Extracellular vesicles derived from inflammatory-educated stem cells reverse brain inflammation-implication of miRNAs.

Inflammation plays a key role in the development of age-related diseases. In Alzheimer's disease, neuronal cell death is attributed to amyloidbeta oligomers that trigger microglial activation. Stem cells have shown promise as therapies for inflammatory diseases- because of their paracrine activity combined with their ability to respond to the inflammatory environment. However, the mechanisms underlying stem cell-promoted neurological recovery are poorly understood. To elucidate these mechanisms, we first primed stem cells with the secretome of lipopolysaccharide- or amyloidbeta-activated microglia. Then, we compared the immunomodulatory effects of extracellular vesicles (EVs) secreted from primed and non-primed stem cells. Our results demonstrate that EVs from primed cells are more effective in inhibiting microglia and astrocyte activation, amyloid deposition, demyelination, memory loss and motor and anxiety-like behavioral dysfunction, compared to EVs from non-primed cells. MicroRNA (miRNA) profiling revealed the upregulation of at least 19 miRNAs on primed-stem cell EVs. The miRNA targets were identified, and KEGG pathway analysis showed that the overexpressed miRNAs target key genes on the toll-like receptor-4 (TLR4) signaling pathway. Overall, our results demonstrate that priming mesenchymal stem cells (MSCs) with the secretome of activated microglia results in the release of miRNAs from EVs with enhanced immune regulatory potential able to fight neuroinflammation.

Role of Cannabidiol for Improvement of the Quality of Life in Cancer Patients: Potential and Challenges.

There is currently a growing interest in the use of cannabidiol (CBD) to alleviate the symptoms caused by cancer, including pain, sleep disruption, and anxiety. CBD is often self-administered as an over-the-counter supplement, and patients have reported benefits from its use. However, despite the progress made, the mechanisms underlying CBD's anti-cancer activity remain divergent and unclear. Herein, we provide a comprehensive review of molecular mechanisms to determine convergent anti-cancer actions of CBD from pre-clinical and clinical studies. In vitro studies have begun to elucidate the molecular targets of CBD and provide evidence of CBD's anti-tumor properties in cell and mouse models of cancer. Furthermore, several clinical trials have been completed testing CBD's efficacy in treating cancer-related pain. However, most use a mixture of CBD and the psychoactive, tetrahydrocannabinol (THC), and/or use variable dosing that is not consistent between individual patients. Despite these limitations, significant reductions in pain and opioid use have been reported in cancer patients using CBD or CBD+THC. Additionally, significant improvements in quality-of-life measures and patients' overall satisfaction with their treatment have been reported. Thus, there is growing evidence suggesting that CBD might be useful to improve the overall quality of life of cancer patients by both alleviating cancer symptoms and by synergizing with cancer therapies to improve their efficacy. However, many questions remain unanswered regarding the use of CBD in cancer treatment, including the optimal dose, effective combinations with other drugs, and which biomarkers/clinical presentation of symptoms may guide its use.

Real-time artificial intelligence-based histologic classification of colorectal polyps with augmented visualization.

BACKGROUND AND AIMS: Artificial intelligence (AI)-based computer-aided diagnostic (CADx) algorithms are a promising approach for real-time histology (RTH) of colonic polyps. Our aim is to present a novel in situ CADx approach that seeks to increase transparency and interpretability of results by generating an intuitive augmented visualization of the model's predicted histology over the polyp surface. METHODS: We developed a deep learning model using semantic segmentation to delineate polyp boundaries and a deep learning model to classify subregions within the segmented polyp. These subregions were classified independently and were subsequently aggregated to generate a histology map of the polyp's surface. We used 740 high-magnification narrow-band images from 607 polyps in 286 patients and over 65,000 subregions to train and validate the model. RESULTS: The model achieved a sensitivity of .96, specificity of .84, negative predictive value (NPV) of .91, and high-confidence rate (HCR) of .88, distinguishing 171 neoplastic polyps from 83 non-neoplastic polyps of all sizes. Among 93 neoplastic and 75 non-neoplastic polyps ≤5 mm, the model achieved a sensitivity of .95, specificity of .84, NPV of .91, and HCR of .86. CONCLUSIONS: The CADx model is capable of accurately distinguishing neoplastic from non-neoplastic polyps and provides a histology map of the spatial distribution of localized histologic predictions along the delineated polyp surface. This capability may improve interpretability and transparency of AI-based RTH and offer intuitive, accurate, and user-friendly guidance in real time for the clinical management and documentation of optical histology results.

