Microglia-targeted inhibition of miR-17 via mannose-coated lipid nanoparticles improves pathology and behavior in a mouse model of Alzheimer's disease.

Neuroinflammation and accumulation of Amyloid Beta (Aß) accompanied by deterioration of special memory are hallmarks of Alzheimer's disease (AD). Effective preventative and treatment options for AD are still needed. Microglia in AD brains are characterized by elevated levels of microRNA-17 (miR-17), which is accompanied by defective autophagy, Aß accumulation, and increased inflammatory cytokine production. However, the effect of targeting miR-17 on AD pathology and memory loss is not clear. To specifically inhibit miR-17 in microglia, we generated mannose-coated lipid nanoparticles (MLNPs) enclosing miR-17 antagomir (Anti-17 MLNPs), which are targeted to mannose receptors readily expressed on microglia. We used a 5XFAD mouse model (AD) that recapitulates many AD-related phenotypes observed in humans. Our results show that Anti-17 MLNPs, delivered to 5XFAD mice by intra-cisterna magna injection, specifically deliver Anti-17 to microglia. Anti-17 MLNPs downregulated miR-17 expression in microglia but not in neurons, astrocytes, and oligodendrocytes. Anti-17 MLNPs attenuated inflammation, improved autophagy, and reduced Aß burdens in the brains. Additionally, Anti-17 MLNPs reduced the deterioration in spatial memory and decreased anxiety-like behavior in 5XFAD mice. Therefore, targeting miR-17 using MLNPs is a viable strategy to prevent several AD pathologies. This selective targeting strategy delivers specific agents to microglia without the adverse off-target effects on other cell types. Additionally, this approach can be used to deliver other molecules to microglia and other immune cells in other organs.

Engineered extracellular vesicle-based gene therapy for the treatment of discogenic back pain.

Painful musculoskeletal disorders such as intervertebral disc (IVD) degeneration associated with chronic low back pain (termed "Discogenic back pain", DBP), are a significant socio-economic burden worldwide and contribute to the growing opioid crisis. Yet there are very few if any successful interventions that can restore the tissue's structure and function while also addressing the symptomatic pain. Here we have developed a novel non-viral gene therapy, using engineered extracellular vesicles (eEVs) to deliver the developmental transcription factor FOXF1 to the degenerated IVD in an in vivo model. Injured IVDs treated with eEVs loaded with FOXF1 demonstrated robust sex-specific reductions in pain behaviors compared to control groups. Furthermore, significant restoration of IVD structure and function in animals treated with FOXF1 eEVs were observed, with significant increases in disc height, tissue hydration, proteoglycan content, and mechanical properties. This is the first study to successfully restore tissue function while modulating pain behaviors in an animal model of DBP using eEV-based non-viral delivery of transcription factor genes. Such a strategy can be readily translated to other painful musculoskeletal disorders.

Early hippocampal high-amplitude rhythmic spikes predict post-traumatic epilepsy in mice.

Oscillations, a highly conserved brain function across mammalian species, are pivotal in brain physiology and pathology. Traumatic brain injury (TBI) often leads to subacute and chronic brain oscillatory alterations associated with complications like post-traumatic epilepsy (PTE) in patients and animal models. Our recent work longitudinally recorded local field potential from the contralateral hippocampus of 12 strains of recombinant inbred Collaborative Cross (CC) mice alongside classical laboratory inbred C57BL/6J mice after lateral fluid percussion injury. In this study, we profiled the acute (<12 hr post-injury) and subacute (12-48 hr post-injury) hippocampal oscillatory responses to TBI and evaluated their predictive value for PTE. We found dynamic high-amplitude rhythmic spikes with elevated power density and reduced entropy that prevailed during the acute phase in CC031 mice who later developed PTE. This characteristic early brain oscillatory alteration is absent in CC031 sham controls or other CC and reference C57BL/6J strains that did not develop PTE after TBI. Our work provides quantitative measures linking early brain oscillation to PTE at a population level in mice under controlled experimental conditions. These findings will offer insights into circuit mechanisms and potential targets for neuromodulatory intervention.

Context is key: glucocorticoid receptor and corticosteroid therapeutics in outcomes after traumatic brain injury.

