Dynamic Weight Bearing Research Articles

Visual exposure to green light therapy reduces knee joint pain and alters the lipidome in osteoarthritic rats.

Visual exposure to dim, green, light has been found to reduce pain levels in patients living with migraine, low back pain, and fibromyalgia. Preclinical studies discovered that the analgesic effect of green light was due to the central release of endogenous opioids and a reduction in inflammatory cytokines in the cerebrospinal fluid. The present study assessed the effect of green light therapy (GLT) on joint pain in a rat model of osteoarthritis (OA) and investigated the role of endolipids. Male and female Wistar rats (207-318 g) received an intra-articular injection of sodium monoiodoacetate (3 mg in 50 μL saline) into the knee to induce OA. On day 9, animals were placed in a room illuminated by either white (neutral-white 4000K; 20 lux) or green (wavelength: 525 nm; luminance: 20 lux) light for 5 days (8 hours per day). Joint nociception was assessed by von Frey hair algesiometry, dynamic weight bearing, and in vivo single unit extracellular recordings from knee joint mechanonociceptors. Compared to white light, GLT significantly reduced secondary mechanical hypersensitivity in both sexes and improved hindlimb weight bearing in females only. There was no effect of GLT on joint nociceptor activity in either sex. Serum lipidomics indicated an increase in circulating analgesic endolipids in response to GLT, particularly the N-acyl-glycines. Partial blockade of the endocannabinoid system with the G protein receptor-18/cannabinoid-1 receptor antagonist AM281 (500 μg/kg i.p.) attenuated GLT-induced analgesia. These data show for the first time that GLT acts to reduce OA pain by upregulating circulating analgesic endolipids, which then engage the endocannabinoid system.

Evaluation of Pain-Associated Behavioral Changes in Monoiodoacetate-Induced Osteoarthritic Rats Using Dynamic Weight Bearing Analysis.

Pain is the primary clinical indication of osteoarthritis (OA), and behavioral assessments in rodent pain models are widely used to understand pain patterns. These preclinical pain assessments can also help us to understand the effectiveness of emerging therapeutics for prolonged OA pain management. Along with evoked methods like mechanical allodynia and thermal hyperalgesia, non-evoked methods such as dynamic weight bearing (DWB) analysis are valuable tools for behavioral assessments of pain. Both these methods were utilized to study pain-induced behavioral changes in a monoiodoacetate (MIA)-induced osteoarthritic pain model, which is a well-established preclinical OA pain model. However, the utility of DWB analysis as an indicator of long-term pain sensitivity (more than 4 weeks) remains largely unexplored. Understanding the long-term sensitivity of DWB is valuable to study the effectiveness of novel prolonged pain-relieving therapeutics. Here, we studied the dynamic behavioral changes in MIA-induced OA rats over a period of 16 weeks using DWB measurements. Female Sprague Dawley rats were injected in the right knee joint with MIA (3 mg) using X-ray guidance. Multiple dynamic postural evaluations such as ipsilateral weight percentage, paw area, contralateral/ipsilateral weight ratio and area ratio were assessed to understand the behavioral changes. The data showed that the ipsilateral weight bearing percentage alone is not sufficient to assess pain-related behavior beyond 6 weeks. This study shows the advantages and limitations of dynamic weight bearing as an assessment tool for the long-term progression of pain behavior in MIA-induced OA rats.

Oleanolic acid-3-glucoside, a synthetic oleanane-type saponin, ameliorates methylmercury-induced dysfunction of synaptic transmission in mice