A multifunctional nanoparticle as a prophylactic and therapeutic approach targeting respiratory syncytial virus.

Respiratory Syncytial Virus (RSV) has been a major health concern globally for decades, yet no effective prophylactic or treatment regimen is available. The key viral proteins responsible for RSV pathology include the fusion protein (F), the immunomodulatory non-structural-protein 1 (NS1) and the phosphoprotein (P) involved in viral replication. Herein, we developed a novel shell-core multifunctional nanosystem with dual payload: a plasmid construct encoding for shRNAs against NS1 and P, and an anti-fusion peptide (HR2D). Anti-ICAM1 antibody conjugated on the nanoparticle (NP) surface is used to target RSV infected cells. Our data show the potential of this nanosystem as a prophylactic and/or a therapeutic regimen against RSV infection. Furthermore, therapy of RSV infected mice with this nanosystem, in addition to reducing viral load, modulated expression of Th2 and allergy-associated cytokines such as IL4, IL-13 and IL-17 indicating a direct role of this nanosystem in the mechanisms involved in the immunoregulation of disease pathogenesis.

The design and characterization of a gravitational microfluidic platform for drug sensitivity assay in colorectal perfused tumoroid cultures.

The tumor microenvironment plays a critical role in tumor initiation, progression, metastasis, and drug resistance. However, models recapitulating the complex 3D structure, heterogeneous cell environment, and cell-cell interactions found in vivo are lacking. Herein, we report on a gravitational microfluidic platform (GMP) retrofitted with MEMS sensors, which is integrated with 3D nanofiber scaffold-aided tumoroid culture. The results showed that this GMP for tumoroid growth mimics the tumor microenvironment more precisely than static culture models of colon cancer, including higher drug resistance, enhanced cancer stem cell properties, and increased secretion of pro-tumor cytokines. In addition, the GMP includes an integrated surface acoustic wave-based biosensing to monitor cell growth and pH changes to assess drug efficacy. Thus, this simple-to-use perfused GMP tumoroid culture system for in vitro and ex vivo studies may accelerate the drug development process and be a tool in personalized cancer treatment.

Treatment with shCCL20-CCR6 nanodendriplexes and human mesenchymal stem cell therapy improves pathology in mice with repeated traumatic brain injury.

Traumatic brain injury (TBI) is a devastating neurological disorder, although the underlying pathophysiology is poorly understood. TBI causes blood-brain barrier (BBB) disruption, immune cell trafficking, neuroinflammation and neurodegeneration. CCL20 is an important chemokine mediating neuroinflammation. Human mesenchymal stem cell (hMSC) therapy is a promising regenerative approach but the inflammatory microenvironment in the brain tends to decrease the efficacy of the hMSC transplantation. Reducing the inflammation prior to hMSC therapy improves the outcome. We developed a combined nano-cell therapy by using dendrimers complexed with plasmids (dendriplexes) targeting CCL20 and its sole receptor CCR6 to reduce inflammation followed by hMSC transplantation. Treatment of TBI mice with shRNA conjugated dendriplexes followed by hMSC administration downregulated the inflammatory markers and significantly increased brain-derived neurotrophic factor (BDNF) expression in the cerebral cortex indicating future possible neurogenesis and improved behavioral deficits. Taken together, this nano-cell therapy ameliorates neuroinflammation and promotes brain tissue repair after TBI.

