Traumatic brain injury induces TDP-43 mislocalization and neurodegenerative effects in tissue distal to the primary injury site in a non-transgenic mouse.

Traumatic brain injury (TBI) initiates tissue and cellular damage to the brain that is immediately followed by secondary injury sequalae with delayed and continual damage. This secondary damage includes pathological processes that may contribute to chronic neurodegeneration and permanent functional and cognitive deficits. TBI is also associated with an increased risk of developing neurodegenerative diseases such as Alzheimer's disease (AD), frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS) as indicated by shared pathological features. For example, abnormalities in the TAR DNA-binding Protein 43 (TDP-43) that includes cytoplasmic mislocalization, cytosolic aggregation, and an increase in phosphorylation and ubiquitination are seen in up to 50% of FTD cases, up to 70% of AD cases, and is considered a hallmark pathology of ALS occurring in > 97% of cases. Yet the prevalence of TDP-43 pathology post-TBI has yet to be fully characterized. Here, we employed a non-transgenic murine controlled cortical injury model of TBI and observed injury-induced hallmark TDP-43 pathologies in brain and spinal cord tissue distal to the primary injury site and did not include the focally damaged tissue within the primary cortical injury site. Analysis revealed a temporal-dependent and significant increase in neuronal TDP-43 mislocalization in the cortical forebrain rostral to and distant from the primary injury site up to 180 days post injury (DPI). TDP-43 mislocalization was also detected in neurons located in the ventral horns of the cervical spinal cord following a TBI. Moreover, a cortical layer-dependent affect was identified, increasing from superficial to deeper cortical layers over time from 7 DPI up to 180 DPI. Lastly, RNAseq analysis confirmed an injury-induced misregulation of several key biological processes implicated in neurons that increased over time. Collectively, this study demonstrates a connection between a single moderate TBI event and chronic neurodegenerative processes that are not limited to the primary injury site and broadly distributed throughout the cortex and corticospinal tract.

Uncovering temporospatial sensitive TBI targeting strategies via in vivo phage display.

The heterogeneous pathophysiology of traumatic brain injury (TBI) is a barrier to advancing diagnostics and therapeutics, including targeted drug delivery. We used a unique discovery pipeline to identify novel targeting motifs that recognize specific temporal phases of TBI pathology. This pipeline combined in vivo biopanning with domain antibody (dAb) phage display, next-generation sequencing analysis, and peptide synthesis. We identified targeting motifs based on the complementarity-determining region 3 structure of dAbs for acute (1 day post-injury) and subacute (7 days post-injury) post-injury time points in a preclinical TBI model (controlled cortical impact). Bioreactivity and temporal sensitivity of the targeting motifs were validated via immunohistochemistry. Immunoprecipitation-mass spectrometry indicated that the acute TBI targeting motif recognized targets associated with metabolic and mitochondrial dysfunction, whereas the subacute TBI motif was largely associated with neurodegenerative processes. This pipeline successfully discovered temporally specific TBI targeting motif/epitope pairs that will serve as the foundation for the next-generation targeted TBI therapeutics and diagnostics.

GdDO3NI Enhanced Magnetic Resonance Imaging Allows Imaging of Hypoxia After Brain Injury.

