Sex differences in the extent of acute axonal pathologies after experimental concussion.

Although human females appear be at a higher risk of concussion and suffer worse outcomes than males, underlying mechanisms remain unclear. With increasing recognition that damage to white matter axons is a key pathologic substrate of concussion, we used a clinically relevant swine model of concussion to explore potential sex differences in the extent of axonal pathologies. At 24 h post-injury, female swine displayed a greater number of swollen axonal profiles and more widespread loss of axonal sodium channels than males. Axon degeneration for both sexes appeared to be related to individual axon architecture, reflected by a selective loss of small caliber axons after concussion. However, female brains had a higher percentage of small caliber axons, leading to more extensive axon loss after injury compared to males. Accordingly, sexual dimorphism in axonal size is associated with more extensive axonal pathology in females after concussion, which may contribute to worse outcomes.

Neuronal somatic plasmalemmal permeability and dendritic beading caused by head rotational traumatic brain injury in pigs-An exploratory study.

Closed-head traumatic brain injury (TBI) is induced by rapid motion of the head, resulting in diffuse strain fields throughout the brain. The injury mechanism(s), loading thresholds, and neuroanatomical distribution of affected cells remain poorly understood, especially in the gyrencephalic brain. We utilized a porcine model to explore the relationships between rapid head rotational acceleration-deceleration loading and immediate alterations in plasmalemmal permeability within cerebral cortex, sub-cortical white matter, and hippocampus. To assess plasmalemmal compromise, Lucifer yellow (LY), a small cell-impermeant dye, was delivered intraventricularly and diffused throughout the parenchyma prior to injury in animals euthanized at 15-min post-injury; other animals (not receiving LY) were survived to 8-h or 7-days. Plasmalemmal permeability preferentially occurred in neuronal somata and dendrites, but rarely in white matter axons. The burden of LY+ neurons increased based on head rotational kinematics, specifically maximum angular velocity, and was exacerbated by repeated TBI. In the cortex, LY+ cells were prominent in both the medial and lateral gyri. Neuronal membrane permeability was observed within the hippocampus and entorhinal cortex, including morphological changes such as beading in dendrites. These changes correlated with reduced fiber volleys and synaptic current alterations at later timepoints in the hippocampus. Further histological observations found decreased NeuN immunoreactivity, increased mitochondrial fission, and caspase pathway activation in both LY+ and LY- cells, suggesting the presence of multiple injury phenotypes. This exploratory study suggests relationships between plasmalemmal disruptions in neuronal somata and dendrites within cortical and hippocampal gray matter as a primary response in closed-head rotational TBI and sets the stage for future, traditional hypothesis-testing experiments.

Axonal Tract Reconstruction Using a Tissue-Engineered Nigrostriatal Pathway in a Rat Model of Parkinson's Disease.

Parkinson's disease (PD) affects 1-2% of people over 65, causing significant morbidity across a progressive disease course. The classic PD motor deficits are caused by the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc), resulting in the loss of their long-distance axonal projections that modulate striatal output. While contemporary treatments temporarily alleviate symptoms of this disconnection, there is no approach able to replace the nigrostriatal pathway. We applied microtissue engineering techniques to create a living, implantable tissue-engineered nigrostriatal pathway (TE-NSP) that mimics the architecture and function of the native pathway. TE-NSPs comprise a discrete population of dopaminergic neurons extending long, bundled axonal tracts within the lumen of hydrogel micro-columns. Neurons were isolated from the ventral mesencephalon of transgenic rats selectively expressing the green fluorescent protein in dopaminergic neurons with subsequent fluorescent-activated cell sorting to enrich a population to 60% purity. The lumen extracellular matrix and growth factors were varied to optimize cytoarchitecture and neurite length, while immunocytochemistry and fast-scan cyclic voltammetry (FSCV) revealed that TE-NSP axons released dopamine and integrated with striatal neurons in vitro. Finally, TE-NSPs were implanted to span the nigrostriatal pathway in a rat PD model with a unilateral 6-hydroxydopamine SNpc lesion. Immunohistochemistry and FSCV established that transplanted TE-NSPs survived, maintained their axonal tract projections, extended dopaminergic neurites into host tissue, and released dopamine in the striatum. This work showed proof of concept that TE-NSPs can reconstruct the nigrostriatal pathway, providing motivation for future studies evaluating potential functional benefits and long-term durability of this strategy. This pathway reconstruction strategy may ultimately replace lost neuroarchitecture and alleviate the cause of motor symptoms for PD patients.