Traumatic brain injury (TBI) is a global health burden, and survivors suffer functional and psychiatric consequences that can persist long after injury. TBI induces a physiological stress response by activating the hypothalamic-pituitary-adrenal (HPA) axis, but the effects of injury on the stress response become more complex in the long term. Clinical and experimental evidence suggests long lasting dysfunction of the stress response after TBI. Additionally, pre- and post-injury stress both have negative impacts on outcome following TBI. This bidirectional relationship between stress and injury impedes recovery and exacerbates TBI-induced psychiatric and cognitive dysfunction. Previous clinical and experimental studies have explored the use of synthetic glucocorticoids as a therapeutic for stress-related TBI outcomes, but these have yielded mixed results. Furthermore, long-term steroid treatment is associated with multiple negative side effects. There is a pressing need for alternative approaches that improve stress functionality after TBI. Glucocorticoid receptor (GR) has been identified as a fundamental link between stress and immune responses, and preclinical evidence suggests GR plays an important role in microglia-mediated outcomes after TBI and other neuroinflammatory conditions. In this review, we will summarize GR-mediated stress dysfunction after TBI, highlighting the role of microglia. We will discuss recent studies which target microglial GR in the context of stress and injury, and we suggest that cell-specific GR interventions may be a promising strategy for long-term TBI pathophysiology.

Deplete and repeat: microglial CSF1R inhibition and traumatic brain injury.

Traumatic brain injury (TBI) is a public health burden affecting millions of people. Sustained neuroinflammation after TBI is often associated with poor outcome. As a result, increased attention has been placed on the role of immune cells in post-injury recovery. Microglia are highly dynamic after TBI and play a key role in the post-injury neuroinflammatory response. Therefore, microglia represent a malleable post-injury target that could substantially influence long-term outcome after TBI. This review highlights the cell specific role of microglia in TBI pathophysiology. Microglia have been manipulated via genetic deletion, drug inhibition, and pharmacological depletion in various pre-clinical TBI models. Notably, colony stimulating factor 1 (CSF1) and its receptor (CSF1R) have gained much traction in recent years as a pharmacological target on microglia. CSF1R is a transmembrane tyrosine kinase receptor that is essential for microglia proliferation, differentiation, and survival. Small molecule inhibitors targeting CSF1R result in a swift and effective depletion of microglia in rodents. Moreover, discontinuation of the inhibitors is sufficient for microglia repopulation. Attention is placed on summarizing studies that incorporate CSF1R inhibition of microglia. Indeed, microglia depletion affects multiple aspects of TBI pathophysiology, including neuroinflammation, oxidative stress, and functional recovery with measurable influence on astrocytes, peripheral immune cells, and neurons. Taken together, the data highlight an important role for microglia in sustaining neuroinflammation and increasing risk of oxidative stress, which lends to neuronal damage and behavioral deficits chronically after TBI. Ultimately, the insights gained from CSF1R depletion of microglia are critical for understanding the temporospatial role that microglia develop in mediating TBI pathophysiology and recovery.

DNA hypomethylation promotes the expression of CASPASE-4 which exacerbates inflammation and amyloid-ß deposition in Alzheimer's disease.

Alzheimer's disease (AD) is the sixth leading cause of death in the USA. It is established that neuroinflammation contributes to the synaptic loss, neuronal death, and symptomatic decline of AD patients. Accumulating evidence suggests a critical role for microglia, innate immune phagocytes of the brain. For instance, microglia release pro-inflammatory products such as IL-1ß which is highly implicated in AD pathobiology. The mechanisms underlying the transition of microglia to proinflammatory promoters of AD remain largely unknown. To address this gap, we performed reduced representation bisulfite sequencing (RRBS) to profile global DNA methylation changes in human AD brains compared to no disease controls. We identified differential DNA methylation of CASPASE-4 (CASP4), which when expressed promotes the generation of IL-1ß and is predominantly expressed in immune cells. DNA upstream of the CASP4 transcription start site was hypomethylated in human AD brains, which was correlated with increased expression of CASP4. Furthermore, microglia from a mouse model of AD (5xFAD) express increased levels of CASP4 compared to wild-type (WT) mice. To study the role of CASP4 in AD, we developed a novel mouse model of AD lacking the mouse ortholog of CASP4 and CASP11, which is encoded by mouse Caspase-4 (5xFAD/Casp4-/-). The expression of CASP11 was associated with increased accumulation of pathologic protein aggregate amyloid-ß (Aß) and increased microglial production of IL-1ß in 5xFAD mice. Utilizing RNA-sequencing, we determined that CASP11 promotes unique transcriptomic phenotypes in 5xFAD mouse brains, including alterations of neuroinflammatory and chemokine signaling pathways. Notably, in vitro, CASP11 promoted generation of IL-1ß from macrophages in response to cytosolic Aß through cleavage of downstream effector Gasdermin D (GSDMD). Therefore, here we unravel the role for CASP11 and GSDMD in the generation of IL-1ß in response to Aß and the progression of pathologic inflammation in AD. Overall, our results demonstrate that overexpression of CASP4 due to differential DNA methylation in AD microglia contributes to the progression of AD pathobiology. Thus, we identify CASP4 as a potential target for immunotherapies for the treatment and prevention of AD.