BACKGROUND: Brain tissue hypoxia is a common consequence of traumatic brain injury (TBI) due to the rupture of blood vessels during impact and it correlates with poor outcome. The current magnetic resonance imaging (MRI) techniques are unable to provide a direct map of tissue hypoxia. PURPOSE: To investigate whether GdDO3NI, a nitroimidazole-based T1 MRI contrast agent allows imaging hypoxia in the injured brain after experimental TBI. STUDY TYPE: Prospective. ANIMAL MODEL: TBI-induced mice (controlled cortical impact model) were intravenously injected with either conventional T1 agent (gadoteridol) or GdDO3NI at 0.3 mmol/kg dose (n = 5 for each cohort) along with pimonidazole (60 mg/kg) at 1 hour postinjury and imaged for 3 hours following which they were euthanized. FIELD STRENGTH/SEQUENCE: 7 T/T2 -weighted spin echo and T1 -weighted gradient echo. ASSESSMENT: Injured animals were imaged with T2 -weighted spin-echo sequence to estimate the extent of the injury. The mice were then imaged precontrast and postcontrast using a T1 -weighted gradient-echo sequence for 3 hours postcontrast. Regions of interests were drawn on the brain injury region, the contralateral brain as well as on the cheek muscle region for comparison of contrast kinetics. Brains were harvested immediately post-imaging for immunohistochemical analysis. STATISTICAL TESTS: One-way analysis of variance and two-sample t-tests were performed with a P < 0.05 was considered statistically significant. RESULTS: GdDO3NI retention in the injury region at 2.5-3 hours post-injection was significantly higher compared to gadoteridol (mean retention fraction 63.95% ± 27.43% vs. 20.68% ± 7.43% for gadoteridol at 3 hours) while it rapidly cleared out of the muscle region. Pimonidazole staining confirmed the presence of hypoxia in both gadoteridol and GdDO3NI cohorts, and the later cohort showed good agreement with MRI contrast enhancement. DATA CONCLUSION: GdDO3NI was successfully shown to visualize hypoxia in the brain post-TBI using T1 -weighted MRI at 2.5-3 hours postcontrast. EVIDENCE LEVEL: 1 TECHNICAL EFFICACY: Stage 1.

Platelet-like particles reduce coagulopathy-related and neuroinflammatory pathologies post-experimental traumatic brain injury.

Coagulopathy may occur following traumatic brain injury (TBI), thereby negatively affecting patient outcomes. Here, we investigate the use of platelet-like particles (PLPs), poly(N-isopropylacrylamide-co-acrylic-acid) microgels conjugated with a fibrin-specific antibody, to improve hemostasis post-TBI. The objective of this study was to diminish coagulopathy in a mouse TBI model (controlled cortical impact) via PLP treatment, and subsequently decrease blood-brain barrier (BBB) permeability and neuroinflammation. Following an acute intravenous injection of PLPs post-TBI, we analyzed BBB permeability, ex vivo coagulation parameters, and neuroinflammation at 24 hr and 7 days post-TBI. Both PLP-treatment and control particle-treatment had significantly decreased BBB permeability and improved clot structure 24 hr post-injury. Additionally, no significant change in tissue sparing was observed between 24 hr and 7 days for PLP-treated cohorts compared to that observed in untreated cohorts. Only PLP-treatment resulted in significant reduction of astrocyte expression at 7 days and percent difference from 24 hr to 7 days. Finally, PLP-treatment significantly reduced the percent difference from 24 hr to 7 days in microglia/macrophage density compared to the untreated control. These results suggest that PLP-treatment addressed acute hypocoagulation and decreased BBB permeability followed by decreased neuroinflammation and fold-change tissue loss by 7 days post-injury. These promising results indicate that PLPs could be a potential therapeutic modality for TBI.

ß-Cyclodextrin-poly (ß-Amino Ester) Nanoparticles Are a Generalizable Strategy for High Loading and Sustained Release of HDAC Inhibitors.

Therapeutic development of histone deacetylase inhibitors (HDACi) has been hampered by a number of barriers to drug delivery, including poor solubility and inadequate tissue penetration. Nanoparticle encapsulation could be one approach to improve the delivery of HDACi to target tissues; however, effective and generalizable loading of HDACi within nanoparticle systems remains a long-term challenge. We hypothesized that the common terminally ionizable moiety on many HDACi molecules could be capitalized upon for loading in polymeric nanoparticles. Here, we describe the simple, efficient formulation of a novel library of ß-cyclodextrin-poly (ß-amino ester) networks (CDN) to achieve this goal. We observed that network architecture was a critical determinant of CDN encapsulation of candidate molecules, with a more hydrophobic core enabling effective self-assembly and a PEGylated surface enabling high loading (up to ∼30% w/w), effective self-assembly of the nanoparticle, and slow release of drug into aqueous media (up to 24 days) for the model HDACi panobinostat. We next constructed a library of CDNs to encapsulate various small, hydrophobic, terminally ionizable molecules (panobinostat, quisinostat, dacinostat, givinostat, bortezomib, camptothecin, nile red, and cytarabine), which yielded important insights into the structural requirements for effective drug loading and CDN self-assembly. Optimized CDN nanoparticles were taken up by GL261 cells in culture and a released panobinostat was confirmed to be bioactive. Panobinostat-loaded CDNs were next administered by convection-enhanced delivery (CED) to mice bearing intracranial GL261 tumors. These studies confirm that CDN encapsulation enables a higher deliverable dose of drug to effectively slow tumor growth. Matrix-assisted laser desorption/ionization (MALDI) analysis on tissue sections confirms higher exposure of tumor to drug, which likely accounts for the therapeutic effects. Taken in sum, these studies present a novel nanocarrier platform for encapsulation of HDACi via both ionic and hydrophobic interactions, which is an important step toward better treatment of disease via HDACi therapy.