Methylmercury (MeHg) is widely distributed in nature and is known to cause neurotoxic effects. This study aimed to examine the anti-MeHg activity of oleanolic acid-3-glucoside (OA3Glu), a synthetic oleanane-type saponin derivative, by evaluating its effects on motor function, pathology, and electrophysiological properties in a mouse model of MeHg poisoning. Mice were orally administered 2 or 4 mg·kg−1·d−1 MeHg with or without 100 µg·kg−1·d−1 OA3Glu 5x/week for four weeks. Motor function was evaluated using beam-walking and dynamic weight-bearing (DWB) tests. High-dose MeHg exposure significantly increased the frequency of stepping off the hind leg while crossing the beam in the beam-walking test, and increased weight on forelegs when moving freely in the DWB test. OA3Glu treatment alleviated motor abnormality caused by high-dose MeHg exposure in both motor function tests. Additionally, OA3Glu treatment reduced the number of contracted Purkinje cells frequently observed in the cerebellum of MeHg-treated groups, although cerebrum histology was similar in all experimental groups. The synaptic potential amplitude in the cerebellum decreased as MeHg exposure increased, which was restored by OA3Glu treatment. Even in the cerebrum, where the effects of MeHg were not observed, the amplitude of the field potential was suppressed with increasing MeHg exposure but was restored with OA3Glu treatment. Taken together, the study findings suggest that OA3Glu improves neurotransmission and movement disorders associated with MeHg exposure via protection of Purkinje cells in the cerebellum while ameliorating pre/post-synaptic deficits in the cerebral cortex in which no changes were observed at the tissue level, potentially providing a treatment to mitigate MeHg toxicity.

Effect of amniotic suspension allograft in a rat destabilization of medial meniscus osteoarthritis model.

Placental-derived allografts have been of interest as a potential nonsurgical treatment to reduce pain and improve function in knee osteoarthritis (OA). The purpose of this study was to evaluate the effect of single and repeat injection of amniotic suspension allograft (ASA) on pain, function, and cytokine levels using a destabilization of the medial meniscus (DMM) rat model of OA. Post-DMM surgery, animals were treated with a single injection of either ASA, vehicle, or triamcinolone, or repeated injection of either ASA or vehicle. Behavioral testing including knee swelling, pain threshold, dynamic weight bearing (DWB), and gait analysis were evaluated during the in-life phase. Postsacrifice, histopathology and serum and synovial fluid analyses were evaluated. Significant improvements in both DWB differentials and pain threshold were seen in response to repeated injection of ASA, while a single injection of ASA and triamcinolone resulted in significant improvements in pain threshold. Histopathology analysis found no significant differences regardless of treatment compared to vehicle, except for an increase in synovitis following repeated injection of ASA. A single injection of ASA and triamcinolone resulted in increased anti-inflammatory cytokines; repeated ASA injection resulted in significant increases in several immune-modulating factors relevant to OA. When comparing the impact of single and repeat ASA treatments on behavioral testing, repeated injection provided significant additional improvements in both pain and function. This study provides evidence demonstrating the impact of a second injection while also providing additional data for evaluating the use of ASA as a nonsurgical treatment for knee OA.

Osteosarcoma-Induced Pain Is Mediated by Glial Cell Activation in the Spinal Dorsal Horn, but Not Capsaicin-Sensitive Nociceptive Neurons: A Complex Functional and Morphological Characterization in Mice.

Bone cancer and its related chronic pain are huge clinical problems since the available drugs are often ineffective or cannot be used long term due to a broad range of side effects. The mechanisms, mediators and targets need to be identified to determine potential novel therapies. Here, we characterize a mouse bone cancer model induced by intratibial injection of K7M2 osteosarcoma cells using an integrative approach and investigate the role of capsaicin-sensitive peptidergic sensory nerves. The mechanical pain threshold was assessed by dynamic plantar aesthesiometry, limb loading by dynamic weight bearing, spontaneous pain-related behaviors via observation, knee diameter with a digital caliper, and structural changes by micro-CT and glia cell activation by immunohistochemistry in BALB/c mice of both sexes. Capsaicin-sensitive peptidergic sensory neurons were defunctionalized by systemic pretreatment with a high dose of the transient receptor potential vanilloid 1 (TRPV1) agonist resiniferatoxin (RTX). During the 14- and 28-day experiments, weight bearing on the affected limb and the paw mechanonociceptive thresholds significantly decreased, demonstrating secondary mechanical hyperalgesia. Signs of spontaneous pain and osteoplastic bone remodeling were detected both in male and female mice without any sex differences. Microglia activation was shown by the increased ionized calcium-binding adapter molecule 1 (Iba1) immunopositivity on day 14 and astrocyte activation by the enhanced glial fibrillary acidic protein (GFAP)-positive cell density on day 28 in the ipsilateral spinal dorsal horn. Interestingly, defunctionalization of the capsaicin-sensitive afferents representing approximately 2/3 of the nociceptive fibers did not alter any functional parameters. Here, we provide the first complex functional and morphological characterization of the K7M2 mouse osteosarcoma model. Bone-cancer-related chronic pain and hyperalgesia are likely to be mediated by central sensitization involving neuroinflammation via glial cell activation in the spinal dorsal horn, but not the capsaicin-sensitive sensory neuronal system.