Neuroimmune interactions and immunoengineering strategies in peripheral nerve repair.

Peripheral nerve injuries result in disrupted cellular communication between the central nervous system and somatic distal end targets. The peripheral nervous system is capable of independent and extensive regeneration; however, meaningful target muscle reinnervation and functional recovery remain limited and may result in chronic neuropathic pain and diminished quality of life. Macrophages, the primary innate immune cells of the body, are critical contributors to regeneration of the injured peripheral nervous system. However, in some clinical scenarios, macrophages may fail to provide adequate support with optimal timing, duration, and location. Here, we review the history of immunosuppressive and immunomodulatory strategies to treat nerve injuries. Thereafter, we enumerate the ways in which macrophages contribute to successful nerve regeneration. We argue that implementing macrophage-based immunomodulatory therapies is a promising treatment strategy for nerve injuries across a wide range of clinical presentations.

Relationships between injury kinematics, neurological recovery, and pathology following concussion.

Mild traumatic brain injury affects millions of individuals annually primarily through falls, traffic collisions, or blunt trauma and can generate symptoms that persist for years. Closed-head rotational loading is the most common cause of mild traumatic brain injury and is defined by a rapid rotational acceleration of brain tissue within an intact skull. Injury kinematics-the mechanical descriptors of injury-inducing motion-explain movement of the head, which govern energy transfer, and, therefore, determine injury severity. However, the relationship between closed-head rotational injury kinematics-such as angular velocity, angular acceleration, and injury duration-and outcome after mild traumatic brain injury is not completely understood. To address this gap in knowledge, we analysed archived surgical records of 24 swine experiencing a diffuse closed-head rotational acceleration mild traumatic brain injury against 12 sham animals. Kinematics were contrasted against acute recovery outcomes, specifically apnea time, extubation time, standing time, and recovery duration. Compared to controls, animals experiencing a mild traumatic brain injury were far more likely to have apnea (P < 0.001), shorter time to extubation (P = 0.023), and longer time from extubation to standing (P = 0.006). Using least absolute shrinkage and selection operator-based regressions, kinematic parameters, including maximum negative angular velocity and time from peak angular velocity to maximum angular deceleration, were selected to explain variation in apnea time, standing time, and recovery duration. Simplified linear models employing the least absolute shrinkage and selection operator-selected variables explained a modest degree of variation in apnea time (adjusted R 2 = 0.18), standing time (adjusted R 2 = 0.19), and recovery duration (adjusted R 2 = 0.27). Neuropathology was correlated with multiple injury kinematics, with maximum angular acceleration exhibiting the strongest correlation (R 2 = 0.66). Together, these data suggest the interplay between multiple injury kinematics, including maximum negative angular velocity (immediately preceding cessation of head motion) and time from peak angular velocity to maximum angular deceleration, best explain acute recovery metrics and neuropathology after mild traumatic brain injury in swine. Future experiments that independently manipulate individual kinematic parameters could be instrumental in developing translational diagnostics for clinical mild traumatic brain injury.

Diverse changes in microglia morphology and axonal pathology during the course of 1 year after mild traumatic brain injury in pigs.

Over 2.8 million people experience mild traumatic brain injury (TBI) in the United States each year, which may lead to long-term neurological dysfunction. The mechanical forces that are caused by TBI propagate through the brain to produce diffuse axonal injury (DAI) and trigger secondary neuroinflammatory cascades. The cascades may persist from acute to chronic time points after injury, altering the homeostasis of the brain. However, the relationship between the hallmark axonal pathology of diffuse TBI and potential changes in glial cell activation or morphology have not been established in a clinically relevant large animal model at chronic time points. In this study, we assessed the tissue from pigs subjected to rapid head rotation in the coronal plane to generate mild TBI. Neuropathological assessments for axonal pathology, microglial morphological changes, and astrocyte reactivity were conducted in specimens out to 1-year post-injury. We detected an increase in overall amyloid precursor protein pathology, as well as periventricular white matter and fimbria/fornix pathology after a single mild TBI. We did not detect the changes in corpus callosum integrity or astrocyte reactivity. However, detailed microglial skeletal analysis revealed changes in morphology, most notably increases in the number of microglial branches, junctions, and endpoints. These subtle changes were most evident in periventricular white matter and certain hippocampal subfields, and were observed out to 1-year post-injury in some cases. These ongoing morphological alterations suggest persistent change in neuroimmune homeostasis. Additional studies are needed to characterize the underlying molecular and neurophysiological alterations, as well as potential contributions to neurological deficits.