Recent Advances in Stem Cell Therapies to Address Neuroinflammation, Stem Cell Survival, and the Need for Rehabilitative Therapies to Treat Traumatic Brain Injuries.

Traumatic brain injuries (TBIs) are a significant health problem both in the United States and worldwide with over 27 million cases being reported globally every year. TBIs can vary significantly from a mild TBI with short-term symptoms to a moderate or severe TBI that can result in long-term or life-long detrimental effects. In the case of a moderate to severe TBI, the primary injury causes immediate damage to structural tissue and cellular components. This may be followed by secondary injuries that can be the cause of chronic and debilitating neurodegenerative effects. At present, there are no standard treatments that effectively target the primary or secondary TBI injuries themselves. Current treatment strategies often focus on addressing post-injury symptoms, including the trauma itself as well as the development of cognitive, behavioral, and psychiatric impairment. Additional therapies such as pharmacological, stem cell, and rehabilitative have in some cases shown little to no improvement on their own, but when applied in combination have given encouraging results. In this review, we will abridge and discuss some of the most recent research advances in stem cell therapies, advanced engineered biomaterials used to support stem transplantation, and the role of rehabilitative therapies in TBI treatment. These research examples are intended to form a multi-tiered perspective for stem-cell therapies used to treat TBIs; stem cells and stem cell products to mitigate neuroinflammation and provide neuroprotective effects, biomaterials to support the survival, migration, and integration of transplanted stem cells, and finally rehabilitative therapies to support stem cell integration and compensatory and restorative plasticity.

Stromal Cell-Derived Factor-1a Autocrine/Paracrine Signaling Contributes to Spatiotemporal Gradients in the Brain.

INTRODUCTION: Stromal cell derived factor-1a (SDF-1a) and its receptor CXCR4 modulate stem cell recruitment to neural injury sites. SDF-1a gradients originating from injury sites contribute to chemotactic cellular recruitment. To capitalize on this injury-induced cell recruitment, further investigation of SDF-1a/CXCR4 signaling dynamics are warranted. Here, we studied how exogenous SDF-1a delivery strategies impact spatiotemporal SDF-1a levels and the role autocrine/paracrine signaling plays. METHODS: We first assessed total SDF-1a and CXCR4 levels over the course of 7 days following intracortical injection of either bolus SDF-1a or SDF-1a loaded nanoparticles in CXCR4-EGFP mice. We then investigated cellular contributors to SDF-1a autocrine/paracrine signaling via time course in vitro measurements of SDF-1a and CXCR4 gene expression following exogenous SDF-1a application. Lastly, we created mathematical models that could recapitulate our in vivo observations. RESULTS: In vivo, we found sustained total SDF-1a levels beyond 3 days post injection, indicating endogenous SDF-1a production. We confirmed in vitro that microglia, astrocytes, and brain endothelial cells significantly change SDF-1a and CXCR4 expression after exposure. We found that diffusion-only based mathematical models were unable to capture in vivo SDF-1a spatial distribution. Adding autocrine/paracrine mechanisms to the model allowed for SDF-1a temporal trends to be modeled accurately, indicating it plays an essential role in SDF-1a sustainment. CONCLUSIONS: We conclude that autocrine/paracrine dynamics play a role in endogenous SDF-1a levels in the brain following exogenous delivery. Implementation of these dynamics are necessary to improving SDF-1a delivery strategies. Further, mathematical models introduced here may be utilized in predicting future outcomes based upon new biomaterial designs.

In Vivo Phage Display as a Biomarker Discovery Tool for the Complex Neural Injury Microenvironment.