Genetic diversity drives extreme responses to traumatic brain injury and post-traumatic epilepsy.

Traumatic brain injury (TBI) is a complex and heterogeneous condition that can cause wide-spectral neurological sequelae such as behavioral deficits, sleep abnormalities, and post-traumatic epilepsy (PTE). However, understanding the interaction of TBI phenome is challenging because few animal models can recapitulate the heterogeneity of TBI outcomes. We leveraged the genetically diverse recombinant inbred Collaborative Cross (CC) mice panel and systematically characterized TBI-related outcomes in males from 12 strains of CC and the reference C57BL/6J mice. We identified unprecedented extreme responses in multiple clinically relevant traits across CC strains, including weight change, mortality, locomotor activity, cognition, and sleep. Notably, we identified CC031 mouse strain as the first rodent model of PTE that exhibit frequent and progressive post-traumatic seizures after moderate TBI induced by lateral fluid percussion. Multivariate analysis pinpointed novel biological interactions and three principal components across TBI-related modalities. Estimate of the proportion of TBI phenotypic variability attributable to strain revealed large range of heritability, including >70% heritability of open arm entry time of elevated plus maze. Our work provides novel resources and models that can facilitate genetic mapping and the understanding of the pathobiology of TBI and PTE.

Divergent Spatial Learning, Enhanced Neuronal Transcription, and Blood-Brain Barrier Disruption Develop During Recovery from Post-Injury Sleep Fragmentation.

Traumatic brain injury (TBI) causes pathophysiology that may significantly decrease quality of life over time. A major propagator of this response is chronic, maladaptive neuroinflammation, which can be exacerbated by stressors such as sleep fragmentation (SF). This study determined whether post-TBI SF had lasting behavioral and inflammatory effects even with a period of recovery. To test this, male and female mice received a moderate lateral fluid percussion TBI or sham surgery. Half the mice were left undisturbed, and half were exposed to daily SF for 30 days. All mice were then undisturbed between 30 and 60 days post-injury (DPI), allowing mice to recover from SF (SF-R). SF-R did not impair global Barnes maze performance. Nonetheless, TBI SF-R mice displayed retrogression in latency to reach the goal box within testing days. These nuanced behavioral changes in TBI SF-R mice were associated with enhanced expression of neuronal processing/signaling genes and indicators of blood-brain barrier (BBB) dysfunction. Aquaporin-4 (AQP4) expression, a marker of BBB integrity, was differentially altered by TBI and TBI SF-R. For example, TBI enhanced cortical AQP4 whereas TBI SF-R mice had the lowest cortical expression of perivascular AQP4, dysregulated AQP4 polarization, and the highest number of CD45+ cells in the ipsilateral cortex. Altogether, post-TBI SF caused lasting, divergent behavioral responses associated with enhanced expression of neuronal transcription and BBB disruption even after a period of recovery from SF. Understanding lasting impacts from post-TBI stressors can better inform both acute and chronic post-injury care to improve long-term outcome post-TBI.

DNA hypomethylation promotes the expression of CASPASE-4 which exacerbates neuroinflammation and amyloid-ß deposition in Alzheimer's disease The Ohio State University College of Medicine.