Laboratory Assessment of a Headband-Mounted Sensor for Measurement of Head Impact Rotational Kinematics.

Head impact sensors measure head kinematics in sports, and sensor accuracy is crucial for investigating the potential link between repetitive head loading and clinical outcomes. Many validation studies mount sensors to human head surrogates and compare kinematic measures during loading from a linear impactor. These studies are often unable to distinguish intrinsic instrumentation limitations from variability caused by sensor coupling. The aim of the current study was to evaluate intrinsic sensor error in angular velocity in the absence of coupling error for a common head impact sensor. Two Triax SIM-G sensors were rigidly attached to a preclinical rotational injury device and subjected to rotational events to assess sensor reproducibility and accuracy. Peak angular velocities between the SIM-G sensors paired for each test were correlated (R2 > 0.99, y = 1.00x, p < 0.001). SIM-G peak angular velocity correlated with the reference (R2 = 0.96, y = 0.82x, p < 0.001); however, SIM-G underestimated the magnitude by 15.0% ± 1.7% (p < 0.001). SIM-G angular velocity rise time (5% to 100% of peak) correlated with the reference (R2 = 0.97, y = 1.06x, p < 0.001) but exhibited a slower fall time (100% to 5% of peak) by 9.0 ± 3.7 ms (p < 0.001). Assessing sensor performance when rigidly coupled is a crucial first step to interpret on-field SIM-G rotational kinematic data. Further testing in increasing biofidelic conditions is needed to fully characterize error from other sources, such as coupling.

Mossy cell hypertrophy and synaptic changes in the hilus following mild diffuse traumatic brain injury in pigs.

BACKGROUND: Each year in the USA, over 2.4 million people experience mild traumatic brain injury (TBI), which can induce long-term neurological deficits. The dentate gyrus of the hippocampus is notably susceptible to damage following TBI, as hilar mossy cell changes in particular may contribute to post-TBI dysfunction. Moreover, microglial activation after TBI may play a role in hippocampal circuit and/or synaptic remodeling; however, the potential effects of chronic microglial changes are currently unknown. The objective of the current study was to assess neuropathological and neuroinflammatory changes in subregions of the dentate gyrus at acute to chronic time points following mild TBI using an established model of closed-head rotational acceleration induced TBI in pigs. METHODS: This study utilized archival tissue of pigs which were subjected to sham conditions or rapid head rotation in the coronal plane to generate mild TBI. A quantitative assessment of neuropathological changes in the hippocampus was performed via immunohistochemical labeling of whole coronal tissue sections at 3 days post-injury (DPI), 7 DPI, 30 DPI, and 1 year post-injury (YPI), with a focus on mossy cell atrophy and synaptic reorganization, in context with microglial alterations (e.g., density, proximity to mossy cells) in the dentate gyrus. RESULTS: There were no changes in mossy cell density between sham and injured animals, indicating no frank loss of mossy cells at the mild injury level evaluated. However, we found significant mossy cell hypertrophy at 7 DPI and 30 DPI in anterior (> 16% increase in mean cell area at each time; p = <  0.001 each) and 30 DPI in posterior (8.3% increase; p = <  0.0001) hippocampus. We also found dramatic increases in synapsin staining around mossy cells at 7 DPI in both anterior (74.7% increase in synapsin labeling; p = <  0.0001) and posterior (82.7% increase; p = < 0.0001) hippocampus. Interestingly, these morphological and synaptic alterations correlated with a significant change in microglia in proximity to mossy cells at 7 DPI in anterior and at 30 DPI in the posterior hippocampus. For broader context, while we found that there were significant increases in microglia density in the granule cell layer at 30 DPI (anterior and posterior) and 1 YPI (posterior only) and in the molecular layer at 1 YPI (anterior only), we found no significant changes in overall microglial density in the hilus at any of the time points evaluated post-injury. CONCLUSIONS: The alterations of mossy cell size and synaptic inputs paired with changes in microglia density around the cells demonstrate the susceptibility of hilar mossy cells after even mild TBI. This subtle hilar mossy cell pathology may play a role in aberrant hippocampal function post-TBI, although additional studies are needed to characterize potential physiological and cognitive alterations.

