentially important regulators of secondary injury after SCI, not only locally but also systemically and in the brain. We have previously shown that SCI in young adult male mice leads to robust changes in plasma EV count and microRNA (miR) content. Here, our goal was to investigate the impact of biological sex and aging on EVs and brain after SCI.
Methods: Young adult age-matched male and female C57BL/6 mice were subjected to moderate thoracic spinal cord contusion. At 19 months post-injury, total plasma EVs were isolated by ultracentrifugation. Particle count and size distribution were assessed by nanoparticle tracking analysis, while individual EV protein expression was quantified by Western blot. EVs microRNA (miR) cargo was examined using the Fireplex® assay. The transcriptional changes in the brain were assessed by a NanoString nCounter Neuropathology panel. Neuropathology in the brain regions was further examined by Western blot (WB) and flow cytometry (FC). A battery of behavioral tests was performed for assessment of locomotor and cognitive function as well as depression. All animal experiments and surgical procedures were performed according to protocol approved by the Institutional Animal Care and Use Committee (IACUC) from the University of Maryland School of Medicine.
Results: NanoString analysis demonstrated that males had greater transcriptional changes than females in the brain following chronic SCI. Furthermore, males had a pronounced reduction in genes related to vesicle trafficking, growth factor signaling and myelination in the cortex. In contrast, females showed an improved oxidative stress and unfolded protein response profile. Decreased expression of anti-inflammatory cytokines (Il6, Cxcr4, Tgfb1, Il10ra) in the hippocampus may be detrimental to neuronal survival and adult neurogenesis. Across both regions and sexes, gene expression related to homeostatic microglia were reduced (C1qa and Egr1). Examination of the injured spinal cord with FC showed higher lymphocyte counts in SCI/Female mice compared to their male counterparts, along with higher production of reactive oxygen species (ROS). In the brain, SCI/Female mice showed higher levels of TNF and MitoSpyRed positive microglia as well as ROS production. WB and NTA showed that EV markers, CD63 and CD81, are elevated in the plasma of male mice after SCI. Furthermore, particle concentration in the cortex also increased after injury, with SCI/Female mice showing significantly higher counts than SCI/Male. Cargo analysis of the plasma and tissue derived EVs showed dramatic changes of miR content driven by injury and sex differences. Alterations in EV miRs paralleled those reported with neurodegenerative disease, depression and inflammatory processes. SCI impaired locomotor and cognitive function and caused depression-like behavior in both sexes. However, neither behavior test showed sex differences.
Conclusions: Collectively, these studies are the first to describe changes in circulating EVs after chronic SCI and in aged animals and support a potential EV-mediated mechanism for SCI-induced brain changes.