Alzheimer's Disease (AD) is the 6th leading cause of death in the US. It is established that neuroinflammation contributes to the synaptic loss, neuronal death, and symptomatic decline of AD patients. Accumulating evidence suggests a critical role for microglia, innate immune phagocytes of the brain. For instance, microglia release proinflammatory products such as IL-1ß which is highly implicated in AD pathobiology. The mechanisms underlying the transition of microglia to proinflammatory promoters of AD remain largely unknown. To address this gap, we performed Reduced Representation Bisulfite Sequencing (RRBS) to profile global DNA methylation changes in human AD brains compared to no disease controls. We identified differential DNA methylation of CASPASE-4 (CASP4), which when expressed, can be involved in generation of IL-1ß and is predominantly expressed in immune cells. DNA upstream of the CASP4 transcription start site was hypomethylated in human AD brains, which was correlated with increased expression of CASP4. Furthermore, microglia from a mouse model of AD (5xFAD) express increased levels of CASP4 compared to wild-type (WT) mice. To study the role of CASP4 in AD, we developed a novel mouse model of AD lacking the mouse ortholog of CASP4, CASP11, which is encoded by mouse Caspase-4 (5xFAD/Casp4-/-). The expression of CASP11 was associated with increased accumulation of pathologic protein aggregate amyloid-ß (Aß) and increased microglial production of IL-1ß in 5xFAD mice. Utilizing RNA sequencing, we determined that CASP11 promotes unique transcriptomic phenotypes in 5xFAD mouse brains, including alterations of neuroinflammatory and chemokine signaling pathways. Notably, in vitro, CASP11 promoted generation of IL-1ß from macrophages in response to cytosolic Aß through cleavage of downstream effector Gasdermin D (G SDMD). We describe a role for CASP11 and GSDMD in the generation of IL-1ß in response to Aß and the progression of pathologic inflammation in AD. Overall, our results demonstrate that overexpression of CASP4 due to differential methylation in AD microglia contributes to the progression of AD pathobiology, thus identifying CASP4 as a potential target for immunotherapies for the treatment of AD.

Multiparity Differentially Affects Specific Aspects of the Acute Neuroinflammatory Response to Traumatic Brain Injury in Female Mice.

Pregnancy is associated with profound acute and long-term physiological changes, but the effects of such changes on brain injury outcomes are unclear. Here, we examined the effects of previous pregnancy and maternal experience (parity) on acute neuroinflammatory responses to lateral fluid percussion injury (FPI), a well-defined experimental traumatic brain injury (TBI) paradigm. Multiparous (2-3 pregnancies and motherhood experiences) and age-matched nulliparous (no previous pregnancy or motherhood experience) female mice received either FPI or sham injury and were euthanized 3 days post-injury (DPI). Increased cortical Iba1, GFAP, and CD68 immunolabeling was observed following TBI independent of parity and microglia morphology did not differ between TBI groups. However, multiparous females had fewer CD45+ cells near the site of injury compared to nulliparous females, which was associated with preserved aquaporin-4 polarization, suggesting that parity may influence leukocyte recruitment to the site of injury and maintenance of blood brain barrier permeability following TBI. Additionally, relative cortical Il6 gene expression following TBI was dependent on parity such that TBI increased Il6 expression in nulliparous, but not multiparous, mice. Together, this work suggests that reproductive history may influence acute neuroinflammatory outcomes following TBI in females.

Triggering receptor expressed on myeloid cells 2 deficiency exacerbates injury-induced inflammation in a mouse model of tauopathy.

Traumatic brain injury (TBI) promotes several Alzheimer's disease-like pathological features, including microtubule-associated protein tau (MAPT) accumulation within neurons. Macrophage activation in the injured hTau mouse model of tauopathy raises the question whether there is a relationship between MAPT pathology and alterations in macrophage activation following TBI. Triggering receptor expressed on myeloid cells 2 (TREM2) is a critical regulator of microglia and macrophage phenotype, but its mechanisms on TBI remain unclear. To address the association with TREM2 in TBI and MAPT pathology, we studied TREM2 deficiency in hTau mice (hTau;Trem2-/- ) 3 (acute phase) and 120 (chronic phase) days after experimental TBI. At three days following injury, hTau;Trem2-/- mice exhibited reduced macrophage activation both in the cortex and hippocampus. However, to our surprise, hTau;Trem2-/- mice exposed to TBI augments macrophage accumulation in the corpus callosum and white matter near the site of tissue damage in a chronic phase, which results in exacerbated axonal injury, tau aggregation, and impaired neurogenesis. We further demonstrate that TREM2 deficiency in hTau injured mice promotes neuronal dystrophy in the white matter due to impaired phagocytosis of apoptotic cells. Remarkably, hTau;Trem2-/- exposed to TBI failed to restore blood-brain barrier integrity. These findings imply that TREM2 deficiency accelerates inflammation and neurodegeneration, accompanied by attenuated microglial phagocytosis and continuous blood-brain barrier (BBB) leakage, thus exacerbating tauopathy in hTau TBI mice.