The heterogeneous injury pathophysiology of traumatic brain injury (TBI) is a barrier to developing highly sensitive and specific diagnostic tools. Phage display, a protein-protein screening technique routinely used in drug development, has the potential to be a powerful biomarker discovery tool for TBI. However, analysis of these large and diverse phage libraries is a bottleneck to moving through the discovery pipeline in a timely and efficient manner. This article describes a unique discovery pipeline involving domain antibody (dAb) phage in vivo biopanning and next-generation sequencing (NGS) analysis to identify targeting motifs that recognize distinct aspects of TBI pathology. To demonstrate this process, we conduct in vivo biopanning on the controlled cortical impact mouse model of experimental TBI at 1 and 7 days postinjury. Phage accumulation in target tissues is quantified via titers before NGS preparation and analysis. This phage display biomarker discovery pipeline for TBI successfully achieves discovery of temporally specific TBI targeting motifs and may further TBI biomarker research for other characteristics of injury. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Phage production and purification Support Protocol: Controlled cortical impact model Basic Protocol 2: Injection and elution of phage Basic Protocol 3: Amplicon sequencing and sequence analysis.

Hyaluronic Acid Biomaterials for Central Nervous System Regenerative Medicine.

Hyaluronic acid (HA) is a primary component of the brain extracellular matrix and functions through cellular receptors to regulate cell behavior within the central nervous system (CNS). These behaviors, such as migration, proliferation, differentiation, and inflammation contribute to maintenance and homeostasis of the CNS. However, such equilibrium is disrupted following injury or disease leading to significantly altered extracellular matrix milieu and cell functions. This imbalance thereby inhibits inherent homeostatic processes that support critical tissue health and functionality in the CNS. To mitigate the damage sustained by injury/disease, HA-based tissue engineering constructs have been investigated for CNS regenerative medicine applications. HA's effectiveness in tissue healing and regeneration is primarily attributed to its impact on cell signaling and the ease of customizing chemical and mechanical properties. This review focuses on recent findings to highlight the applications of HA-based materials in CNS regenerative medicine.

Sex-Dependent Macromolecule and Nanoparticle Delivery in Experimental Brain Injury.

The development of effective therapeutics for brain disorders is challenging, in particular, the blood-brain barrier (BBB) severely limits access of the therapeutics into the brain parenchyma. Traumatic brain injury (TBI) may lead to transient BBB permeability that affords a unique opportunity for therapeutic delivery via intravenous administration ranging from macromolecules to nanoparticles (NPs) for developing precision therapeutics. In this regard, we address critical gaps in understanding the range/size of therapeutics, delivery window(s), and moreover, the potential impact of biological factors for optimal delivery parameters. Here we show, for the first time, to the best of our knowledge, that 24-h postfocal TBI female mice exhibit a heightened macromolecular tracer and NP accumulation compared with male mice, indicating sex-dependent differences in BBB permeability. Furthermore, we report for the first time the potential to deliver NP-based therapeutics within 3 days after focal injury in both female and male mice. The delineation of injury-induced BBB permeability with respect to sex and temporal profile is essential to more accurately tailor time-dependent precision and personalized nanotherapeutics. Impact statement In this study, we identified a sex-dependent temporal profile of blood/brain barrier disruption in a preclinical mouse model of traumatic brain injury (TBI) that contributes to starkly different macromolecule and nanoparticle delivery profiles post-TBI. The implications and potential impact of this work are profound and far reaching as it indicates that a demand of true personalized medicine for TBI is necessary to deliver the right therapeutic at the right time for the right patient.

Extracellular matrix proteins are time-dependent and regional-specific markers in experimental diffuse brain injury.