CHRONIC STRESS RESULTS IN INCREASED PAIN PERCEPTION IN OSTEOARTHRITIS IN VIVO

Osteoarthritis (OA) affects the whole joint and leads to chronic pain. The sympathetic nervous system (SNS) seems to be involved in OA pathogenesis, as indicated by in vitro studies as well as by our latest work demonstrating that sympathectomy in mice results in increased subchondral bone volume in the OA knee joint. We assume that chronic stress may lead to opposite effects, such as an increased bone loss in OA due to an elevated sympathetic tone. Therefore, we analyzed experimental OA progression in mice exposed to chronic stress. OA was induced in male C57BL/6J mice by surgical destabilization of the medial meniscus (DMM) and Sham as well as non-operated mice served as controls. Half of these groups were exposed to chronic unpredictable mild stress (CUMS). After 12 weeks, chronic stress efficiency was assessed using behavioral tests. In addition to measuring body weight and length, changes in subchondral bone were analyzed by μCT. Dynamic Weight Bearing system was used to monitor OA-related pain. Histological scoring will be conducted to investigate the severity cartilage degeneration and synovial inflammation. CUMS resulted in increased anxiety and significant decrease in body weight gain in all CUMS groups compared to non-CUMS groups. CUMS also increased serum corticosterone in healthy mice, with even higher levels in CUMS mice after DMM surgery. CUMS had no significant effect on subchondral bone, but subarticular bone mineral density and trabecular thickness were increased. Moreover, CUMS resulted in significant potentiation of DMM-associated pain. Our results suggest that the autonomic imbalance with increased sympathetic nervous activity induced by chronic stress exacerbates the severity of OA pain perception. We expect significantly increased cartilage degeneration as well as more severe synovial inflammation in CUMS DMM mice compared to DMM mice.

TissueGene-C induces long-term analgesic effects through regulation of pain mediators and neuronal sensitization in a rat monoiodoacetate-induced model of osteoarthritis pain

TissueGene-C (TG-C), a combination of human allogeneic chondrocytes and irradiated GP2-293 cells engineered to overexpress transforming growth factor-β1 (TGF-β1), has been developed as a novel cell-based gene therapy and a candidate for disease modifying osteoarthritis drug (DMOAD). We aim to investigate analgesic mechanism of TG-C in a pre-clinical animal model with monoiodoacetate (MIA)-induced pain. We used a rat MIA model of osteoarthritis (OA) pain. We examined that TG-C can regulate pain by inhibiting the upregulation of various pain mediators in both knee joint tissue and dorsal root ganglia (DRG) (n=112) and alleviating pain behavior (n=41) and neuronal hyperexcitability in DRG (n=60), afferent nerve fiber (n=24), and spinal cord (n=35). TG-C significantly alleviated pain-related behavior by restoring altered dynamic weight bearing and reduced mechanical threshold of the affected hindlimb. TG-C significantly suppressed the expression of nerve growth factor (NGF) and calcitonin gene-related peptide (CGRP) in inflamed joint tissue. TG-C significantly suppressed the upregulation of tropomyosin receptor kinase A (TrkA) and nerve injury/regeneration protein (GAP43) and activation of Iba1-positive microglial cells in DRG. TG-C significantly recovered neuronal hyperexcitability by restoring RMP and firing threshold and frequency of DRG neurons, attenuating firing rates of mechanosensitive C- or Aδ-nerve fiber innervating knee joint, and lowering increased miniature and evoked excitatory postsynaptic currents (mEPSCs and eEPSCs) in the spinal cord. Our results demonstrated that TG-C exerted potent analgesic effects in a rat MIA model of OA pain by inhibiting the upregulation of pain mediators and modulating neuronal sensitization.