CCL20-CCR6 axis modulated traumatic brain injury-induced visual pathologies.

BACKGROUND: Traumatic brain injury (TBI) is a major cause of death and disability in the USA and the world; it constitutes 30% of injury-related deaths (Taylor et al., MMWR Surveill Summ 66:1-16, 2017). Contact sports athletes often experience repetitive TBI (rTBI), which exerts a cumulative effect later in life. Visual impairment is a common after-effect of TBI. Previously, we have shown that C-C chemokine 20 (CCL20) plays a critical role in neurodegeneration and inflammation following TBI (Das et al., J Neuroinflammation 8:148, 2011). C-C chemokine receptor 6 (CCR6) is the only receptor that CCL20 interacts with. The objective of the present study was to investigate the role of CCL20-CCR6 axis in mediating rTBI-induced visual dysfunction (TVD). METHODS: Wild type (WT) or CCR6 knock out (CCR6-/-) mice were subjected to closed head rTBI. Pioglitazone (PG) is a peroxisome proliferator-activated receptor γ (PPARγ) agonist which downregulates CCL20 production. Subsets of WT mice were treated with PG following final rTBI. A subset of mice was also treated with anti-CCL20 antibody to neutralize the CCL20 produced after rTBI. Histopathological assessments were performed to show cerebral pathologies, retinal pathologies, and inflammatory changes induced by rTBI. RESULTS: rTBI induced cerebral neurodegeneration, retinal degeneration, microgliosis, astrogliosis, and CCL20 expression. CCR6-/- mice showed reduced retinal degeneration, microgliosis, and inflammation. Treatment with CCL20 neutralization antibody or PG showed reduced CCL20 expression along with reduced retinal degeneration and inflammation. rTBI-induced GFAP-positive glial activation in the optic nerve was not affected by knocking out CCR6. CONCLUSION: The present data indicate that rTBI-induced retinal pathology is mediated at least in part by CCL20 in a CCR6-dependent manner.

Perturbation Analysis of a Multiple Layer Guided Love Wave Sensor in a Viscoelastic Environment.

Surface acoustic wave sensors have the advantage of fast response, low-cost, and wireless interfacing capability and they have been used in the medical analysis, material characterization, and other application fields that immerse the device under a liquid environment. The theoretical analysis of the single guided layer shear horizontal acoustic wave based on the perturbation theory has seen developments that span the past 20 years. However, multiple guided layer systems under a liquid environment have not been thoroughly analyzed by existing theoretical models. A dispersion equation previously derived from a system of three rigidly coupled elastic mass layers is extended and developed in this study with multiple guided layers to analyze how the liquid layer's properties affect the device's sensitivity. The combination of the multiple layers to optimize the sensitivity of an acoustic wave sensor is investigated in this study. The Maxwell model of viscoelasticity is applied to represent the liquid layer. A thorough analysis of the complex velocity due to the variations of the liquid layer's properties and thickness is derived and discussed to optimize multilayer Surface acoustic wave (SAW) sensor design. Numerical simulation of the sensitivity with a liquid layer on top of two guided layers is investigated in this study as well. The parametric investigation was conducted by varying the thicknesses for the liquid layer and the guided layers. The effect of the liquid layer viscosity on the sensitivity of the design is also presented in this study. The two guided layer device can achieve higher sensitivity than the single guided layer counterpart in a liquid environment by optimizing the second guided layer thickness. This perturbation analysis is valuable for Love wave sensor optimization to detect the liquid biological samples and analytes.

Finite Element Analysis for Surface Acoustic Wave Device Characteristic Properties and Sensitivity.