In vivo Mouse Intervertebral Disc Degeneration Models and Their Utility as Translational Models of Clinical Discogenic Back Pain: A Comparative Review.

Low back pain is a leading cause of disability worldwide and studies have demonstrated intervertebral disc (IVD) degeneration as a major risk factor. While many in vitro models have been developed and used to study IVD pathophysiology and therapeutic strategies, the etiology of IVD degeneration is a complex multifactorial process involving crosstalk of nearby tissues and systemic effects. Thus, the use of appropriate in vivo models is necessary to fully understand the associated molecular, structural, and functional changes and how they relate to pain. Mouse models have been widely adopted due to accessibility and ease of genetic manipulation compared to other animal models. Despite their small size, mice lumbar discs demonstrate significant similarities to the human IVD in terms of geometry, structure, and mechanical properties. While several different mouse models of IVD degeneration exist, greater standardization of the methods for inducing degeneration and the development of a consistent set of output measurements could allow mouse models to become a stronger tool for clinical translation. This article reviews current mouse models of IVD degeneration in the context of clinical translation and highlights a critical set of output measurements for studying disease pathology or screening regenerative therapies with an emphasis on pain phenotyping. First, we summarized and categorized these models into genetic, age-related, and mechanically induced. Then, the outcome parameters assessed in these models are compared including, molecular, cellular, functional/structural, and pain assessments for both evoked and spontaneous pain. These comparisons highlight a set of potential key parameters that can be used to validate the model and inform its utility to screen potential therapies for IVD degeneration and their translation to the human condition. As treatment of symptomatic pain is important, this review provides an emphasis on critical pain-like behavior assessments in mice and explores current behavioral assessments relevant to discogenic back pain. Overall, the specific research question was determined to be essential to identify the relevant model with histological staining, imaging, extracellular matrix composition, mechanics, and pain as critical parameters for assessing degeneration and regenerative strategies.

Chronic Cortical Inflammation, Cognitive Impairment, and Immune Reactivity Associated with Diffuse Brain Injury Are Ameliorated by Forced Turnover of Microglia.

Traumatic brain injury (TBI) is associated with an increased risk of cognitive, psychiatric, and neurodegenerative complications that may develop after injury. Increased microglial reactivity following TBI may underlie chronic neuroinflammation, neuropathology, and exaggerated responses to immune challenges. Therefore, the goal of this study was to force turnover of trauma-associated microglia that develop after diffuse TBI and determine whether this alleviated chronic inflammation, improved functional recovery and attenuated reduced immune reactivity to lipopolysaccharide (LPS) challenge. Male mice received a midline fluid percussion injury (mFPI) and 7 d later were subjected to a forced microglia turnover paradigm using CSF1R antagonism (PLX5622). At 30 d postinjury (dpi), cortical gene expression, dendritic complexity, myelin content, neuronal connectivity, cognition, and immune reactivity were assessed. Myriad neuropathology-related genes were increased 30 dpi in the cortex, and 90% of these gene changes were reversed by microglial turnover. Reduced neuronal connectivity was evident 30 dpi and these deficits were attenuated by microglial turnover. TBI-associated dendritic remodeling and myelin alterations, however, remained 30 dpi independent of microglial turnover. In assessments of functional recovery, increased depressive-like behavior, and cognitive impairment 30 dpi were ameliorated by microglia turnover. To investigate microglial priming and reactivity 30 dpi, mice were injected intraperitoneally with LPS. This immune challenge caused prolonged lethargy, sickness behavior, and microglial reactivity in the TBI mice. These extended complications with LPS in TBI mice were prevented by microglia turnover. Collectively, microglial turnover 7 dpi alleviated behavioral and cognitive impairments associated with microglial priming and immune reactivity 30 dpi.SIGNIFICANCE STATEMENT A striking feature of traumatic brain injury (TBI), even mild injuries, is that over 70% of individuals have long-term neuropsychiatric complications. Chronic inflammatory processes are implicated in the pathology of these complications and these issues can be exaggerated by immune challenge. Therefore, our goal was to force the turnover of microglia 7 d after TBI. This subacute 7 d postinjury (dpi) time point is a critical transitional period in the shift toward chronic inflammatory processes and microglia priming. This forced microglia turnover intervention in mice attenuated the deficits in behavior and cognition 30 dpi. Moreover, microglia priming and immune reactivity after TBI were also reduced with microglia turnover. Therefore, microglia represent therapeutic targets after TBI to reduce persistent neuroinflammation and improve recovery.