Advanced Dynamic Weight Bearing as an Observer-independent Measure of Hyperacute Hypersensitivity in Mice

ABSTRACT Background Standard methods assessing pain in rodents are often observer dependent, potentially resulting in biased outcomes. Advanced dynamic weight bearing (ADWB) offers an observer-independent approach that can provide objective, reliable data in preclinical pain research. Aims The aim of this study was to characterize the use of ADWB in assessing murine responses to allyl isothiocyanate (AITC)-induced hyperacute hypersensitivity and identify best practices for use of the device. Methods Male C57BL/6J mice received intraplantar injections of saline or 0.1% AITC solution and were assessed using the ADWB system; simultaneous observer-dependent durations of paw licking and biting were measured. ADWB data were analyzed using the proprietary software from Bioseb and correlated to observer-dependent results, with parameters assessed to optimize data collected. Results ADWB detected pain-directed changes in weight and surface area distribution in AITC-treated mice, with paw weight and surface area placement correlating to paw licking and biting. Optimization of adjustable threshold parameters allowed for reduced coefficients of variability and increased duration of validated data. Conclusions The ADWB assay provides an efficient and unbiased measure of chemical-induced hyperacute hypersensitivity in mice. ADWB detection parameters influence amount of validated data and variability, a consideration for data analysis in future studies.

Evaluation of tarsal injuries in C57BL/6J male mice.

Tarsal joint abnormalities have been observed in aged male mice on a C57BL background. This joint disease consists of calcaneal displacement, inflammation, and proliferation of cartilage and connective tissue, that can progress to ankylosis of the joint. While tarsal pathology has been described previously in C57BL/6N substrains, as well as in STR/ort and B10.BR strain, no current literature describes this disease occurring in C57BL/6J mice. More importantly the behavioral features that may result from such a change to the joint have yet to be evaluated. This condition was observed in older male mice of the C57BL/6J lineage, around the age of 20 weeks or older, at a frequency of 1% of the population. To assess potential phenotypic sequela, this study sought to evaluate body weight, frailty assessment, home cage wheel running, dynamic weight bearing, and mechanical allodynia with and without the presence of pain relief with morphine. Overall mice with tarsal injuries had significantly higher frailty scores (p< 0.05) and weighed less (p<0.01) compared to unaffected mice. Affected mice had greater overall touch sensitivity (p<0.05) and they placed more weight on their forelimbs (p<0.01) compared to their hind limbs. Lastly, when housed with a running wheel, affected mice ran for a shorter length of time (p<0.01) but tended to run a greater distance within the time they did run (p<0.01) compared to unaffected mice. When tested just after being given morphine, the affected mice performed more similarly to unaffected mice, suggesting there is a pain sensation to this disease process. This highlights the importance of further characterizing inbred mouse mutations, as they may impact research programs or specific study goals.

A STUDY OF SHORT FOOT EXERCISE AND CRYO BALL THERAPY IN YOUNG INDIVIDUAL WITH FLEXIBLE FLAT FOOT: A CASE STUDY

Flatfoot, pes planus, or fallen arch is a common disorder that arises from a decrease of the medial longitudinal arch and it is commonly observed in clinical practice which causes pain and difficulty during physical activities. It is the result of the weakness of intrinsic and extrinsic foot muscles. In particular, the tibialis posterior muscle plays an essential role in maintaining the Medial Longitudinal Arch during dynamic weight bearing and balance. In addition, this muscle acts longer than other supination muscles during the stance phase in the gait cycle. So, the strengthening exercises of the tibialis posterior and cryotherapy improves pain and foot function among young adults with flat foot.

Qilian Formula Inhibits Tumor Cell Growth in a Bone Metastasis Model of Lung Cancer.