Biomaterial-mediated reprogramming of monocytes via microparticle phagocytosis for sustained modulation of macrophage phenotype.

Monocyte-derived macrophages orchestrate tissue regeneration by homing to sites of injury, phagocytosing pathological debris, and stimulating other cell types to repair the tissue. Accordingly, monocytes have been investigated as a translational and potent source for cell therapy, but their utility has been hampered by their rapid acquisition of a pro-inflammatory phenotype in response to the inflammatory injury microenvironment. To overcome this problem, we designed a cell therapy strategy where monocytes are exogenously reprogrammed by intracellularly loading the cells with biodegradable microparticles containing an anti-inflammatory drug in order to modulate and maintain an anti-inflammatory phenotype over time. To test this concept, poly(lactic-co-glycolic) acid microparticles were loaded with the anti-inflammatory drug dexamethasone (Dex) and administered to primary human monocytes for four hours to facilitate phagocytic uptake. After removal of non-phagocytosed microparticles, microparticle-loaded monocytes differentiated into macrophages and stored the microparticles intracellularly for several weeks in vitro, releasing drug into the extracellular environment over time. Cells loaded with intracellular Dex microparticles showed decreased expression and secretion of inflammatory factors even in the presence of pro-inflammatory stimuli up to 7 days after microparticle uptake compared to untreated cells or cells loaded with blank microparticles, without interfering with phagocytosis of tissue debris. This study represents a new strategy for long-term maintenance of anti-inflammatory macrophage phenotype using a translational monocyte-based cell therapy strategy without the use of genetic modification. Because of the ubiquitous nature of monocyte-derived macrophage involvement in pathology and regeneration, this strategy holds potential as a treatment for a vast number of diseases and disorders. STATEMENT OF SIGNIFICANCE: We report a unique and translational strategy to overcome the challenges associated with monocyte- and macrophage-based cell therapies, in which the cells rapidly take on inflammatory phenotypes when administered to sites of injury. By intracellularly loading monocytes with drug-loaded microparticles prior to administration via phagocytosis, we were able to inhibit inflammation while preserving functional behaviors of human primary macrophages derived from those monocytes up to seven days later. To our knowledge, this study represents the first report of reprogramming macrophages to an anti-inflammatory phenotype without the use of genetic modification.

Bundled Three-Dimensional Human Axon Tracts Derived from Brain Organoids.

Reestablishing cerebral connectivity is a critical part of restoring neuronal network integrity and brain function after trauma, stroke, and neurodegenerative diseases. Creating transplantable axon tracts in the laboratory is an unexplored strategy for overcoming the common barriers limiting axon regeneration in vivo, including growth-inhibiting factors and the limited outgrowth capacity of mature neurons in the brain. We describe the generation, phenotype, and connectivity of constrained three-dimensional human axon tracts derived from brain organoids. These centimeter-long constructs are encased in an agarose shell that permits physical manipulation and are composed of discrete cellular regions spanned by axon tracts, mirroring the separation of cerebral gray and white matter. Features of cerebral cortex also are emulated, as evidenced by the presence of neurons with different cortical layer phenotypes. This engineered neural tissue represents a first step toward potentially reconstructing brain circuits by physically replacing neuronal populations and long-range axon tracts in the brain.

Acute drivers of neuroinflammation in traumatic brain injury.

Neuroinflammation is initiated as a result of traumatic brain injury and can exacerbate evolving tissue pathology. Immune cells respond to acute signals from damaged cells, initiate neuroinflammation, and drive the pathological consequences over time. Importantly, the mechanism(s) of injury, the location of the immune cells within the brain, and the animal species all contribute to immune cell behavior following traumatic brain injury. Understanding the signals that initiate neuroinflammation and the context in which they appear may be critical for understanding immune cell contributions to pathology and regeneration. Within this paper, we review a number of factors that could affect immune cell behavior acutely following traumatic brain injury.