Wolframin is a novel regulator of tau pathology and neurodegeneration.

Selective neuronal vulnerability to protein aggregation is found in many neurodegenerative diseases including Alzheimer's disease (AD). Understanding the molecular origins of this selective vulnerability is, therefore, of fundamental importance. Tau protein aggregates have been found in Wolframin (WFS1)-expressing excitatory neurons in the entorhinal cortex, one of the earliest affected regions in AD. The role of WFS1 in Tauopathies and its levels in tau pathology-associated neurodegeneration, however, is largely unknown. Here we report that WFS1 deficiency is associated with increased tau pathology and neurodegeneration, whereas overexpression of WFS1 reduces those changes. We also find that WFS1 interacts with tau protein and controls the susceptibility to tau pathology. Furthermore, chronic ER stress and autophagy-lysosome pathway (ALP)-associated genes are enriched in WFS1-high excitatory neurons in human AD at early Braak stages. The protein levels of ER stress and autophagy-lysosome pathway (ALP)-associated proteins are changed in tau transgenic mice with WFS1 deficiency, while overexpression of WFS1 reverses those changes. This work demonstrates a possible role for WFS1 in the regulation of tau pathology and neurodegeneration via chronic ER stress and the downstream ALP. Our findings provide insights into mechanisms that underpin selective neuronal vulnerability, and for developing new therapeutics to protect vulnerable neurons in AD.

Sleep fragmentation engages stress-responsive circuitry, enhances inflammation and compromises hippocampal function following traumatic brain injury.

Traumatic brain injury (TBI) impairs the ability to restore homeostasis in response to stress, indicating hypothalamic-pituitary-adrenal (HPA)-axis dysfunction. Many stressors result in sleep disturbances, thus mechanical sleep fragmentation (SF) provides a physiologically relevant approach to study the effects of stress after injury. We hypothesize SF stress engages the dysregulated HPA-axis after TBI to exacerbate post-injury neuroinflammation and compromise recovery. To test this, male and female mice were given moderate lateral fluid percussion TBI or sham-injury and left undisturbed or exposed to daily, transient SF for 7- or 30-days post-injury (DPI). Post-TBI SF increases cortical expression of interferon- and stress-associated genes characterized by inhibition of the upstream regulator NR3C1 that encodes glucocorticoid receptor (GR). Moreover, post-TBI SF increases neuronal activity in the hippocampus, a key intersection of the stress-immune axes. By 30 DPI, TBI SF enhances cortical microgliosis and increases expression of pro-inflammatory glial signaling genes characterized by persistent inhibition of the NR3C1 upstream regulator. Within the hippocampus, post-TBI SF exaggerates microgliosis and decreases CA1 neuronal activity. Downstream of the hippocampus, post-injury SF suppresses neuronal activity in the hypothalamic paraventricular nucleus indicating decreased HPA-axis reactivity. Direct application of GR agonist, dexamethasone, to the CA1 at 30 DPI increases GR activity in TBI animals, but not sham animals, indicating differential GR-mediated hippocampal action. Electrophysiological assessment revealed TBI and SF induces deficits in Schaffer collateral long-term potentiation associated with impaired acquisition of trace fear conditioning, reflecting dorsal hippocampal-dependent cognitive deficits. Together these data demonstrate that post-injury SF engages the dysfunctional post-injury HPA-axis, enhances inflammation, and compromises hippocampal function. Therefore, external stressors that disrupt sleep have an integral role in mediating outcome after brain injury.

Lateral Fluid Percussion Injury Causes Sex-Specific Deficits in Anterograde but Not Retrograde Memory.

