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Background: Traumatic brain injury (TBI) is an underrecognized public health threat. Survivors of TBI often suffer long-term neurocognitive deficits leading to the progressive onset of neurodegenerative disease. Recent data suggests that the gut-brain axis is complicit in this process. However, no study has specifically addressed whether fecal microbiota transfer (FMT) attenuates neurologic deficits after TBI. Hypothesis: We hypothesized that fecal microbiota transfer would attenuate neurocognitive, anatomic, and pathologic deficits after TBI. Methods: C57Bl/6 mice were subjected to severe TBI (n ¼ 20) or sham-injury (n ¼ 20) via an open-head controlled cortical impact. Post-injury, this cohort of mice underwent weekly oral gavage with a slurry of healthy mouse stool or vehicle alone beginning 1 h post-TBI followed by behavioral testing and neuropathologic analysis. 16S ribosomal RNA sequencing of fecal samples was performed to characterize gut microbial community structure pre-and post-injury. Zero maze and open field testing were used to evaluate post-traumatic anxiety, exploratory behavior, and generalized activity. 3D, contrast enhanced, magnetic resonance imaging was used to determine differences in cortical volume loss and white matter connectivity. Prior to euthanasia, brains were harvested for neuropathologic analysis. Results: Fecal microbiome analysis revealed a large variance between TBI, and sham animals treated with vehicle, while FMT treated TBI mice had restoration of gut dysbiosis back to levels of control mice. Neurocognitive testing demonstrated a rescue of normal anxiety-like and exploratory behavior in TBI mice treated with FMT. FMT treated TBI mice spent a greater percentage of time (22%, P ¼ 0.0001) in the center regions of the Open Field as compared to vehicle treated TBI mice (13%). Vehicle-treated TBI animals also spent less time (19%) in the open areas of zero maze than FMT treated TBI mice (30%, P ¼ 0.0001). Comparing in TBI mice treated with FMT, MRI demonstrated a marked attenuation in ventriculomegaly (P < 0.002) and a significant change in fractional anisotropy (i.e., loss of white matter connectivity) (P < 0.0001). Histologic analysis of brain sections revealed a FMT-injury dependent interaction in the microglia/macrophage-specific ionized calcium-binding protein, Iba1 (P ¼ 0.002). Conclusion: These data suggest that restoring a pre-injury gut microbial community structure may be a promising therapeutic intervention after TBI.

The Centers for Disease Control and Injury Prevention estimate that almost 2 million people sustain a traumatic brain injury (TBI) every year in the United States. In fact, TBI is a contributing factor to over a third of all injury-related mortality. Nonetheless, the cellular and molecular mechanisms underlying the pathophysiology of TBI are poorly understood. Thus, preclinical models of TBI capable of replicating the injury mechanisms pertinent to TBI in human patients are a critical research need. The controlled cortical impact (CCI) model of TBI utilizes a mechanical device to directly impact the exposed cortex. While no model can full recapitulate the disparate injury patterns and heterogeneous nature of TBI in human patients, CCI is capable of inducing a wide range of clinically applicable TBI. Furthermore, CCI is easily standardized allowing investigators to compare results across experiments as well as across investigative groups. The following protocol is a detailed description of applying a severe CCI with a commercially available impacting device in a murine model of TBI.

Aged traumatic brain injury (TBI) patients suffer increased mortality and long-term neurocognitive and neuropsychiatric morbidity compared with younger patients. Microglia, the resident innate immune cells of the brain, are complicit in both. We hypothesized that aged microglia would fail to return to a homeostatic state after TBI and adopt a long-term injury-associated state within aged brains compared with young brains after TBI. Young and aged male C57BL/6 mice underwent TBI via controlled cortical impact versus sham injury and were sacrificed 4 months post-TBI. We used single-cell RNA sequencing to examine age-associated cellular responses after TBI. Brains were harvested, and CD45+ cells were isolated via fluorescence-activated cell sorting. cDNA libraries were prepared using the 10x Genomics Chromium Single Cell 3′ Reagent Kit, followed by sequencing on a HiSeq 4,000 instrument and computational analyses. Post-injury, aged mice demonstrated a disparate microglial gene signature and an increase in infiltrating T cells compared with young adult mice. Notably, aged mice post-injury had a subpopulation of age-specific, immune-inflammatory microglia resembling the gene profile of neurodegenerative disease-associated microglia with enriched pathways involved in leukocyte recruitment and brain-derived neurotrophic factor signaling. Meanwhile, post-injury, aged mice demonstrated heterogeneous T-cell infiltration with gene profiles corresponding to CD8 effector memory, CD8 naive-like, CD8 early active T cells, and Th1 cells with enriched pathways, such as macromolecule synthesis. Taken together, our data showed that the aged brain had an age-specific gene signature change in both T-cell infiltrates and microglia, which may contribute to its increased vulnerability to TBI and the long-term sequelae of TBI.

Background: Traumatic brain injury (TBI) is an underrecognized public health threat. The constitutive activation of microglia after TBI has been linked to long-term neurocognitive deficits and the progression of neurodegenerative disease. Evolving evidence indicates a critical role for the gut-brain axis in this process. Specifically, TBI has been shown to induce the depletion of commensal gut bacteria. The resulting gut dysbiosis is associated with neuroinflammation and disease. Hypothesis: We hypothesized that fecal microbiota transplantation would attenuate microglial activation and improve neuropathology after TBI. Methods: C57Bl/6 mice were subjected to severe TBI (n = 10) or sham injury (n = 10) via an open-head controlled cortical impact. The mice underwent fecal microbiota transplantation (FMT) or vehicle alone via oral gavage once weekly for 4 weeks after injury. At 59 days after TBI, mice underwent three-dimensional, contrast-enhanced magnetic resonance imaging. Following imaging, mice were killed, brains harvested at 60 DPI, and CD45+ cells isolated via florescence-activated cell sorting. cDNA libraries were prepared using the 10x Genomics Chromium Single Cell 3′ Reagent kit followed by sequencing on a HiSeq4000 instrument, and computational analysis was performed. Results: Fecal microbiota transplantation resulted in a >marked reduction of ventriculomegaly (P < 0.002) and preservation of white matter connectivity at 59 days after TBI (P < 0.0001). In addition, microglia from FMT-treated mice significantly reduced inflammatory gene expression and enriched pathways involving the heat-shock response compared with mice treated with vehicle alone. Conclusions: We hypothesized that restoring gut microbial community structure via FMT would attenuate microglial activation and reduce neuropathology after TBI. Our data demonstrated significant preservation of cortical volume and white matter connectivity after an injury compared with mice treated with vehicle alone. This preservation of neuroanatomy after TBI was associated with a marked reduction in inflammatory gene expression within the microglia of FMT-treated mice. Microglia from FMT-treated mice enriched pathways in the heat-shock response, which is known to play a neuroprotective role in TBI and other neurodegenerative disease processes.