Post-acute sequelae of COVID-19 (PASC) encompasses persistent neurological disease, including olfactory and cognitive dysfunction. The basis for this dysfunction is poorly understood. Here, we report neurological dysfunction for at least 120 d postinfection in mice infected with a virulent nonneurotropic mouse-adapted SARS-CoV-2. Long after recovery from nasal infection, we observed diminished tyrosine hydroxylase expression in olfactory bulb glomeruli and in substantia nigra. Similar changes were observed in brains of COVID-19 deceased patients. Vulnerability of dopaminergic neurons in these brain areas was accompanied by increased proinflammatory cytokines, and neurobehavioral changes. RNAseq analysis unveiled persistent microglia activation, similar to human neurodegenerative diseases. Treatment with antivirals (nirmatrelvir and molnupiravir) at the time of infection minimally prevented neurological abnormalities, consistent with patient data. In contrast, antivirals plus corticosteroids resulted in nearly complete recovery of neurological function. Remarkably, initiation of combined therapy even three days after infection improved outcomes. Together these results demonstrate that neurological dysfunction in SARS-CoV-2 infected mice resembles human neurodegenerative disease and indicate that minimizing inflammation early after SARS-CoV-2 infection may be critical for decreasing neurological PASC. The requirement for decreasing inflammation soon after infection may also explain why antiviral therapy has had inconsistent effects in patients.

Arthritis is an inflammatory state within joints resulting in cartilage damage, pain, and loss of mobility. Recent advances in arthritis research specifically demonstrate that the joint capsule (e.g., synovium) is an important source of this inflammation, but there are no human models that replicate essential synovial architecture. To address this, the Joint Space Analysis System, or JSAS, was created. Anterior synovium was obtained intra-operatively from patients undergoing Total Knee Arthroplasty (TKA). Synovium was dissected and sectioned into 3 mm biopsy cores. Cores were placed in the upper well of a 5 µm or 0.4 µm transwell with 300 µl of DMEM with10% FBS. In the bottom well, 600 µL of media was added, and exchanged every 2-3 days. Viability was assessed up to 7 days in hyperoxic (50%), atmospheric/standard (~21%), and physiologic (5%) incubation conditions. Stimuli in the bottom well included monocyte chemoattractant protein 1 (MCP-1/CCL2), lipopolysaccharide (LPS), N-acetyl cysteine, S. aureus, and B. burgdorferi. Media was stored for ELISA, and tissue was stored for formalin fixed paraffin embedded (FFPE) analysis. In standard conditions, synovium remained fully viable for 3 days. Stimulus modified the structure and function of intimal lining and sublining synovial cells, including loss of the resident macrophage border, sublining expansion, upregulation of pathogenic fibroblasts, and production of cytokines IL-1β and TNFα. Immune cells and fibroblasts migrated to the bottom chamber (5 µm pores) per flow cytometry analysis. Mobile B. burgdorferi migrated into tissue at the 0.4 µm pore size while non-motile S. aureus did not. Relevant cytokines were expressed in sufficient quantity for ELISA. JSAS is a modular system capable of studying acute alterations to human synovium, allowing for the complexity of 3D structures in a pre-clinical model while maintaining biologically relevant structure and function.

Ankle osteoarthritis (OA) is predominantly post-traumatic in origin. It differs in important ways biologically, mechanically, and clinically from hip and knee OA. Despite recognition of its long-term burden, treatment strategies for ankle OA remain largely reactive and centered on end-stage reconstruction. The Arthritis Foundation and the American Orthopaedic Foot & Ankle Society (AOFAS) co-sponsored an Ankle Arthritis Think Tank to address this deficiency in Napa, CA, on January 22, 2026. The focus was to solve patient frustration of limited treatment options and relatively poor outcomes when comparing to hip and knee osteoarthritis. The 2026 meeting brought together a multidisciplinary group of scientists and clinicians to present our current related state of knowledge and to define translational research priorities capable of advancing prevention and treatment of ankle OA within the next decade. Four structured sessions addressed topics relating to ankle OA: (1) Biologic and Biomechanical Pathogenesis, (2) Diagnostic and Management Challenges, (3) Therapeutic Strategies, and (4) Research Methodology. A moderated closing discussion focused on identifying actionable and fundable research pathways. Session presentations and discussion are synthesized here into a unified review.