INTRODUCTION: The extracellular matrix (ECM) provides structural support for neuronal, glial, and vascular components of the brain, and regulates intercellular signaling required for cellular morphogenesis, differentiation and homeostasis. We hypothesize that the pathophysiology of diffuse brain injury impacts the ECM in a multi-dimensional way across brain regions and over time, which could facilitate damage and repair processes. METHODS: Experimental diffuse TBI was induced in male Sprague-Dawley rats (325-375 g) by midline fluid percussion injury (FPI); uninjured sham rats serve as controls. Tissue from the cortex, thalamus, and hippocampus was collected at 15 min, 1, 2, 6, and 18 hr postinjury as well as 1, 3, 7, and 14 days postinjury. All samples were quantified by Western blot for glycoproteins: fibronectin, laminin, reelin, and tenascin-C. Band intensities were normalized to sham and relative to ß-actin. RESULTS: In the cortex, fibronectin decreased significantly at 15 min, 1 hr, and 2 hr postinjury, while tenascin-C decreased significantly at 7 and 14 days postinjury. In the thalamus, reelin decreased significantly at 2 hr, 3 and 14 days postinjury. In the hippocampus, tenascin-C increased significantly at 15 min and 7 days postinjury. CONCLUSION: Acute changes in the levels of these glycoproteins suggest involvement in circuit dismantling, whereas postacute levels may indicate a restorative or regenerative response associated with recovery from TBI.

Leveraging the Dynamic Blood-Brain Barrier for Central Nervous System Nanoparticle-based Drug Delivery Applications.

Neurological diseases and injuries have profound impact on a patient's lifespan and functional capabilities, but often lack effective intervention strategies to address the underlying neuropathology. The blood-brain barrier (BBB) is a major hurdle in the effective delivery of therapeutics to the brain. Recent discoveries in BBB maintenance reveal a dynamic system where time of day, disease progression, and even biological variables all strongly influence its permeability and flux of molecules. Nanoparticles can be used to improve the efficacy of therapeutics by increasing circulation time, bioavailability, selectivity, and controlling the rate of payload release. Considering these recent findings, the next generation of pharmacological paradigms are evolving to leverage nanotechnology to turn therapeutic intervention to meet the needs of a specific patient (i.e. personalized medicine).

Multi-Biomarker Detection Following Traumatic Brain Injury.

The Centers for Disease Control and Prevention estimates almost two million traumatic brain injuries (TBIs) occur annually in the U.S., resulting in nearly $80 billion of economic burden. Despite its prevalence, current TBI diagnosis methods mainly rely on cognitive assessments vulnerable to subjective interpretation, thus highlighting the critical need to develop effective unbiased diagnostic methods. The presented study aims to assess the feasibility of a rapid multianalyte TBI blood diagnostic. Specifically, two electrochemical impedance techniques were used to evaluate four biomarkers: glial fibrillary acidic protein, neuron specific enolase (NSE), S-100ß, and tumor necrosis factor-α. First, these biomarkers were characterized in purified solutions (detection limit, DL = 2-5 pg/mL), then verified in spiked whole blood and plasma solutions (90% whole blood DL = 14-67 pg/mL). Finally, detection of two of these biomarkers was validated in a controlled cortical impact model of TBI in rats, where a statistical difference between NSE and S-100ß concentrations differed several days postinjury (p = 0.02 and p = 0.06, respectively). A statistical difference between mild and moderate injury was found at the various time points. The proposed diagnostic method enabled preliminary quantification of TBI-relevant biomarkers in complex media without the use of expensive electrode coatings or membranes. Collectively, these data demonstrate the feasibility of using electrochemical impedance techniques to rapidly detect TBI biomarkers and lay the groundwork for development of a novel method for quantitative diagnostics of TBI.

Biomimetic Strategies To Treat Traumatic Brain Injury by Leveraging Fibrinogen.

There were over 27 million new cases of traumatic brain injuries (TBIs) in 2016 across the globe. TBIs are often part of complicated trauma scenarios and may not be diagnosed initially because the primary clinical focus is on stabilizing the patient. Interventions used to stabilize trauma patients may inadvertently impact the outcomes of TBIs. Recently, there has been a strong interest in the trauma community toward administrating fibrinogen-containing solutions intravenously to help stabilize trauma patients. While this interventional shift may benefit general trauma scenarios, fibrinogen is associated with potentially deleterious effects for TBIs. Here, we deconstruct what components of fibrinogen may be beneficial as well as potentially harmful following TBI and extrapolate this to biomimetic approaches to treat bleeding and trauma that may also lead to better outcomes following TBI.

Current trends in biomarker discovery and analysis tools for traumatic brain injury.