Cognitive impairment is a common symptom after traumatic brain injury (TBI). Memory, in particular, is often disrupted during chronic post-injury recovery. To understand the sex-specific effects of brain injury on retrograde and anterograde memory, we examined paired associate learning (PAL), spatial learning and memory, and fear memory after lateral fluid percussion TBI. We hypothesized that male and female mice would display unique memory deficits after TBI. PAL task acquisition was initiated via touchscreen operant conditioning 22 weeks before sham injury or TBI. Post-injury PAL testing occurred 7 weeks post-injury. Barnes maze and fear conditioning were completed at 14- and 15-weeks post-injury, respectively. Contrary to our expectations, behavioral outcomes were not primarily influenced by TBI. Instead, sex-specific differences were observed in all tasks which exposed task-specific trends in male TBI mice. Male mice took longer to complete the PAL task, but this was not affected by TBI and did not compromise the ability to make a correct choice. Latency to reach the goal box decreased across testing days in Barnes maze, but male TBI mice lagged in improvement compared to all other groups. Use of two learning indices revealed that male TBI mice were deficient in transferring information from 1 day to the next. Finally, acquisition and contextual retention of fear memory were similar between all groups. Cued retention of the tone-shock pairing was influenced by both injury and sex. Male sham mice displayed the strongest cued retention of fear memory, evidenced by increased freezing behavior across the test trial. In contrast, male TBI mice displayed reduced freezing behavior with repetitive tone exposure. An inverse relationship in freezing behavior to tone exposure was detected between female sham and TBI mice, although the difference was not as striking. Together, these studies show that retrograde memory is intact after lateral TBI. However, male mice are more vulnerable to post-injury anterograde memory deficits. These behaviors were not associated with gross pathological change near the site injury or in subcortical brain regions associated with memory formation. Future studies that incorporate pre- and post-injury behavioral analysis will be integral in defining sex-specific memory impairment after TBI.

Robust Transcriptional Profiling and Identification of Differentially Expressed Genes With Low Input RNA Sequencing of Adult Hippocampal Neural Stem and Progenitor Populations.

Multipotent neural stem cells (NSCs) are found in several isolated niches of the adult mammalian brain where they have unique potential to assist in tissue repair. Modern transcriptomics offer high-throughput methods for identifying disease or injury associated gene expression signatures in endogenous adult NSCs, but they require adaptation to accommodate the rarity of NSCs. Bulk RNA sequencing (RNAseq) of NSCs requires pooling several mice, which impedes application to labor-intensive injury models. Alternatively, single cell RNAseq can profile hundreds to thousands of cells from a single mouse and is increasingly used to study NSCs. The consequences of the low RNA input from a single NSC on downstream identification of differentially expressed genes (DEGs) remains insufficiently explored. Here, to clarify the role that low RNA input plays in NSC DEG identification, we directly compared DEGs in an oxidative stress model of cultured NSCs by bulk and single cell sequencing. While both methods yielded DEGs that were replicable, single cell sequencing using the 10X Chromium platform yielded DEGs derived from genes with higher relative transcript counts compared to non-DEGs and exhibited smaller fold changes than DEGs identified by bulk RNAseq. The loss of high fold-change DEGs in the single cell platform presents an important limitation for identifying disease-relevant genes. To facilitate identification of such genes, we determined an RNA-input threshold that enables transcriptional profiling of NSCs comparable to standard bulk sequencing and used it to establish a workflow for in vivo profiling of endogenous NSCs. We then applied this workflow to identify DEGs after lateral fluid percussion injury, a labor-intensive animal model of traumatic brain injury. Our work joins an emerging body of evidence suggesting that single cell RNA sequencing may underestimate the diversity of pathologic DEGs. However, our data also suggest that population level transcriptomic analysis can be adapted to capture more of these DEGs with similar efficacy and diversity as standard bulk sequencing. Together, our data and workflow will be useful for investigators interested in understanding and manipulating adult hippocampal NSC responses to various stimuli.

Traumatic Brain Injury Causes Chronic Cortical Inflammation and Neuronal Dysfunction Mediated by Microglia.