Background:Metallosis is a known complication of joint arthroplasty, but its role in failures of spinal fusion procedures likely remains underrecognized. The release of metallic debris from implant interfaces may impair osteogenesis by triggering local inflammatory responses. To date, no in vivo models have been developed to investigate this process in the context of spinal fusion. Using a unique modelling system, we sought to determine whether high local concentrations of metal particles impair spinal fusion, and whether particle load correlates with inflammatory response. Methods:Twelve rabbits underwent posterolateral lumbar spinal fusion using autologous iliac crest bone grafts. Six weeks postoperatively, animals received bilateral paraspinal injections of Ti/Cr/ Co alloy at high, low, or zero concentrations immediately dorsal to the graft site. At twelve weeks, specimens were euthanized, and the grafts were assessed by radiography, manual palpation, and histopathology. A custom image analysis algorithm was used to quantify metal debris in histologic sections, and inflammatory responses were evaluated. Fusion outcomes and metal burden were compared between groups using unpaired t-tests. Results:Five of eight evaluable specimens exhibited non-fusion, most of which contained higher levels of visible metal debris than fused counterparts upon histologic analysis. Fused grafts, in contrast, demonstrated minimal particle burden and continuous trabecular bridging. Lymphocytes were found to be localized near metal particles, and fibrotic tissue was observed to replace bone at non-fused sites. No significant differences in inflammatory cell counts were observed between groups. Conclusion:Elevated metal particle burden in the posterolateral fusion environment was associated with impaired osteogenesis and increased localized inflammation, particularly in non-fused specimens. These results establish this model's utility for investigating metallosis-driven spinal fusion failure and suggest its potential as a predictive platform for assessing particle-related risk in spinal instrumentation. Clinical Relevance:This study provides a reproducible in vivo model to explore how metallic debris may contribute to spinal fusion failure. It offers a platform for testing future interventions targeting metallosis-related complications in spinal fusion surgery.

Joint trauma often leads to articular cartilage degeneration and post-traumatic osteoarthritis (PTOA). Pivotal determinants include trauma-induced excessive tissue strains that damage cartilage cells. As a downstream effect, these damaged cells can trigger cartilage degeneration via oxidative stress, cell death, and proteolytic tissue degeneration. N-acetylcysteine (NAC) has emerged as an antioxidant capable of inhibiting oxidative stress, cell death, and cartilage degeneration post-impact. However, the temporal effects of NAC are not fully understood and remain difficult to assess solely by physical experiments. Thus, we developed a computational finite element analysis framework to simulate a drop-tower impact of cartilage in Abaqus, and subsequent oxidative stress-related cell damage, and NAC treatment upon cartilage proteoglycan content in Comsol Multiphysics, based on prior ex vivo experiments. Model results provide evidence that immediate NAC treatment can reduce proteoglycan loss by mitigating oxidative stress, cell death (improved proteoglycan biosynthesis), and enzymatic proteoglycan depletion. Our simulations also indicate that delayed NAC treatment may not inhibit cartilage proteoglycan loss despite reduced cell death after impact. These results enhance understanding of the temporal effects of impact-related cell damage and treatment that are critical for the development of effective treatments for PTOA. In the future, our modeling framework could increase understanding of time-dependent mechanisms of oxidative stress and downstream effects in injured cartilage and aid in developing better treatments to mitigate PTOA progression.

Objective: Joint injury precipitates post-traumatic osteoarthritis (PTOA) via chondrocyte mitochondrial oxidative damage. Carbon monoxide (CO) is a small molecule with potent antioxidant and mitochondrial benefits in other tissues that have not been explored in healthy chondrocytes. We hypothesized that CO would subvert the mitochondrial effects of articular cartilage injuries upon resident chondrocytes. Design: We evaluated intra-articular delivery of a novel carbon monoxide-containing foam (COF). We used in vitro impact injuries to explore mitochondrial and redox endpoints after CO exposure. We then applied intra-articular injections of COF or control room air foam (RAF) to assess safety, efficacy, and other intra-articular responses. Results: COF increased the expression of HO1 and mitofusin-1 within 1 h and this increase was sustained for 12 h in vitro. COF increased chondrocyte mitochondrial respiration by 40% and increased reduced (not oxidized) thiols by 50% following in vitro injury to osteochondral explants. After cartilage injury, COF prevented the formation of 3-nitrotyrosine and the loss of articular chondrocyte mitochondria. When injected intra-articularly, COF was retained for 24 h post-injection in mouse stifle joints. It increased HO1 in those joints, enhanced reduced thiol levels in rabbit stifle joints, and exhibited no toxicity 1 and 4 weeks after injection. Conclusions: This study supports the hypothesis that CO functions as an antioxidant for articular chondrocytes by supporting mitochondria and intracellular GSH in the presence or absence of cartilage injury. Challenges in delivering exogenous CO have limited its preclinical development, but new CO-releasing materials like COF may enable new examinations of this promising small molecule.