Bone metastasis is frequently common in advanced lung cancer with the major issue of a pathological fracture. Previous studies suggested that Astragalus membranaceus (Qi) and Ampelopsis japonica (Lian), which are used as folk medicine in China, have potential effects on inhibiting tumor growth and protecting bones, respectively. In this study, an experiment on the inhibitory effect of the Qilian formula (AAF) in vivo was designed to examine tumor growth in bone and osteoclast formation. The bone metastasis xenograft models were established by implanting NCI-H460-luc2 lung cancer cells into the right tibiae bones of mice. After confirming the model's viability through optical imaging 7 days post-implantation, 2 groups, namely the AAF group and the control group, were administered 0.3 mL of AAF extract (9 g/kg/day) or normal saline via intragastric delivery for a duration of 4 weeks. Throughout the study, we longitudinally assessed tumor burden, bone destruction, and weight-bearing capacity in vivo using reporter gene bioluminescence imaging (BLI), micro-CT, and dynamic weight-bearing (DWB) tests. Mechanistic insights were gained through Hematoxylin-eosin (H&E) staining, immunohistochemical (IHC) analysis, western blotting, and flow cytometry. Qilian formula produced significant inhibition to the progress of bone destruction and tumor burden in the right tibiae bone in the treatment group. It was further evidenced by molecular imaging in vivo via small animal micro-CT and BLI with parametric quantification, characterizing significantly lower uptake of BLI signal in the treated tumor lesions and improving the pathological changes in the microstructure of bone. Furthermore, DWB tests revealed that Qilian formula treatment significantly maintained the weight-bearing capacity. According to immunohistochemical analysis, the effect of the Qilian formula appeared to involve the suppression of osteoclast formation by lower expression of the tartrate-resistant acid phosphatase. Cell apoptosis and death induction were evidenced by a higher percentage of Bal2、BAX and caspase 3 expressions of Qilian formula-treated tumor tissues. Our study demonstrated a significant inhibitory effect of the Qilian formula on the progression of osteolytic invasion in vivo by suppressing osteoclastogenesis and promoting apoptotic cell death.

Effects of ethanol on mechanical allodynia and dynamic weight bearing in male and female mice with spared nerve injury.

Men and women with chronic pain report increased alcohol use and are more likely to be diagnosed with alcohol use disorder. The relationship between alcohol use and pain is bidirectional. Alcohol is used as an analgesic, but chronic alcohol intake increases pain. Sex differences in the relationship between chronic pain and alcohol are reported in the clinical and preclinical literature, but due to this bidirectional relationship, it is challenging to investigate the mechanisms that contribute to these differences. Thus, animal models of chronic pain are needed to characterize the efficacy of ethanol as an analgesic in males and females. The current experiments tested the hypothesis that ethanol differentially reduces pain behaviors in male and female mice in chronic neuropathic pain. The spared nerve injury (SNI) model was used to investigate the analgesic effects of multiple doses of ethanol (0.5, 1, 2, g/kg i.p.) in male and female mice using von Frey and dynamic weight-bearing (DWB) assays. In both male and female mice, SNI led to robust allodynia and shifts in dynamic weight bearing. In male SNI mice, all three doses of ethanol fully reversed mechanical allodynia and shifts in DWB. In SNI females, only the highest dose (2.0g/kg) was fully antiallodynic in the von Frey assay, while shifts in weight bearing were reversed at the 1.0 and 2.0g/kg doses. The differences between male and females were not due to lower blood ethanol concentrations in female mice. These data indicate that while ethanol has antiallodynic and antinociceptive effects in male and female mice, the doses and time course of these effects are distinct. Studies investigating the relationship between pain and ethanol exposure in mice should consider sex as a key variable. These data also inform reported sex differences in rodent models of chronic pain and in chronic pain patients.

R399E, A Mutated Form of Growth and Differentiation Factor 5, for Disease Modification of Osteoarthritis.

To preclinically characterize a mutant form of growth and differentiation factor 5, R399E, with reduced osteogenic properties as a potential disease-modifying osteoarthritis (OA) drug. Cartilage, synovium, and meniscus samples from patients with OA were used to evaluate anabolic and antiinflammatory properties of R399E. In the rabbit joint instability model, 65 rabbits underwent transection of the anterior cruciate ligament plus partial meniscectomy. Three intraarticular (IA) R399E doses were administered biweekly 6 times, and static incapacitance was determined to assess joint pain. OA was evaluated 13 weeks after surgery. In sheep, medial meniscus transection was performed to induce OA, dynamic weight bearing was measured in-life, and OA was assessed after 13 weeks. Intermittent exposure to R399E (1 week per month) was sufficient to induce cell proliferation and release of anabolic markers in 3-dimensional chondrocyte cultures. R399E also inhibited the release of interleukin-1β (IL-1β), IL-6, and prostaglandin E2 from cartilage with synovium, meniscal cell, and synoviocyte cultures. In rabbits, the mean difference (95% confidence interval [95% CI]) in weight bearing for R399E compared to vehicle was -5.8 (95% confidence interval [95% CI] -9.54, -2.15), -7.2 (95% CI -10.93, -3.54), and -7.7 (95% CI -11.49, -3.84) for the 0.6, 6, and 60 μg doses, respectively, 6 hours after the first IA injection, and was statistically significant through the entire study for all doses. Cartilage surface structure improved with the 6-μg dose. Structural and symptomatic improvement with the same dose was confirmed in the sheep model of OA. R399E influences several pathologic processes contributing to OA, highlighting its potential as a disease-modifying therapy.