Traumatic brain injury (TBI) can lead to significant neuropsychiatric problems and neurodegenerative pathologies, which develop and persist years after injury. Neuroinflammatory processes evolve over this same period. Therefore, we aimed to determine the contribution of microglia to neuropathology at acute [1 d postinjury (dpi)], subacute (7 dpi), and chronic (30 dpi) time points. Microglia were depleted with PLX5622, a CSF1R antagonist, before midline fluid percussion injury (FPI) in male mice and cortical neuropathology/inflammation was assessed using a neuropathology mRNA panel. Gene expression associated with inflammation and neuropathology were robustly increased acutely after injury (1 dpi) and the majority of this expression was microglia independent. At 7 and 30 dpi, however, microglial depletion reversed TBI-related expression of genes associated with inflammation, interferon signaling, and neuropathology. Myriad suppressed genes at subacute and chronic endpoints were attributed to neurons. To understand the relationship between microglia, neurons, and other glia, single-cell RNA sequencing was completed 7 dpi, a critical time point in the evolution from acute to chronic pathogenesis. Cortical microglia exhibited distinct TBI-associated clustering with increased type-1 interferon and neurodegenerative/damage-related genes. In cortical neurons, genes associated with dopamine signaling, long-term potentiation, calcium signaling, and synaptogenesis were suppressed. Microglial depletion reversed the majority of these neuronal alterations. Furthermore, there was reduced cortical dendritic complexity 7 dpi, reduced neuronal connectively 30 dpi, and cognitive impairment 30 dpi. All of these TBI-associated functional and behavioral impairments were prevented by microglial depletion. Collectively, these studies indicate that microglia promote persistent neuropathology and long-term functional impairments in neuronal homeostasis after TBI.SIGNIFICANCE STATEMENT Millions of traumatic brain injuries (TBIs) occur in the United States alone each year. Survivors face elevated rates of cognitive and psychiatric complications long after the inciting injury. Recent studies of human brain injury link chronic neuroinflammation to adverse neurologic outcomes, suggesting that evolving inflammatory processes may be an opportunity for intervention. Here, we eliminate microglia to compare the effects of diffuse TBI on neurons in the presence and absence of microglia and microglia-mediated inflammation. In the absence of microglia, neurons do not undergo TBI-induced changes in gene transcription or structure. Microglial elimination prevented TBI-induced cognitive changes 30 d postinjury (dpi). Therefore, microglia have a critical role in disrupting neuronal homeostasis after TBI, particularly at subacute and chronic timepoints.

A Levee to the Flood: Pre-injury Neuroinflammation and Immune Stress Influence Traumatic Brain Injury Outcome.

Increasing evidence demonstrates that aging influences the brain's response to traumatic brain injury (TBI), setting the stage for neurodegenerative pathology like Alzheimer's disease (AD). This topic is often dominated by discussions of post-injury aging and inflammation, which can diminish the consideration of those same factors before TBI. In fact, pre-TBI aging and inflammation may be just as critical in mediating outcomes. For example, elderly individuals suffer from the highest rates of TBI of all severities. Additionally, pre-injury immune challenges or stressors may alter pathology and outcome independent of age. The inflammatory response to TBI is malleable and influenced by previous, coincident, and subsequent immune insults. Therefore, pre-existing conditions that elicit or include an inflammatory response could substantially influence the brain's ability to respond to traumatic injury and ultimately affect chronic outcome. The purpose of this review is to detail how age-related cellular and molecular changes, as well as genetic risk variants for AD affect the neuroinflammatory response to TBI. First, we will review the sources and pathology of neuroinflammation following TBI. Then, we will highlight the significance of age-related, endogenous sources of inflammation, including changes in cytokine expression, reactive oxygen species processing, and mitochondrial function. Heightened focus is placed on the mitochondria as an integral link between inflammation and various genetic risk factors for AD. Together, this review will compile current clinical and experimental research to highlight how pre-existing inflammatory changes associated with infection and stress, aging, and genetic risk factors can alter response to TBI.

Comparison between midline and lateral fluid percussion injury in mice reveals prolonged but divergent cortical neuroinflammation.

Animal models are critical for determining the mechanisms mediating traumatic brain injury-induced (TBI) neuropathology. Fluid percussion injury (FPI) is a widely used model of brain injury typically applied either midline or parasagittally (lateral). Midline FPI induces a diffuse TBI, while lateral FPI induces both focal cortical injury (ipsilateral hemisphere) and diffuse injury (contralateral hemisphere). Nonetheless, discrete differences in neuroinflammation and neuropathology between these two versions of FPI remain unclear. The purpose of this study was to compare acute (4-72 h) and subacute (7 days) neuroinflammatory responses between midline and lateral FPI. Midline FPI resulted in longer righting reflex times than lateral FPI. At acute time points, the inflammatory responses to the two different injuries were similar. For instance, there was evidence of monocytes and cytokine mRNA expression in the brain with both injuries acutely. Midline FPI had the highest proportion of brain monocytes and highest IL-1ß/TNFα mRNA expression 24 h later. NanoString nCounter analysis 7 days post-injury revealed robust and prolonged expression of inflammatory-related genes in the cortex after midline FPI compared to lateral FPI; however, Iba-1 cortical immunoreactivity was increased with lateral FPI. Thus, midline and lateral FPI caused similar cortical neuroinflammatory responses acutely and mRNA expression of inflammatory genes was detectable in the brain 7 days later. The primary divergence was that inflammatory gene expression was greater and more diverse subacutely after midline FPI. These results provide novel insight to variations between midline and lateral FPI, which may recapitulate unique temporal pathogenesis.