BACKGROUND:Evidence that oxidative stress contributes to the progression of posttraumatic intervertebral disc degeneration suggests targeting oxidant metabolism in disc cells as a strategy to mitigate degeneration in injured discs. We previously identified amobarbital, a drug that suppresses mitochondrial activity in a cell culture model with the chemical induction of oxidative stress, as a promising candidate for this purpose. PURPOSE:The objective of this study was to investigate the preventive effects of amobarbital on the progression of disc degeneration using a rabbit disc herniation model. STUDY DESIGN/SETTING:The effects of amobarbital on the prevention of disc degeneration were evaluated in both ex vivo and in vivo herniated discs. METHODS:Spine motion segments or animals were randomly assigned for intact control, vehicle control (hydrogel only), and amobarbital. Disc herniation was initiated by puncturing the annulus fibrosus with a needle, and amobarbital was delivered to the nucleus pulposus (NP) in a Pluronic F-127/hyaluronic acid hydrogel. A modified histological classification was applied to evaluate for degenerative cellular and matrix changes in both the annulus fibrosus and NP. Additionally, immunohistochemical staining for apoptosis and oxidative stress response, and radiography for disc height index were examined in the ex vivo and in vivo models, respectively. RESULTS:Amobarbital injection allowed uniform distribution in the whole NP and showed sustained release for 3 to 4 days. In an organ culture model, amobarbital treatment after a discal injury prohibited morphologic changes of notochordal cells, structural changes of extracellular matrix, endplate chondrocyte migration, and cell apoptosis compared with the vehicle control. Similarly, the preventive effects of amobarbital on disc degeneration were confirmed in an animal study, especially at 2 weeks, with maintained disc height. CONCLUSIONS:In summary, amobarbital injection loaded in F127/hyaluronic acid hydrogel reduced cellular and structural degenerative changes against herniated discal injuries. Therefore, amobarbital has great potential for treating degenerative disc degeneration. CLINICAL SIGNIFICANCE:Amobarbital encapsulated in hydrogel in this model allowed for effective local delivery into the NP and sustained release of amobarbital, which is promising for potential clinical application. Delivering amobarbital in NP cells may be a therapeutic option to retard intervertebral disc degeneration.

Diabetic wound healing is uniquely challenging to manage due to chronic inflammation and heightened microbial growth from elevated interstitial glucose. Carbon monoxide (CO), widely acknowledged as a toxic gas, is also known to provide unique therapeutic immune-modulating effects. To facilitate delivery of CO, we have designed hyaluronic-acid-based CO gas-entrapping materials (CO-GEMs) for topical and prolonged gas delivery to the wound bed. We demonstrate that CO-GEMs promote the healing response in murine diabetic wound models (full-thickness wounds and pressure ulcers) compared to N2-GEMs and untreated controls.