Objective and Quantitative Evaluation of Spontaneous Pain-Like Behaviors Using Dynamic Weight-Bearing System in Mouse Models of Postsurgical Pain.

BackgroundThe paucity of objective and reliable measurements of pain-like behaviors has impeded the translatability of mouse models of postsurgical pain. The advanced dynamic weight-bearing (DWB) system enables evaluation of spontaneous pain-like behaviors in pain models. This study investigated the suitability and efficiency of the DWB system for assessing spontaneous pain-like behaviors and analgesic therapies in murine models of postsurgical pain.MethodsMale adult C57BL/6JJcl mice were subjected to multiple surgical pain models with distinct levels of invasiveness, including a superficial incisional pain model involving only hind paw skin incision, deep incisional pain model that also involved incision and elevation of the underlying hind paw muscles, and orthopedic pain model involving tibial bone fracture and fixation with a pin (fracture and pinning [F/P] model). Spontaneous pain-like behaviors post-surgery were evaluated using weight distribution, pawprint area of the operated paw in the DWB system, and guarding pain score. Mechanical hypersensitivity was assessed using the von Frey test. The therapeutic effects of analgesics (diclofenac and buprenorphine for the deep incision model and diclofenac for the F/P model) were evaluated using the DWB system and von Frey test.ResultsThe von Frey test demonstrated contradictory results between superficial and deep incisional pain models. The DWB system captured weight distribution changes in the operated hind paw, in accordance with the invasiveness and time course of wound healing in these surgical pain models. The reduction in weight-bearing on the operated paw correlated with guarding score, degree of paw swelling, and local expression of inflammatory mediators. DWB enabled accurate evaluation of the pharmacological effects of analgesics for detecting attenuation of surgery-induced weight-bearing changes in these models.ConclusionThe DWB system serves as an objective and reliable method for quantifying pain-like behaviors and evaluating the therapeutic effects of analgesics in mouse models of postsurgical pain models.

Comprehensive Evaluation of Long-term Dynamic Mechanical Behavior in a Rat MIA OA Model

Osteoarthritis (OA) is highly prevalent across the world with 30% of the US population who are 45 and older estimated to have knee osteoarthritis. The primary clinical indication of OA is pain. Mono-iodo acetate (MIA) induced osteoarthritis is commonly used to study pain in pre-clinical models. Currently mechanical allodynia and thermal hyperalgesia are used to measure the pain outcomes in most pre-clinical models however, the non-evoked dynamic weight bearing analysis provides more clinically relevant behavioral assessment of pain. The goal of the study is to comprehensively understand the changes in dynamic weight bearing behavior of rats with MIA induced OA over a period of 16 weeks. Osteoarthritis was induced in female Sprague-Dawley rats by C-arm guided injections of MIA (3 mg) into the intra-articular region of the right knee. Non-evoked dynamic weight bearing was evaluated using a pressure sensor system (Bioseb DWB 2.0), mechanical allodynia using Von Frey filaments, and transcriptional changes at the dorsal root ganglion using BioRad gene array. MIA model showed mechanical allodynia of the ipsilateral paw for over 3 months. The dynamic weight bearing parameters reveal continuous compensatory weight preference towards the uninjured limbs for the same period. Unlike Von Frey, significantly different dynamic behavioral parameters begin to appear within 24 hours post MIA injection. Moreover, parameters related to weight and area distribution showed temporal changes over the course of the study. The study shows that dynamic weight bearing assessment is an effective tool to understand the long-term progression of pain behavior in a MIA model. Grant support from NIH R01AR075143.

