Background: Exercise remains beneficial at any age, but the physiological response to aerobic exercise declines with aging. This study examines differentially regulated pathways in aging skeletal muscle that may be responsible for this decreased adaptation to aerobic exercise. Methods: To assess differential effects of aerobic exercise with age, Young (Y, 10-12 weeks) and Old (O, 23-25 months) mice were subjected to 8 weeks of treadmill running at similar relative workloads (70% VO 2max ) or no regimented exercise. All mice were maintained in reverse light/dark cycles with exercise performed at similar times following acclimatization. Mice were then evaluated for maximal running speed, distance, and fatiguability with repeated tetanic stimulation. Gastrocnemius (G) muscle was then harvested for single nuclei (sn)RNA sequencing (Azenta) and other G muscle was assessed for fiber type by immunofluorescence. To assess the importance of hypoxia signaling in exercise response with aging, we generated mice with inducible, skeletal muscle fiber-specific knock-in of PHD2 (muscle (m)PHD2), which is a critical upstream inhibitor of hypoxia signaling. mPHD2 and control mice were similarly exercised or underwent no regimented exercise and then similarly evaluated. IOX2, a PHD2 inhibitor, or vehicle control was administered to Y and O mice in a separate experiment during the training period and these mice were similarly evaluated. Results: Y, trained mice experienced a significant increase in maximal distance running, decreased fatigue with repeated tetanic stimulation, and a fiber type transition to oxidative fibers in comparison to age-matched, untrained controls. O mice improved to a lesser degree in running distance and speed but did not improve significantly in fatiguability or fiber type transition. snRNA sequencing analysis of G from Y and O mice, both with and without exercise training, demonstrated many of the most differentially regulated genes with exercise in young mice, which were not similarly regulated in old mice, were hypoxia signaling factors and produced by myofibers. Using our genetically modified mice, immunoblotting demonstrated prolyl hydroxylase domain 2 (PHD2), which limits hypoxia signaling, was 5-fold higher in skeletal muscle at baseline and following exercise in O in contrast to Y mice. Similar to O mice, mPHD2 mice, which exhibit 6-fold higher levels of PHD2 in skeletal muscle versus controls, did not improve maximal distance running, fatiguability, or exhibit a fiber type transition to oxidative fibers whereas littermate control mice did. Administration of IOX2, a PHD2 inhibitor, increased maximal running distance and fiber type adaptation in response to exercise training in O mice. Conclusion: Loss of skeletal muscle hypoxia signaling due to increased PHD2 may be responsible for diminished response to endurance exercise in aging. Activation of hypoxia signaling by inhibition of PHD2 holds promise for rescuing exercise adaptability in old muscle. IS is supported by National Institute of Aging R01 AG078307 and P30AG031679. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic heterotopic ossification (HO) disorder that currently lacks a practical and definitive preventative approach. FOP is driven by gain-of-function variants in ACVR1, increasing dysregulated BMP signaling pathway, thus resulting in progressive and permanent replacement of skeletal muscle and connective tissues with heterotopic bone, ultimately leading to severe debilitation and premature death. Here, we describe the discovery of RK783, a small-molecule that inhibit BMP type 1 receptor kinase developed for treating FOP. This compound, the result of a rigorous process that involved screening approximately 140,000 compounds in silico with ligand-based structure followed by inhibitory activity and pharmacokinetics studies, offers a promising new direction in treating FOP. RK783 preferentially suppressed both basal and stimulated BMP-Smad1/5/9 signaling in vitro without affecting the signaling of Smad2/3. In vivo, the efficacy of RK783 was demonstrated using two FOP mice models, a conditional knock-in ACVR1-R206H and a transgenic ACVR1-Q207D mouse model, where oral dosing suppressed infiltration of immune cells and differentiation of fibroblast-adipose progenitor (FAP) cells, thus preventing ectopic cartilage and HO formation in muscles. Optimized dosing revealed that high and frequent treatment within the first couple of days after HO induction is critical to successfully suppress HO by RK738. These data suggest that RK783 can be used as an acute medication to prevent HO in FOP.