In orthopedic research, many studies have applied vitamin E as a protective antioxidant or used tert-butyl hydroperoxide to induce oxidative injury to chondrocytes. These studies often support the hypothesis that joint pathology causes oxidative stress and increased lipid peroxidation that might be prevented with lipid antioxidants to improve cell survival or function and joint health; however, lipid antioxidant supplementation was ineffective against osteoarthritis in clinical trials and animal data have been equivocal. Moreover, increased circulating vitamin E is associated with increased rates of osteoarthritis. This disconnect between benchtop and clinical results led us to hypothesize that oxidative stress-driven paradigms of chondrocyte redox function do not capture the metabolic and physiologic effects of lipid antioxidants and prooxidants on articular chondrocytes. We used ex vivo and in vivo cartilage models to investigate the effect of lipid antioxidants on healthy, primary, articular chondrocytes and applied immuno-spin trapping techniques to provide a broad indicator of high levels of oxidative stress independent of specific reactive oxygen species. Key findings demonstrate lipid antioxidants were pro-mitochondrial while lipid prooxidants decreased mitochondrial measures. In the absence of injury, radical formation was increased by lipid antioxidants; however, in the presence of injury, radical formation was decreased. In unstressed conditions, this relationship between chondrocyte mitochondria and redox regulation was reproduced in vivo with overexpression of glutathione peroxidase 4. In mice aged 18 months or more, overexpression of glutathione peroxidase 4 significantly decreased the presence of pro-mitochondrial peroxisome proliferation activated receptor gamma and deranged the relationship between mitochondria and the redox environment. This complex interaction suggests strategies targeting articular cartilage may benefit from adopting more nuanced paradigms of articular chondrocyte redox metabolism.

Posttraumatic osteoarthritis (PTOA) is a well-recognized public health burden without any disease modifying treatment. This occurs despite noted advances in surgical care in the past 50 years. Mitochondrial oxidative damage pathways initiate PTOA after severe injuries like intraarticular fracture that often require surgery and contribute to PTOA after less severe injuries that may or may not require surgery like meniscal injuries. When considering the mitochondrial and redox environment of the injured joint, we hypothesized that activation of heme metabolism, previously associated with healing in many settings, would cause prototypic mitochondrial reprogramming effects in cartilage ideally suited for use at the time of injury repair. Activation of heme metabolism can be accomplished through the gasotransmitter carbon monoxide (CO), which activates hemeoxygenase-1 (HO1) and subsequent heme metabolism. In this study, we employed unique carbon monoxide (CO)-containing foam (COF) to stimulate heme metabolism and restore chondrocyte oxygen metabolism in vitro and in vivo . Doxycycline-inducible, chondrocyte-specific HO1 overexpressing transgenic mice show similar mitochondrial reprogramming after induction compared to COF. CO is retained at least 24 h after COF injection into stifle joints and induces sustained increases in heme metabolism. Lastly, intraarticular injection of COF causes key redox outcomes without any adverse safety outcomes in rabbit stifle joints ex vivo and in vivo . We propose that activation of heme metabolism is an ideal adjuvant to trauma care that replenishes chondrocyte mitochondrial metabolism and restores redox homeostasis.

Elbow trauma can lead to joint contracture and reduced range of motion (ROM). Nonsurgical interventions can improve ROM, but in some cases capsule release surgery is required. Although surgery can improve ROM, it often does not restore full ROM. Thus, alternatives are needed. One approach is to target activated myofibroblasts, which are commonly associated with fibrotic tissue. Mechanical and biochemical cues drive a feedback loop that can result in normal or pathological healing. We hypothesize that this feedback loop exists in joint contracture and can be manipulated so that myofibroblast activity is reduced, normal healing is achieved, and ROM is improved. We previously demonstrated that blebbistatin can inhibit myofibroblast contractile forces and reduce collagen synthesis in vitro. Thus, the purpose of this study was to assess the use of blebbistatin in an animal model of elbow contracture, which was induced in 7 groups of 4 rats each (n = 28). All elbows were mechanically and histologically tested. The uninjured contralateral elbows of each rat were used as a control group. Capsule release surgery significantly improved (p < 0.01) outcomes 1 week after surgery compared to injury alone and was not significantly different from uninjured elbows. Three weeks after surgery, outcomes worsened, indicating joint stiffening consistent with what is observed clinically. The addition of blebbistatin did not significantly improve outcomes. Future work will investigate relationships among treatment, fibrotic tissue deposition, myofibroblast activity, and biomechanics to determine if blebbistatin is a useful adjunctive therapy for treating joint contracture.

