antiCD49d Ab treatment ameliorates age-associated inflammatory response and mitigates CD8+ T-cell cytotoxicity after traumatic brain injury.

Patients aged 65 years and older account for an increasing proportion of patients with traumatic brain injury (TBI). Older TBI patients experience increased morbidity and mortality compared to their younger counterparts. Our prior data demonstrated that by blocking α4 integrin, anti-CD49d antibody (aCD49d Ab) abrogates CD8+ T-cell infiltration into the injured brain, improves survival, and attenuates neurocognitive deficits. Here, we aimed to uncover how aCD49d Ab treatment alters local cellular responses in the aged mouse brain. Consequently, mice incur age-associated toxic cytokine and chemokine responses long-term post-TBI. aCD49d Ab attenuates this response along with a T helper (Th)1/Th17 immunological shift and remediation of overall CD8+ T cell cytotoxicity. Furthermore, aCD49d Ab reduces CD8+ T cells exhibiting higher effector status, leading to reduced clonal expansion in aged, but not young, mouse brains with chronic TBI. Together, aCD49d Ab is a promising therapeutic strategy for treating TBI in the older people. Graphic abstract: Aged brains after TBI comprise two pools of CD8 + T cells . The aged brain has long been resided by a population of CD8 + T cells that's exhaustive and dysfunctional. Post TBI, due to BBB impairment, functional CD8 + T cells primarily migrate into the brain parenchyma. Aged, injury-associated microglia with upregulated MHC class I molecules can present neoantigens such as neuronal and/or myelin debris in the injured brains to functional CD8+ T, resulting in downstream CD8+ T cell cytotoxicity. aCD49d Ab treatment exerts its function by blocking the migration of functional effector CD8 + T cell population, leading to less cytotoxicity and resulting in improved TBI outcomes in aged mice.

ANTI-CD49D ANTIBODY TREATMENT IMPROVES SURVIVAL AND ATTENUATES NEUROCOGNITIVE DEFICITS AFTER TRAUMATIC BRAIN INJURY IN AGED MICE.

ABSTRACT: Patients 65 years and older account for an increasing proportion of traumatic brain injury (TBI) patients. Aged TBI patients experience increased morbidity and mortality compared with young TBI patients. We previously demonstrated a marked accumulation of CD8 + T-cells within the brains of aged TBI mice compared with young TBI mice. Therefore, we hypothesized that blocking peripheral T-cell infiltration into the injured brain would improve neurocognitive outcomes in aged mice after TBI. Young and aged male C57BL/6 mice underwent TBI via controlled cortical impact versus sham injury. Two hours after injuries, mice received an anti-CD49d antibody (aCD49d Ab) to block peripheral lymphocyte infiltration or its isotype control. Dosing was repeated every 2 weeks. Mortality was tracked. Neurocognitive testing for anxiety, associative learning, and memory was assessed. Motor function was evaluated. Plasma was collected for cytokine analysis. Flow cytometry was used to phenotype different immune cells within the brains. Consequently, aCD49d Ab treatment significantly improved post-TBI survival, anxiety level, associative learning, memory, and motor function in aged mice 2 months after TBI compared with isotype control treated mice. aCD49d Ab treatment augmented T H 2 response in the plasma of aged mice 2 months after TBI compared with isotype control-treated mice. Notably, aCD49d Ab treatment significantly reduced activated CD8 + cytotoxic T-cells within aged mouse brains after TBI. Contrastingly, no difference was detected in young mice after aCD49d Ab treatment. Collectively, aCD49 Ab treatment reduced T-cells in the injured brain, improved survival, and attenuated neurocognitive and gait deficits. Hence, aCD49d Ab may be a promising therapeutic intervention in aged TBI subjects-a population often excluded in TBI clinical trials.

MICROGLIA AND INFILTRATING T-CELLS ADOPT LONG-TERM, AGE-SPECIFIC, TRANSCRIPTIONAL CHANGES AFTER TRAUMATIC BRAIN INJURY IN MICE.

ABSTRACT: 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.

POSTINJURY FECAL MICROBIOME TRANSPLANT DECREASES LESION SIZE AND NEUROINFLAMMATION IN TRAUMATIC BRAIN INJURY.

ABSTRACT: 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.

Fecal Microbiota Transfer Attenuates Gut Dysbiosis and Functional Deficits After Traumatic Brain Injury.

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 = 20) or sham-injury (n = 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 = 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 = 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 = 0.002). CONCLUSION: These data suggest that restoring a pre-injury gut microbial community structure may be a promising therapeutic intervention after TBI.

Differential Fecal Microbiome Dysbiosis after Equivalent Traumatic Brain Injury in Aged Versus Young Adult Mice.

Traumatic brain injury (TBI) has a bimodal age distribution with peak incidence at age 24 and age 65 with worse outcomes developing in aged populations. Few studies have specifically addressed age at the time of injury as an independent biologic variable in TBI-associated secondary pathology. Within the framework of our published work, identifying age related effects of TBI on neuropathology, cognition, memory and motor function we analyzed fecal pellets collected from young and aged TBI animals to assess for age-induced effects in TBI induced dysbiosis. In this follow up, work we hypothesized increased dysbiosis after TBI in aged (80-week-old, N=10) versus young (14-week-old, N=10) mice. C57BL/6 males received a sham incision or TBI via open-head controlled cortical impact. Fresh stool pellets were collected 1-day pre-TBI, then 1, 7, and 28-days post-TBI for 16S rRNA gene sequencing and taxonomic analysis. Data revealed an age induced increase in disease associated microbial species which were exacerbated by injury. Consistent with our hypothesis, aged mice demonstrated a high number of disease associated changes to the gut microbiome pre- and post-injury. Our data suggest divergent microbiome phenotypes in injury between young and aged reflecting a previously unknown interaction between age, TBI, and the gut-brain axis implying the need for different treatment strategies.

Differential neuropathology and functional outcome after equivalent traumatic brain injury in aged versus young adult mice.

The CDC estimate that nearly 3 million Americans sustain a traumatic brain injury (TBI) each year. Even when medical comorbidities are accounted for, age is an independent risk factor for poor outcome after TBI. Nonetheless, few studies have examined the pathophysiology of age-linked biologic outcomes in TBI. We hypothesized that aged mice would demonstrate more severe neuropathology and greater functional deficits as compared to young adult mice after equivalent traumatic brain injuries. Young adult (14-week-old) and aged (80-week-old) C57BL/6 male mice underwent an open-head controlled cortical impact to induce TBI or a sham injury. At 30-days post-injury groups underwent behavioral phenotyping, magnetic resonance imaging, and histologic analyses. Contrary to our hypothesis, young adult TBI mice exhibited more severe neuropathology and greater loss of white matter connectivity as compared to aged mice after TBI. These findings correlated to differential functional outcomes in anxiety response, learning, and memory between young adult and aged mice after TBI. Although the mechanisms underlying this age-effect remain unclear, attenuated signs of secondary brain injury in aged TBI mice point towards different inflammatory and repair processes between age groups. These data suggest that age may need to be an a priori consideration in future clinical trial design.

Murine Model of Controlled Cortical Impact for the Induction of Traumatic Brain Injury.

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.