Modulation of macrophage phenotype via phagocytosis of drug-loaded microparticles.

Monocyte-derived macrophages play a critical role in directing wound pathology following injury. Depending on their phenotype, macrophages also promote tissue regeneration. However, the therapeutic administration of macrophages with a controlled phenotype is challenging because macrophages are highly plastic and quickly revert to a detrimental, inflammatory phenotype in response to the environment of a damaged tissue. To address this issue, we developed a novel strategy to modulate macrophage phenotype intracellularly through phagocytosis of drug-loaded microparticles. Poly(lactic-co-glycolic acid) microparticles loaded with the anti-inflammatory drug dexamethasone (Dex) were phagocytosed by monocytes and stored intracellularly for at least 5 days. After differentiation into macrophages, cell phenotype was characterized over time with high-throughput gene expression analysis via NanoString. We found that the microparticles modulated macrophage phenotype for up to 7 days after microparticle uptake, with decreases in inflammation-related genes at early timepoints and upregulation of homing- and phagocytosis-related genes at multiple timepoints in a manner similar to cells treated with continuous free Dex. These data suggest that intracellularly loading macrophages with Dex microparticles via phagocytosis could be a unique methodology to selectively modulate macrophage phenotype over time. This strategy would allow therapeutic administration of macrophages for the treatment of a number of inflammatory disease and disorders. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1213-1224, 2019.

Concussion Induces Hippocampal Circuitry Disruption in Swine.

Hippocampal-dependent deficits in learning and memory formation are a prominent feature of traumatic brain injury (TBI); however, the role of the hippocampus in cognitive dysfunction after concussion (mild TBI) is unknown. We therefore investigated functional and structural changes in the swine hippocampus following TBI using a model of head rotational acceleration that closely replicates the biomechanics and neuropathology of closed-head TBI in humans. We examined neurophysiological changes using a novel ex vivo hippocampal slice paradigm with extracellular stimulation and recording in the dentate gyrus and CA1 occurring at 7 days following non-impact inertial TBI in swine. Hippocampal neurophysiology post-injury revealed reduced axonal function, synaptic dysfunction, and regional hyperexcitability at one week following even "mild" injury levels. Moreover, these neurophysiological changes occurred in the apparent absence of intra-hippocampal neuronal or axonal degeneration. Input-output curves demonstrated an elevated excitatory post-synaptic potential (EPSP) output for a given fiber volley input in injured versus sham animals, suggesting a form of homeostatic plasticity that manifested as a compensatory response to decreased axonal function in post-synaptic regions. These data indicate that closed-head rotational acceleration-induced TBI, the common cause of concussion in humans, may induce significant alterations in hippocampal circuitry function that have not resolved at 7 days post-injury. This circuitry dysfunction may underlie some of the post-concussion symptomatology associated with the hippocampus, such as post-traumatic amnesia and ongoing cognitive deficits.

Rapid neuroinflammatory response localized to injured neurons after diffuse traumatic brain injury in swine.

Despite increasing appreciation of the critical role that neuroinflammatory pathways play in brain injury and neurodegeneration, little is known about acute microglial reactivity following diffuse traumatic brain injury (TBI) - the most common clinical presentation that includes all concussions. Therefore, we investigated acute microglial reactivity using a porcine model of closed-head rotational velocity/acceleration-induced TBI that closely mimics the biomechanical etiology of inertial TBI in humans. We observed rapid microglial reactivity within 15min of both mild and severe TBI. Strikingly, microglial activation was restrained to regions proximal to individual injured neurons - as denoted by trauma-induced plasma membrane disruption - which served as epicenters of acute reactivity. Single-cell quantitative analysis showed that in areas free of traumatically permeabilized neurons, microglial density and morphology were similar between sham or following mild or severe TBI. However, microglia density increased and morphology shifted to become more reactive in proximity to injured neurons. Microglial reactivity around injured neurons was exacerbated following repetitive TBI, suggesting further amplification of acute neuroinflammatory responses. These results indicate that neuronal trauma rapidly activates microglia in a highly localized manner, and suggest that activated microglia may rapidly influence neuronal stability and/or pathophysiology after diffuse TBI.