Purpose: Mechanical injury to articular cartilage precipitates post-traumatic osteoarthritis (PTOA), a leading cause of disability. Our group has shown that sublethal mechanical injury leads to pathogenic mitochondrial dysfunction. These changes were prevented by the antioxidant N-acetylcysteine or via mitochondrial complex I inhibition with amobarbital in vivo. In these studies, cell death was exacerbated by atmospheric oxygen (O2) in vitro but not explored further. We wanted to examine the direct effects of hyperoxia upon chondrocyte redox behavior in our large animal osteochondral explant model. We employed carbon monoxide (CO) to disable heme-containing proteins, including many O2 metabolizing systems. We have developed a gas entrapping foam that allows delivery of CO in a controlled fashion, and produces antioxidant and anti-inflammatory effects in rodent models of inflammatory bowel disease. Though CO has long been known as “the silent killer”, it is also a critical gasotransmitter for mitochondrial and redox biology. Upregulation of heme oxygenase-1 (HO-1), an enzyme expressed by chondrocytes that produces CO, is important to PTOA prevention in rodents; however, its normal function is not well understood within the joint. We hypothesize that hyperoxia disturbs primary chondrocyte redox status and exacerbates traumatic injury in a heme-dependent manner.

Purpose: Ataxia telangiectasia mutated kinase (ATM) inhibitors are potent radiosensitizers that regulate DNA damage responses and redox metabolism, but they have not been translated clinically because of the potential for excess normal tissue toxicity. Pharmacologic ascorbate (P-AscH(-); intravenous administration achieving mM plasma concentrations) selectively enhances H2O2-induced oxidative stress and radiosensitization in tumors while acting as an antioxidant and mitigating radia-tion damage in normal tissues including the bowel. We hypothesized that P-AscH(-) could enhance the therapeutic index of ATM inhibitor-based chemoradiation by simultaneously enhancing the intended effects of ATM inhibitors in tumors and mit-igating off-target effects in adjacent normal tissues. Methods and Materials: Clonogenic survival was assessed in human (human colon tumor [HCT]116, SW480, HT29) and murine (CT26, MC38) colorectal tumor lines and normal cells (human umbilical vein endothelial cell, FHs74) after radiation +/- DNA repair inhibitors f P-AscH(-). Tumor growth delay was assessed in mice with HCT116 or MC38 tumors after fraction-ated radiation (5 Gy x 3) +/- the ATM inhibitor KU60019 +/- P-AscH(-). Intestinal injury, oxidative damage, and transforming growth factor b immunoreactivity were quantified using immunohistochemistry after whole abdominal radiation (10 Gy) +/- KU60019 +/- P-AscH(-). Cell cycle distribution and ATM subcellular localization were assessed using flow cytometry and immu-nohistochemistry. The role of intracellular H2O2 fluxes was assessed using a stably expressed doxycycline-inducible catalase transgene. Results: KU60019 with P-AscH(-) enhanced radiosensitization in colorectal cancer models in vitro and in vivo by H2O2- dependent oxidative damage to proteins and enhanced DNA damage, abrogation of the postradiation G2 cell cycle checkpoint, and inhibition of ATM nuclear localization. In contrast, concurrent P-AscH(-) markedly reduced intestinal toxicity and oxidative damage with KU60019. Conclusions: We provide evidence that redox modulating drugs, such as P-AscH(-), may facilitate the clinical translation of ATM inhibitors by enhancing tumor radiosensitization while simultaneously protecting normal tissues. (c) 2022 Elsevier Inc. All rights reserved.

Background Radiotherapy for tumor treatment in or near bones often causes osteopenia and/or osteoporosis, and the resulting increased bone fragility can lead to pathologic fractures. Bone mineral density (BMD) is often used to screen for fracture risk, but no conclusive relationship has been established between BMD and the microstructural/ biomechanical changes in irradiated bone. Understanding the effects of radiation dosing regimen on the bone structure-strength relationship would improve the ability to reduce fracture-related complications resulting from cancer treatment. Methods Thirty-two C57B6J mice aged 10 - 12 weeks old were randomized to single dose (1 x 25 Gy) and fractionated dose (5 x 5 Gy) irradiation groups. Right hindlimbs were irradiated while the contralateral hindlimbs served as the non-irradiated control. Twelve weeks after irradiation, BMD and bone microstructure were assessed with micro-computed tomography, and mechanical strength/stiffness was assessed with a torsion test. The effects of radiation dosing regimen on bone microstructure and strength were assessed using ANOVA, and bone strength-structure relationships were investigated through correlation analysis of microstructural and mechanical parameters. Results Fractionated irradiation induced significantly greater losses in BMD in the femur (23% - male mice, p=0.016; 19% - female mice) and the tibia (18% - male mice; 6% - female mice) than the single-dose radiation. The associated reductions in trabecular bone volume (-38%) and trabecular number (-34% to -42%), and the increase in trabecular separation (23% to 29%) were only significant in the male mice with fractionated dosing. There was a significant reduction in fracture torque in the femurs of male (p=0.021) and female (p=0.0017) mice within the fractionated radiation group, but not in the single dose radiation groups. There was moderate correlation between bone microstructure and mechanical strength in the single-dose radiation group (r = 0.54 to 0.73), but no correlation in the fractionated dosing group (r=0.02 to 0.03). Conclusion Our data indicate more detrimental changes in bone microstructure and mechanical parameters in the fractionated irradiation group compared to the single dose group. This may suggest the potential for protecting bone if a needed therapeutic radiation dose can be delivered in a single session rather than administered in fractions.