BACKGROUND AND OBJECTIVES:Anatomic features of neuromas have been explored in imaging studies. However, there has been limited research into these features using resected, ex vivo human neuroma specimens. The aim of this study was to investigate the influence that time may have on neuroma growth and size, and the clinical significance of these parameters. METHODS:Patients who underwent neuroma excision between 2022 through 2023 were prospectively included in this study. Neuroma specimens were obtained after operative resection. Standardized neuroma size measurements, expressed as a neuroma-to-nerve ratio (NNR), were conducted with ImageJ software. Pain data (numeric rating scale, 0-10) were prospectively recorded during preoperative evaluation, and patient factors were collected from chart reviews. RESULTS:Fifty terminal neuroma specimens from 31 patients were included, with 94.0% of the neuromas obtained from individuals with amputations. Most neuromas were excised from the lower extremities (n = 44, 88.0%). The neuromas had a median NNR of 2.45, and the median injury to neuroma excision interval was 6.3 years. Larger NNRs were associated with a longer injury to neuroma excision interval and with a smaller native nerve diameter. In addition, sensory nerves were associated with a larger NNR compared with mixed nerves. NNR was not associated with preoperative pain or with anatomical nerve distribution. CONCLUSION:This study suggests that neuromas seem to continue to grow over time and that smaller nerves may form relatively larger neuromas. In addition, sensory nerves develop relatively larger neuromas compared with mixed nerves. Neuroma size does not appear to correlate with pain severity. These findings may stimulate future research efforts and contribute to a better understanding of symptomatic neuroma development.

ABSTRACT Neuromas are a substantial cause of morbidity and reduction in quality of life. This is not only caused by a disruption in motor and sensory function from the underlying nerve injury but also by the debilitating effects of neuropathic pain resulting from symptomatic neuromas. A wide range of surgical and therapeutic modalities have been introduced to mitigate this pain. Nevertheless, no single treatment option has been successful in completely resolving the associated constellation of symptoms. While certain novel surgical techniques have shown promising results in reducing neuroma-derived and phantom limb pain, their effectiveness and the exact mechanism behind their pain-relieving capacities have not yet been defined. Furthermore, surgery has inherent risks, may not be suitable for many patients, and may yet still fail to relieve pain. Therefore, there remains a great clinical need for additional therapeutic modalities to further improve treatment for patients with devastating injuries that lead to symptomatic neuromas. However, the molecular mechanisms and genetic contributions behind the regulatory programs that drive neuroma formation-as well as the resulting neuropathic pain-remain incompletely understood. Here, we review the histopathological features of symptomatic neuromas, our current understanding of the mechanisms that favor neuroma formation, and the putative contributory signals and regulatory programs that facilitate somatic pain, including neurotrophic factors, neuroinflammatory peptides, cytokines, along with transient receptor potential, and ionotropic channels that suggest possible approaches and innovations to identify novel clinical therapeutics.

Background This study analyzed all reported cases of painful traumatic neuromas to better understand their anatomic distribution, etiologies, and surgical treatment. Methods PubMed, Embase, Cochrane, and Web of Science were searched in October 2023 for articles describing painful traumatic neuromas. Results In total, 414 articles reporting 5,562 neuromas were included and categorized into head/neck, trunk, upper extremity, lower extremity, and autonomic nerves. Distribution was as follows: Head/neck: 82 articles reported on 393 neuromas (93.2% iatrogenic) most frequently involving the lingual (44.3%), cervical plexus (14.9%), great auricular (8.5%), inferior/superior alveolar (8.3%), and occipital (7.2%) nerves. Trunk: 47 articles reported on 554 neuromas (92.9% iatrogenic) most commonly involving the intercostal (35.4%), genitofemoral (14.3%), and pudendal (12.9%) nerves. Upper extremity: 159 articles reported on 2079 neuromas (53.3% after amputation) most frequently involving the digital (46.9%), superficial radial (18.3%), and median (7.0%) nerves. Lower extremity: 128 articles reported on 2,531 neuromas (53.0% after amputation) most commonly involving the sural (17.9%), superficial peroneal (17.3%), and saphenous (16.0%) nerves. Autonomic nerves: 15 articles reported on 53 neuromas (100% iatrogenic) most frequently involving the biliary tract (73.9%) and vagus nerve (14.9%). Compared with the extremities, neuromas in the head/neck and trunk had significantly longer symptom duration before surgical treatment and the nerve end was significantly less frequently reconstructed after neuroma excision. Conclusion Painful neuromas are predominantly reported in the extremities yet may occur throughout the body primarily after iatrogenic injury. Knowledge of their anatomic distribution from head to toe will encourage awareness to avoid injury and expedite diagnosis to prevent treatment delay.