NIH HEAL Initiative: National Institute of Neurological Disorders and Stroke Preclinical Program for Non-Addictive Pain Therapeutic Development

The National Institutes of Health Helping to End Addiction Long-term, or NIH HEAL Initiative aims to focus efforts on advancing scientific solutions to stem the opioid crisis, improving prevention and treatment of opioid misuse/addiction, and enhancing pain management. NINDS is charged with accelerating the discovery and development of new non-addictive pharmacologic and non-pharmacologic pain therapeutics as part of the HEAL Initiative. PSPP provides researchers from academia and industry, within the US and internationally, an efficient, rigorous, one-stop in vivo resource to screen and profile therapeutic candidates including small molecules, biologics, natural products, and devices for the treatment of pain. Under PSPP direction, preclinical testing is performed by a National Institute of Neurological Disorders and Stroke (NINDS) contract facility, PsychoGenics Inc., in a blinded and confidential manner at no cost to the PSPP participants. Participant assets are evaluated in a suite of in vivo pain-related models, for example, chemotherapy-induced neuropathic pain, osteoarthritis pain, and headache. In these models, PSPP uses both evoked (e.g., paw withdrawal threshold, acetone) and non-evoked (e.g., dynamic weight bearing) endpoints to rigorously assess efficacy of the asset in both male and female animals. Importantly, abuse liability is also assessed using a conditioned place preference paradigm or using intravenous self-administration. A key feature of the PSPP is the flexibility to continuously acquire and validate innovative models and endpoints that more closely represent human pain conditions. Notably, in addition to enabling evaluation of small molecules, biologics and natural products, models and endpoints can be appropriately adapted or incorporated to assess devices. We will describe the advances made to date towards establishing program goals of evaluating assets in a rigorous and reproducible manner.

MiR‐106b‐25 Dysregulation in Complex Regional Pain Syndrome Contributes to T Cell Dysfunction

BackgroundComplex regional pain syndrome (CRPS) is a debilitating chronic pain disorder with no effective treatments. Growing evidence implicates aberrant immune regulation in the skin and T cell dysfunction in CRPS pathology. MicroRNA (miRNA) show promise in identifying biomarkers and disease mechanisms. miRNA cluster miR‐106b‐25 is dysregulated in CRPS patient whole blood and serum derived small extracellular vesicles (sEVs) compared to healthy controls. miR‐106b‐25 members are predicted to target several immune genes related to T cell function including CD69. We hypothesize that miR‐106b‐25 cluster plays a role in T‐cell dysfunction by regulating members of CD69 signaling pathway. Here we examine miR‐106b‐25 mediated signaling in sEVs, whole blood, and skin from the tibia fracture model (TFM) of CRPS.MethodsMouse TFM was generated, and pain hypersensitivity was confirmed by von Frey and dynamic weight bearing. Serum, whole blood, and hind limb skin were collected from TFM and control mice. sEVs were isolated by differential centrifugation and characterized using nanoparticle tracking analysis, transmission electron microscopy and western blotting. miRNA from sEVs were assessed through HTG EdgeSeq. T cells were isolated from single cell suspensions of hind limb skin. Whole blood samples were obtained from CRPS patients or healthy controls. miRNA and gene expression changes in blood and T cells were assessed by qPCR. Luciferase reporter assay was used to confirm miR‐25 binding to CD69. Jurkat cells were transfected with miR‐25 mimic or inhibitor to assess regulation of gene expression by qPCR and flow cytometry. T cell phenotypes and expression of CD69 and other residency markers were determined by flow cytometry.ResultsCharacterization of sEVs showed no difference between TFM and control mice. miRNA analysis of TFM sEVs confirmed dysregulation of 30 miRNA previously associated with CRPS patients including miR‐106b‐25. There was an inverse correlation of miR‐25 and CD69 in blood samples from CRPS patients compared to controls. Reporter assay confirmed miR‐25 binding to CD69. miR‐25 nucleofection in Jurkat cells downregulated CD69 as measured by flow cytometry. Analysis of miR‐106b‐25 and target gene expression in TFM whole blood and skin T cells and T cell flow cytometry experiments are ongoing.ConclusionsmiRNA signatures in CRPS patients and TFM mice show common alterations including miR‐106b‐25. miR‐25 can regulate CD69 expression in vitro. miR‐25 may contribute to localized T cell dysfunction in the skin in CRPS.