The most vital step in the development of novel and existing surface acoustic wave (SAW)-based sensors and transducers is their design and optimization. Demand for SAW devices has been steadily increasing due to their low cost, portability, and versatility in electronics, telecommunications, and biosensor applications. However, a full characterization of surface acoustic wave biosensors in a three-dimensional (3D) finite element model has not yet been developed. In this study, a novel approach is developed for analyzing shear horizontal Love wave resonator devices. The developed modeling methodology was verified using fabricated devices. A thorough analysis of the 3D model and the experimental device was performed in this study including scattering parameters (S-parameters), reflection coefficient parameters, transmission parameters, and phase velocity. The simulated results will be used as a design guideline for future device design and optimization, which has thus far resulted in close matching between prediction and experimental results. This manuscript is the first to demonstrate a 3D finite element model to correlate the sensitivity of the SAW device with the magnitude of the phase shift, the real and imaginary part of the response, insertion loss, and the frequency shift. The results show that the imaginary part of the response shift has a higher sensitivity compared to other parameters.

Withania Somnifera (Ashwagandha) and Withaferin A: Potential in Integrative Oncology.

Ashwagandha (Withania Somnifera, WS), belonging to the family Solanaceae, is an Ayurvedic herb known worldwide for its numerous beneficial health activities since ancient times. This medicinal plant provides benefits against many human illnesses such as epilepsy, depression, arthritis, diabetes, and palliative effects such as analgesic, rejuvenating, regenerating, and growth-promoting effects. Several clinical trials of the different parts of the herb have demonstrated safety in patients suffering from these diseases. In the last two decades, an active component of Withaferin A (WFA) has shown tremendous cytotoxic activity suggesting its potential as an anti-carcinogenic agent in treatment of several cancers. In spite of enormous progress, a thorough elaboration of the proposed mechanism and mode of action is absent. Herein, we provide a comprehensive review of the properties of WS extracts (WSE) containing complex mixtures of diverse components including WFA, which have shown inhibitory properties against many cancers, (breast, colon, prostate, colon, ovarian, lung, brain), along with their mechanism of actions and pathways involved.

Precision Medicine for CRC Patients in the Veteran Population: State-of-the-Art, Challenges and Research Directions.

Colorectal cancer (CRC) accounts for ~9% of all cancers in the Veteran population, a fact which has focused a great deal of the attention of the VA's research and development efforts. A field-based meeting of CRC experts was convened to discuss both challenges and opportunities in precision medicine for CRC. This group, designated as the VA Colorectal Cancer Cell-genomics Consortium (VA4C), discussed advances in CRC biology, biomarkers, and imaging for early detection and prevention. There was also a discussion of precision treatment involving fluorescence-guided surgery, targeted chemotherapies and immunotherapies, and personalized cancer treatment approaches. The overarching goal was to identify modalities that might ultimately lead to personalized cancer diagnosis and treatment. This review summarizes the findings of this VA field-based meeting, in which much of the current knowledge on CRC prescreening and treatment was discussed. It was concluded that there is a need and an opportunity to identify new targets for both the prevention of CRC and the development of effective therapies for advanced disease. Also, developing methods integrating genomic testing with tumoroid-based clinical drug response might lead to more accurate diagnosis and prognostication and more effective personalized treatment of CRC.

Natriuretic peptide receptor A signaling regulates stem cell recruitment and angiogenesis: a model to study linkage between inflammation and tumorigenesis.

