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.

Microglia Adopt Longitudinal Transcriptional Changes After Traumatic Brain Injury.

BACKGROUND: Traumatic brain injury (TBI) is an under-recognized public health threat. Even mild brain injuries can lead to long-term neurologic impairment. Microglia play a fundamental role in the development and progression of this ensuing neurologic impairment. Despite this, a microglia-specific injury signature has yet to be identified. We hypothesized that TBI would lead to long-term changes in the transcriptional profile of microglial pathways associated with the development of subsequent neurologic impairment. MATERIALS AND METHODS: Male C57BL/6 mice underwent TBI via a controlled cortical impact and were followed longitudinally. FACSorted microglia from TBI mice were subjected to Quantiseq 3'-biased RNA sequencing at 7, 30, and 90 d after TBI. K-means clustering on 396 differentially expressed genes was performed, and gene ontology enrichment analysis was used to determine corresponding enriched processes. RESULTS: Differentially expressed genes in microglia exhibited four main patterns of expression over the course of TBI. In particular, we identified four gene clusters which corresponded to the host defense response, synaptic plasticity, lipid remodeling, and membrane polarization. CONCLUSIONS: Transcriptional profiling within individual populations of microglia after TBI remains a critical unmet research need within the field of TBI. This focused study identified several physiologic processes within microglia that may be associated with development of long-term neurologic impairment after TBI. These data demonstrate the capability of longitudinal transcriptional profiling to uncover potential cell-specific targets for the treatment of TBI.

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.

Monocyte depletion attenuates the development of posttraumatic hydrocephalus and preserves white matter integrity after traumatic brain injury.

Monocytes are amongst the first cells recruited into the brain after traumatic brain injury (TBI). We have shown monocyte depletion 24 hours prior to TBI reduces brain edema, decreases neutrophil infiltration and improves behavioral outcomes. Additionally, both lesion and ventricle size correlate with poor neurologic outcome after TBI. Therefore, we aimed to determine the association between monocyte infiltration, lesion size, and ventricle volume. We hypothesized that monocyte depletion would attenuate lesion size, decrease ventricle enlargement, and preserve white matter in mice after TBI. C57BL/6 mice underwent pan monocyte depletion via intravenous injection of liposome-encapsulated clodronate. Control mice were injected with liposome-encapsulated PBS. TBI was induced via an open-head, controlled cortical impact. Mice were imaged using magnetic resonance imaging (MRI) at 1, 7, and 14 days post-injury to evaluate progression of lesion and to detect morphological changes associated with injury (3D T1-weighted MRI) including regional alterations in white matter patterns (multi-direction diffusion MRI). Lesion size and ventricle volume were measured using semi-automatic segmentation and active contour methods with the software program ITK-SNAP. Data was analyzed with the statistical software program PRISM. No significant effect of monocyte depletion on lesion size was detected using MRI following TBI (p = 0.4). However, progressive ventricle enlargement following TBI was observed to be attenuated in the monocyte-depleted cohort (5.3 ± 0.9mm3) as compared to the sham-depleted cohort (13.2 ± 3.1mm3; p = 0.02). Global white matter integrity and regional patterns were evaluated and quantified for each mouse after extracting fractional anisotropy maps from the multi-direction diffusion-MRI data using Siemens Syngo DTI analysis package. Fractional anisotropy (FA) values were preserved in the monocyte-depleted cohort (123.0 ± 4.4mm3) as compared to sham-depleted mice (94.9 ± 4.6mm3; p = 0.025) by 14 days post-TBI. All TBI mice exhibited FA values lower than those from a representative naïve control group with intact white matter tracts and FA~200 mm3). The MRI derived assessment of injury progression suggests that monocyte depletion at the time of injury may be a novel therapeutic strategy in the treatment of TBI. Furthermore, non-invasive longitudinal imaging allows for the evaluation of both TBI progression as well as therapeutic response over the course of injury.

Spontaneous reduction of acute cecal herniation through the foramen of Winslow.