Ethanol Antiallodynia and Reward Seeking in Mice with Spared Nerve Injury

Clinical data suggests that both men and women with chronic pain are more likely to develop alcohol use disorder, to relapse after a period of abstinence, and to report using alcohol to manage chronic pain. These data led to the hypothesis that alcohol-related behaviors are different in those experiencing chronic pain. To test this hypothesis, the analgesic effect of multiple doses of ethanol (0.5, 1, 2 g/kg i.p.) on male and female mice with spared nerve injury (SNI) was investigated using Von Frey and dynamic weight bearing assays. A conditioned place preference (CPP) paradigm was then used to model ethanol seeking after nerve injury. Findings indicate that SNI reduced mechanical thresholds, increased weight bearing on the contralateral hindpaw, and decreased paw print area on the ipsilateral hindpaw. In male SNI mice, all three doses of ethanol fully reversed mechanical hypersensitivity, while in female mice only the highest dose (2.0 g/kg) was fully antiallodynic. Additionally, the highest dose was analgesic in Sham animals in both male and female mice. In the CPP experiments, conditioning with 0.5 g/kg ethanol induced a modest CPP for the ethanol-paired chamber in SNI and Sham male mice. Further, following extinction, painful stimulation reinstated ethanol-seeking behavior only in SNI mice. These data suggest that the analgesic effects of alcohol are distinct in male and female mice and that animals in chronic pain may be more sensitive to stress-induced reinstatement when pain is the stressor. Ongoing work is focused on characterizing ethanol CPP in female mice and investigating neural correlates of ethanol analgesia and reward in mice in chronic pain.

Walk-IT: An Open-Source Modular Low-Cost Smart Rollator.

Rollators are widely used in clinical rehabilitation for gait assessment, but gait analysis usually requires a great deal of expertise and focus from medical staff. Smart rollators can capture gait parameters autonomously while avoiding complex setups. However, commercial smart rollators, as closed systems, can not be modified; plus, they are often expensive and not widely available. This work presents a low cost open-source modular rollator for monitorization of gait parameters and support. The whole system is based on commercial components and its software architecture runs over ROS2 to allow further customization and expansion. This paper describes the overall software and hardware architecture and, as an example of extended capabilities, modules for monitoring dynamic partial weight bearing and for estimation of spatiotemporal gait parameters of clinical interest. All presented tests are coherent from a clinical point of view and consistent with input data.

Weight Bearing CT Analysis of the Transverse Tarsal Joint During Eversion and Inversion

Category:Basic Sciences/Biologics; HindfootIntroduction/Purpose:Current understanding of the kinematics of the transverse tarsal joint continues to evolve. Unfortunately, most prior studies have been in cadavers or under non-physiologic conditions. Weight bearing CT scans may provide more accurate in vivo information about the position of the transverse tarsal joint during eversion and inversion.Methods:Institutional Review Board approval was obtained. Five normal volunteers underwent bilateral weight bearing cone- beam PedCAT scans (CurveBeam LLC, Hatfield, PA) while standing on platforms that positioned both hindfeet in 20o of valgus and then 20o of varus. The CT scans had a field-of-view diameter of 350 mm and a field-of-view height of 200 mm. The scan exposure time was 9 seconds, with a total scan time of 54 seconds. The datasets were reconstructed using filtered back projection with 0.37mm isotropic voxels. The resulting volumes were then segmented, identifying the [x, y, z] coordinates that comprised each bone. Each bone of the hindfoot was segmented on the scan images and the motion between the talus and calcaneus was analyzed. A similar analysis was done for motion between the navicular and cuboid. Two-tailed paired Student's t- tests were used to compare the angle and distance measurements between valgus and varus positions.Results:In the coronal plane, the axis of the talus rotated by 17.0° relative to the axis of the calcaneus as the hindfoot moved from eversion to inversion (p=0.03). The distance between the centers of the talar head and the anterior calcaneus also moved closer to each other by 7.1 mm (p=0.00005). There was no significant change in angle or distance between the navicular and cuboid when moving from eversion to inversion. The angle between the navicular and cuboid axes changed 2.1° (p=0.7), while the distance changed 0.8 mm (p=0.7)Conclusion:Previous work proposed that the transverse tarsal joint locking mechanism occurs from the talonavicular and calcaneocuboid joint axes diverging (less parallel) as the hindfoot inverts. The current weight bearing CT data shows that not only do the talar and calcaneal axes rotate relative to each other during inversion, but the center of the talar head and anterior calcaneus translate to move closer together. Because the navicular and cuboid do not move, transverse tarsal joint locking may occur due to tightening of the joint capsules and ligaments. Further in vivo study is warranted, especially under dynamic weightbearing conditions.