Natriuretic peptide receptor A (NPRA), the signaling receptor for the cardiac hormone, atrial natriuretic peptide (ANP), is expressed abundantly in inflamed/injured tissues and tumors. NPRA deficiency substantially decreases tissue inflammation and inhibits tumor growth. However, the precise mechanism of NPRA function and whether it links inflammation and tumorigenesis remains unknown. Since both injury repair and tumor growth require stem cell recruitment and angiogenesis, we examined the role of NPRA signaling in tumor angiogenesis as a model of tissue injury repair in this study. In in vitro cultures, aortas from NPRA-KO mice show significantly lower angiogenic response compared to wild-type counterparts. The NPRA antagonist that decreases NPRA expression, inhibits lipopolysaccharide-induced angiogenesis. The reduction in angiogenesis correlates with decreased expression of vascular endothelial growth factor and chemokine (C-X-C motif) receptor 4 (CXCR4) implicating a cell recruitment defect. To test whether NPRA regulates migration of cells to tumors, mesenchymal stem cells (MSCs) were administered i.v., and the results showed that MSCs fail to migrate to the tumor microenvironment in NPRA-KO mice. However, coimplanting tumor cells with MSCs increases angiogenesis and tumorigenesis in NPRA-KO mice, in part by promoting expression of CXCR4 and its ligand, stromal cell-derived factor 1α. Taken together, these results demonstrate that NPRA signaling regulates stem cell recruitment and angiogenesis leading to tumor growth. Thus, NPRA signaling provides a key linkage between inflammation and tumorigenesis, and NPRA may be a target for drug development against cancers and tissue injury repair.

Magnetic micelles for DNA delivery to rat brains after mild traumatic brain injury.

Traumatic brain injury (TBI) causes significant mortality, long term disability and psychological symptoms. Gene therapy is a promising approach for treatment of different pathological conditions. Here we tested chitosan and polyethyleneimine (PEI)-coated magnetic micelles (CP-mag micelles or CPMMs), a potential MRI contrast agent, to deliver a reporter DNA to the brain after mild TBI (mTBI). CPMM-tomato plasmid (ptd) conjugate expressing a red-fluorescent protein (RFP) was administered intranasally immediately after mTBI or sham surgery in male SD rats. Evans blue extravasation following mTBI suggested CPMM-ptd entry into the brain via the compromised blood-brain barrier. Magnetofection increased the concentration of CPMMs in the brain. RFP expression was observed in the brain (cortex and hippocampus), lung and liver 48 h after mTBI. CPMM did not evoke any inflammatory response by themselves and were excreted from the body. These results indicate the possibility of using intranasally administered CPMM as a theranostic vehicle for mTBI. From the clinical editor: In this study, chitosan and PEI-coated magnetic micelles (CPMM) were demonstrated as potentially useful vehicles in traumatic brain injury in a rodent model. Magnetofection increased the concentration of CPMMs in the brain and, after intranasal delivery, CPMM did not evoke any inflammatory response and were excreted from the body.

Modulation of Paracellular Permeability in SARS-CoV-2 Blood-to-Brain Transcytosis.

SARS-CoV-2 primarily infects the lungs via the ACE2 receptor but also other organs including the kidneys, the gastrointestinal tract, the heart, and the skin. SARS-CoV-2 also infects the brain, but the hematogenous route of viral entry to the brain is still not fully characterized. Understanding how SARS-CoV-2 traverses the blood-brain barrier (BBB) as well as how it affects the molecular functions of the BBB are unclear. In this study, we investigated the roles of the receptors ACE2 and DPP4 in the SARS-CoV-2 infection of the discrete cellular components of a transwell BBB model comprising HUVECs, astrocytes, and pericytes. Our results demonstrate that direct infection on the BBB model does not modulate paracellular permeability. Also, our results show that SARS-CoV-2 utilizes clathrin and caveolin-mediated endocytosis to traverse the BBB, resulting in the direct infection of the brain side of the BBB model with a minimal endothelial infection. In conclusion, the BBB is susceptible to SARS-CoV-2 infection in multiple ways, including the direct infection of endothelium, astrocytes, and pericytes involving ACE2 and/or DPP4 and the blood-to-brain transcytosis, which is an event that does not require the presence of host receptors.

Cholesterol-rich microdomains as docking platforms for respiratory syncytial virus in normal human bronchial epithelial cells.