This is the case of a 70-year-old woman who presented to the emergency department complaining of 2 hours of acute-onset epigastric pain. She had experienced this pain once before which had spontaneously resolved. Axial imaging demonstrated the cecum in an abnormal position within the lesser sac, as well as compression of the inferior vena cava and portal vein. She was taken emergently to the operating room for laparotomy, where a free-floating cecum and ascending colon was identified without ischemia, and a right hemicolectomy was performed. Foramen of Winslow hernias are rare internal hernias with a high rate of strangulation and bowel ischemia, requiring urgent surgical intervention. Operative treatment depends on the type of herniated viscera. Spontaneous reduction is not well documented.

Nonclassical Monocytes Mediate Secondary Injury, Neurocognitive Outcome, and Neutrophil Infiltration after Traumatic Brain Injury.

Traumatic brain injury (TBI) results in rapid recruitment of leukocytes into the injured brain. Monocytes constitute a significant proportion of the initial infiltrate and have the potential to propagate secondary brain injury or generate an environment of repair and regeneration. Monocytes are a diverse population of cells (classical, intermediate, and nonclassical) with distinct functions, however, the recruitment order of these subpopulations to the injured brain largely remains unknown. Thus, we examined which monocyte subpopulations are required for the generation of early inflammatory infiltrate within the injured brain, and whether their depletion attenuates secondary injury or neurocognitive outcome. Global monocyte depletion correlated with significant improvements in brain edema, motor coordination, and working memory, and abrogated neutrophil infiltration into the injured brain. However, targeted depletion of classical monocytes alone had no effect on neutrophil recruitment to the site of injury, implicating the nonclassical monocyte in this process. In contrast, mice that have markedly reduced numbers of nonclassical monocytes (CX3CR1-/-) exhibited a significant reduction in neutrophil infiltration into the brain after TBI as compared with control mice. Our data suggest a critical role for nonclassical monocytes in the pathology of TBI in mice, including important clinical outcomes associated with mortality in this injury process.

The Role of Microglia in the Etiology and Evolution of Chronic Traumatic Encephalopathy.

Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative disease that presents as a late sequela from traumatic brain injury (TBI). TBI is a growing and under-recognized public health concern with a high degree of morbidity and large associated global costs. While the immune response to TBI is complex, its contribution to the development of CTE remains largely unknown. In this review, we summarize the current understanding of the link between CTE and the resident innate immune system of the brain-microglia. We discuss the neuropathology underlying CTE including the creation and aggregation of phosphorylated tau protein into neurofibrillary tangles and the formation of amyloid beta deposits. We also present how microglia, the resident innate immune cells of the brain, drive the continuous low-level inflammation associated with the insidious onset of CTE. In this review, we conclude that the latency period between the index brain injury and the long-term development of CTE presents an opportunity for therapeutic intervention. Encouraging advances with microtubule stabilizers, cis p-tau antibodies, and the ability to therapeutically alter the inflammatory state of microglia have shown positive results in both animal and human trials. Looking forward, recent advancements in next-generation sequencing technology for the study of genomic, transcriptomic, and epigenetic information will provide an opportunity for significant advancement in our understanding of prorepair and pro-injury gene signatures allowing for targeted intervention in this highly morbid injury process.

Differential Activation of Infiltrating Monocyte-Derived Cells After Mild and Severe Traumatic Brain Injury.

Microglia are the resident innate immune cells of the brain. Although embryologically and functionally distinct, they are morphologically similar to peripheral monocyte-derived cells, resulting in a poor ability to discriminate between the two cell types. The purpose of this study was to develop a rapid and reliable method to simultaneously characterize, quantify, and discriminate between whole populations of myeloid cells from the brain in a murine model of traumatic brain injury. Male C57BL/6 mice underwent traumatic brain injury (n = 16) or sham injury (n = 14). Brains were harvested at 24 h after injury. Multiparameter flow cytometry and sequential gating analysis were performed, allowing for discrimination between microglia and infiltrating leukocytes as well as for the characterization and quantification of individual subtypes within the infiltrating population. The proportion of infiltrating leukocytes within the brain increased with the severity of injury, and the predominant cell types within the infiltrating population were monocyte derived (P = 0.01). In addition, the severity of injury altered the overall makeup of the infiltrating monocyte-derived cells. In conclusion, we describe a flow cytometry-based technique for gross discrimination between infiltrating leukocytes and microglia as well as the ability to simultaneously characterize and quantify individual myeloid subtypes and their maturation states within these populations.