Traumatic brain injury (TBI) affects 1.7 million people in the United States each year, causing lifelong functional deficits in cognition and behavior. The complex pathophysiology of neural injury is a primary barrier to developing sensitive and specific diagnostic tools, which consequentially has a detrimental effect on treatment regimens. Biomarkers of other diseases (e.g. cancer) have provided critical insight into disease emergence and progression that lend to developing powerful clinical tools for intervention. Therefore, the biomarker discovery field has recently focused on TBI and made substantial advancements to characterize markers with promise of transforming TBI patient diagnostics and care. This review focuses on these key advances in neural injury biomarkers discovery, including novel approaches spanning from omics-based approaches to imaging and machine learning as well as the evolution of established techniques.

Exploiting Phage Display for Development of Novel Cellular Targeting Strategies.

Targeting strategies for drug delivery applications rely on targeting moieties (i.e., peptide, antibody) specific to the desired cell surface receptor or protein of interest. However, current targeting strategies are limited to previously identified epitopes/ligand pairs. The field of phage display opens up the targeting moiety options whereby new epitope/ligand pairs may be discovered through well-designed biopanning assays for the target cell population of interest. Here, we provide a detailed protocol to perform phage biopanning assays on adherent cell cultures. The methods described here may be modified to user-specific targeting interests.

Blood-brainbarrier disruption dictates nanoparticle accumulation following experimental brain injury.

Clinically, traumatic brain injury (TBI) results in complex heterogeneous pathology that cannot be recapitulated in single pre-clinical animal model. Therefore, we focused on evaluating utility of nanoparticle (NP)-based therapeutics following three diffuse-TBI models: mildclosed-head injury (mCHI), repetitive-mCHI and midline-fluid percussion injury (FPI). We hypothesized that NP accumulation after diffuse TBI correlates directly with blood-brainbarrier permeability. Mice received PEGylated-NP cocktail (20-500 nm) (intravenously) after single- or repetitive-(1 impact/day, 5 consecutive days) CHI (immediately) and midline-FPI (1 h, 3 h and 6 h). NPs circulated for 1 h before perfusion/brain extraction. NP accumulation was analyzed using fluorescent microscopy in brain regions vulnerable to neuropathology. Minimal/no NP accumulation after mCHI/RmCHI was observed. In contrast, midlineFPI resulted in significant peak accumulation of up to 500 nm NP at 3 h post-injury compared to sham, 1 h, and 6 h groups in the cortex. Therefore, our study provides the groundwork for feasibility of NP-delivery based on NPinjection time and NPsize after mCHI/RmCHI and midline-FPI.

pH driven precipitation of quisinostat onto PLA-PEG nanoparticles enables treatment of intracranial glioblastoma.

Histone deacetylases (HDACs) are known to be key enzymes in cancer development and progression through their modulation of chromatin structure and the expression and post-translational modification of numerous proteins. Aggressive dedifferentiated tumors, like glioblastoma, frequently overexpress HDACs, while HDAC inhibition can lead to cell cycle arrest, promote cellular differentiation and induce apoptosis. Although multiple HDAC inhibitors, such as quisinostat, are of interest in oncology due to their potent in vitro efficacy, their failure in the clinic as monotherapies against solid tumors has been attributed to poor delivery. Thus, we were motivated to develop quisinostat loaded poly(D,L-lactide)-b-methoxy poly(ethylene glycol) nanoparticles (NPs) to test their ability to treat orthotopic glioblastoma. In developing our NP formulation, we identified a novel, pH-driven approach for achieving over 9% (w/w) quisinostat loading. We show quisinostat-loaded NPs maintain drug potency in vitro and effectively slow tumor growth in vivo, leading to a prolonged survival compared to control mice.

Using biomaterials to modulate chemotactic signaling for central nervous system repair.

Chemotaxis enables cellular communication and movement within the body. This review focuses on exploiting chemotaxis as a tool for repair of the central nervous system (CNS) damaged from injury and/or degenerative diseases. Chemokines and factors alone may initiate repair following CNS injury/disease, but exogenous administration may enhance repair and promote regeneration. This review will discuss critical chemotactic molecules and factors that may promote neural regeneration. Additionally, this review highlights how biomaterials can impact the presentation and delivery of chemokines and growth factors to alter the regenerative response.