Respiratory syncytial virus (RSV) is one of the major causes of respiratory infections in children, and it is the main pathogen causing bronchiolitis in infants. The binding and entry mechanism by which RSV infects respiratory epithelial cells has not yet been determined. In this study, the earliest stages of RSV infection in normal human bronchial epithelial cells were probed by tracking virions with fluorescent lipophilic dyes in their membranes. Virions colocalized with cholesterol-containing plasma membrane microdomains, identified by their ability to bind cholera toxin subunit B. Consistent with an important role for cholesterol in RSV infection, cholesterol depletion profoundly inhibited RSV infection, while cholesterol repletion reversed this inhibition. Merger of the outer leaflets of the viral envelope and the cell membrane appeared to be triggered at these sites. Using small-molecule inhibitors, RSV infection was found to be sensitive to Pak1 inhibition, suggesting the requirement of a subsequent step of cytoskeletal reorganization that could involve plasma membrane rearrangements or endocytosis. It appears that RSV entry depends on its ability to dock to cholesterol-rich microdomains (lipid rafts) in the plasma membrane where hemifusion events begin, assisted by a Pak1-dependent process.

Multispectral optoacoustic tomography (MSOT): Monitoring neurovascular changes in a mouse repetitive traumatic brain injury model.

BACKGROUND: Evidence suggests that mild TBI injuries, which comprise > 75% of all TBIs, can cause chronic post-concussive symptoms, especially when experienced repetitively (rTBI). rTBI is a major cause of cognitive deficit in athletes and military personnel and is associated with neurovascular changes. Current methods to monitor neurovascular changes in detail are prohibitively expensive and invasive for patients with mild injuries. NEW METHOD: We evaluated the potential of multispectral optoacoustic tomography (MSOT) to monitor neurovascular changes and assess therapeutic strategies in a mouse model of rTBI. Mice were subjected to rTBI or sham via controlled cortical impact and administered pioglitazone (PG) or vehicle. Oxygenated and deoxygenated hemoglobin were monitored using MSOT. Indocyanine green clearance was imaged via MSOT to evaluate blood-brain-barrier (BBB) integrity. RESULTS: Mice subjected to rTBI show a transient increase in oxygenated/total hemoglobin ratio which can be mitigated by PG administration. rTBI mice also show BBB disruption shortly after injury and reduction of oxygenated/total hemoglobin in the chronic stage, neither of which were affected by PG intervention. COMPARISON WITH EXISTING METHODS: MSOT imaging has the potential as a noninvasive in vivo imaging method to monitor neurovascular changes and assess therapeutics in mouse models of rTBI. In comparison to standard methods of tracking inflammation and BBB disruption, MSOT can be used multiple times throughout the course of injury without the need for surgery. Thus, MSOT is especially useful in research of rTBI models for screening therapeutics, and with further technological improvements may be extended for use in rTBI patients.

Withaferin A Increases the Effectiveness of Immune Checkpoint Blocker for the Treatment of Non-Small Cell Lung Cancer.

Treatment of late-stage lung cancers remains challenging with a five-year survival rate of 8%. Immune checkpoint blockers (ICBs) revolutionized the treatment of non-small cell lung cancer (NSCLC) by reactivating anti-tumor immunity. Despite achieving durable responses, ICBs are effective in only 20% of patients due to immune resistance. Therefore, synergistic combinatorial approaches that overcome immune resistance are currently under investigation. Herein, we studied the immunomodulatory role of Withaferin A (WFA)-a herbal compound-and its effectiveness in combination with an ICB for the treatment of NSCLC. Our in vitro results show that WFA induces immunogenic cell death (ICD) in NSCLC cell lines and increases expression of the programmed death ligand-1 (PD-L1). The administration of N-acetyl cysteine (NAC), a reactive oxygen species (ROS) scavenger, abrogated WFA-induced ICD and PD-L1 upregulation, suggesting the involvement of ROS in this process. Further, we found that a combination of WFA and α-PD-L1 significantly reduced tumor growth in an immunocompetent tumor model. Our results showed that WFA increases CD-8 T-cells and reduces immunosuppressive cells infiltrating the tumor microenvironment. Administration of NAC partially inhibited the anti-tumor response of the combination regimen. In conclusion, our results demonstrate that WFA sensitizes NSCLC to α-PD-L1 in part via activation of ROS.