Energy-intensive kidney reabsorption processes essential for normal whole-body function are maintained by tubular epithelial cell metabolism. Tubular metabolism changes markedly following acute kidney injury (AKI), but which changes are adaptive versus maladaptive remain poorly understood. In publicly available data sets, we noticed a consistent downregulation of the mitochondrial pyruvate carrier (MPC) after AKI, which we experimentally confirmed. To test the functional consequences of MPC downregulation, we generated novel tubular epithelial cell-specific Mpc1 knockout (MPC TubKO) mice. 13C-glucose tracing, steady-state metabolomic profiling, and enzymatic activity assays revealed that MPC TubKO coordinately increased activities of the pentose phosphate pathway and the glutathione and thioredoxin oxidant defense systems. Following rhabdomyolysis-induced AKI, MPC TubKO decreased markers of kidney injury and oxidative damage and strikingly increased survival. Our findings suggest that decreased mitochondrial pyruvate uptake is a central adaptive response following AKI and raise the possibility of therapeutically modulating the MPC to attenuate AKI severity.

Pancreatic secretions become viscous and acidic in Cystic fibrosis (CF), highlighting the role of CFTR in pancreatic fluid and bicarbonate secretion. Forskolin-induced swelling (FIS) assay developed in intestinal organoids measures residual CFTR function. It is not known whether FIS reflects bicarbonate secretion in pancreas, an organ that secretes near-isotonic NaHCO3 levels. To investigate this, we generated pancreatic duct organoids from CF and non-CF pigs. Epithelial and ductal origin was confirmed with epithelial markers, ion transporters and lack of acinar, islet cell markers. CF organoids were small with no identifiable lumen; CFTR was expressed only in non-CF organoids. Utilizing FIS, organoid size increased only in response to chloride, not bicarbonate. This report highlights pancreatic duct organoids isolated for the first time from CF pigs and evidence for chloride and not bicarbonate driving pancreatic organoid swelling. These organoids would be useful to test chloride permeability of CFTR mutations that cause CF pancreatic disease.

Synovium is critical for maintaining joint homeostasis and may contribute to mechanobiological responses during joint movement. We investigated mechanobiological responses of whole synovium from patients with late-stage knee osteoarthritis (OA). Synovium samples were collected during total knee arthroplasty and assigned to histopathology or cyclic 10% tensile strain loading, including (1) static (control); (2) low-frequency (0.3 Hz); and iii) high-frequency (1.0 Hz) for 30-min. After 6-h incubation, tissues were bisected for RNA isolation and immunostaining (3-nitrotyrosine; 3-NT). RNA sequencing was analyzed for differentially expressed genes and pathway enrichment. Cytokines and lactate were measured in conditioned media. Compared to controls, low-frequency strain induced enrichment of pathways related to interferon response, Fc-receptor signaling, and cell metabolism. High-frequency strain induced enrichment of pathways related to NOD-like receptor signaling, high metabolic demand, and redox signaling/stress. Metabolic and redox cell stress was confirmed by increased release of lactate into conditioned media and increased 3-NT formation in the synovial lining. Late-stage OA synovial tissue responses to tensile strain include frequency-dependent increases in inflammatory signaling, metabolism, and redox biology. Based on these findings, we speculate that some synovial mechanobiological responses to strain may be beneficial, but OA likely disturbs synovial homeostasis leading to aberrant responses to mechanical stimuli, which requires further validation.