Acetate-encapsulated Linolenic Acid Liposomes Reduce SARS-CoV-2 and RSV Infection.

Emergent Coronaviridae viruses, such as SARS-CoV-1 in 2003, MERS-CoV in 2012, and SARS-CoV-2 (CoV-2) in 2019, have caused millions of deaths. These viruses have added to the existing respiratory infection burden along with respiratory syncytial virus (RSV) and influenza. There are limited therapies for respiratory viruses, with broad-spectrum treatment remaining an unmet need. Since gut fermentation of fiber produces short-chain fatty acids (SCFA) with antiviral potential, developing a fatty acid-based broad-spectrum antiviral was investigated. Molecular docking of fatty acids showed α-linolenic acid (ALA) is likely to interact with CoV-2-S, NL63-CoV-S, and RSV-F, and an ALA-containing liposome interacted with CoV-2 directly, degrading the particle. Furthermore, a combination of ALA and a SCFA-acetate synergistically inhibited CoV2-N expression and significantly reduced viral plaque formation and IL-6 and IL-1ß transcript expression in Calu-3 cells, while increasing the expression of IFN-ß. A similar effect was also observed in RSV-infected A549 cells. Moreover, mice infected with a murine-adapted SARS-CoV-2 (MA10) and treated with an ALA-liposome encapsulating acetate showed significant reductions in plaque-forming units present in lung tissue and in infection-associated lung inflammation and cytokines. Taken together, these results demonstrate that the ALA liposome-encapsulating acetate can be a promising broad antiviral therapy against respiratory infections.

A mouse model of repeated traumatic brain injury-induced hearing impairment: Early cochlear neurodegeneration in the absence of hair cell loss.

PURPOSE: Traumatic Brain Injury (TBI) is a major cause of death and disability worldwide. Mounting evidence suggests that even mild TBI injuries, which comprise >75% of all TBIs, can cause chronic post-concussive neurological symptoms, especially when experienced repetitively (rTBI). The most common post-concussive symptoms include auditory dysfunction in the form of hearing loss, tinnitus, or impaired auditory processing, which can occur even in the absence of direct damage to the auditory system at the time of injury. The mechanism by which indirect damage causes loss of auditory function is poorly understood, and treatment is currently limited to symptom management rather than preventative care. We reasoned that secondary injury mechanisms, such as inflammation, may lead to damage of the inner ear and parts of the brain used for hearing after rTBI. Herein, we established a model of indirect damage to the auditory system induced by rTBI and characterized the pathology of hearing loss. METHODS: We established a mouse model of rTBI in order to determine a timeline of auditory pathology following multiple mild injuries. Mice were subject to controlled cortical impact at the skull midline once every 48 h, for a total of 5 hits. Auditory function was assessed via the auditory brainstem response (ABR) at various timepoints post injury. Brain and cochleae were collected to establish a timeline of cellular pathology. RESULTS: We observed increased ABR thresholds and decreased (ABR) P1 amplitudes in rTBI vs sham animals at 14 days post-impact (dpi). This effect persisted for up to 60 days (dpi). Auditory temporal processing was impaired beginning at 30 dpi. Spiral ganglion degeneration was evident at 14 dpi. No loss of hair cells was detected at this time, suggesting that neuronal loss is one of the earliest notable events in hearing loss caused by this type of rTBI. CONCLUSIONS: We conclude that rTBI results in chronic auditory dysfunction via damage to the spiral ganglion which occurs in the absence of any reduction in hair cell number. This suggests early neuronal damage that may be caused by systemic mechanisms similar to those leading to the spread of neuronal death in the brain following TBI. This TBI-hearing loss model provides an important first step towards identifying therapeutic targets to attenuate damage to the auditory system following head injury.