Introduction/Aims:Neuromas come in different shapes and sizes; yet the correlationbetween neuroma morphology and symptomatology is unknown. Therefore, we aimto investigate macroscopic traits of excised human neuromas and assess the validityof a morphological classification system and its potential clinical implications. Methods:End-neuroma specimens were collected from prospectively enrolledpatients undergoing symptomatic neuroma surgery. Protocolized images of the speci-mens were obtained intraoperatively. Pain data (Numeric rating scale, 0-10) wereprospectively collected during preoperative interview, patient demographic andcomorbidity factors were collected from chart review. A morphological classificationis proposed, and the inter-rater reliability (IRR) was assessed. Distribution of neuromamorphology with patient factors, was described. Results:Forty-five terminal neuroma specimens from 27 patients were included.Residual limb patients comprised 93% of the population, of which 2 were upper(8.0%) and 23 (92.0%) were lower extremity residual limb patients. The proposedmorphological classification, consisting of three groups (bulbous, fusiform, atypical),demonstrated a strong IRR (Cohen's kappa=0.8). Atypical neuromas demonstratedhigher preoperative pain, compared with bulbous and fusiform. Atypical morphologywas more prevalent in patients with diabetes and peripheral vascular disease Discussion:A validated morphological classification of neuroma is introduced. Thesefindings may assist surgeons and researchers with better understanding of symptom-atic neuroma development and their clinical implications. The potential relationshipof neuroma morphology with the vascular and metabolic microenvironment requiresfurther investigation.

BACKGROUND:Targeted muscle reinnervation (TMR) is an effective surgical treatment for neuropathic pain in amputees. Qualitative descriptions of pain, depicted by pain sketches, could enhance the understanding of symptomatic improvement after surgery. Our aim is to assess whether preoperative pain sketches, drawn by lower extremity (LE) amputees, can predict surgical outcomes after secondary TMR surgery. STUDY DESIGN:Eligible patients were LE amputees who underwent secondary TMR surgery between 2017 and 2023. Pain sketches and pain scores were prospectively collected both before and after surgery. The pain trajectory, as categorized by preoperative pain sketches, was analyzed and assessed for improvement, defined as reaching the minimal clinically important difference. The transition into different pain sketches and the occurrence of phantom drawings were evaluated for their association with improvement. RESULTS:Fifty-eight patients were included, of which 18 (31.1%) depicted diffuse pain, 26 (44.8%) depicted focal pain (FP), and 18 (24.1%) depicted radiating pain (RP) in their preoperative sketch. FP sketches were associated with the lowest pre- and postoperative pain scores and most frequently developed into sketches indicating "no pain." RP sketches were associated with the least pain improvement, the lowest likelihood of achieving the minimal clinically important difference, and were more prevalent in patients with diabetes or depression. RP sketches were associated with phantom drawings; no other sketch types developed into RP sketches at the final follow-up. CONCLUSIONS:In LE amputees who underwent secondary TMR, preoperative pain sketches could serve as a helpful tool in predicting pain outcomes. RP sketches seemed to be associated with worse outcomes and FP sketches with the most improvement.

INTRODUCTION:Targeted muscle reinnervation (TMR) has demonstrated efficacy for treatment of neuropathic pain. This study aims to identify patients for whom delayed TMR may be most effective and to identify associated factors for favorable pain outcomes in this patient population. METHODS:An analysis was conducted on prospectively enrolled amputee patients who underwent delayed TMR at a tertiary care center from 2017 to 2024. Data on demographics, comorbidities, surgical details, and pain outcomes were collected. Patient reported pain severity on a 0-10 scale was prospectively collected. The main pain outcome was pain remission (achieving the minimally clinically important difference (MCID)). Additionally, sustained mild pain (pain score ≤ 3/10 for ≥ 3 months), and pain disappearance (pain score 0/10 for ≥ 3 months) were assessed. Multivariable regression analyses identified factors influencing pain outcomes. RESULTS:Out of 101 patients included in this study, 64 patients (63.4%) achieved pain remission within a two-year post-operative period, and 37 patients (36.6%) did not. Sustained mild pain, which could be achieved in addition to pain remission, was achieved by 45.8% of patients, with 17.8% of these achieving complete pain disappearance. Patients achieving pain remission demonstrated lower pain over the entire post-operative trajectory (p < 0.001). Lower pre-operative pain scores, absence of depression, no pre-operative opioid use, lower Elixhauser Comorbidity Index, and distal amputation levels were correlated with favorable outcomes following delayed TMR (p < 0.05). DISCUSSION:Pre-operative pain severity, psychiatric comorbidities, and opioid use significantly influenced pain outcomes, emphasizing the need for comprehensive patient assessment. These findings will help with patient stratification and pre-operative counseling to support patients who are best suitable for delayed TMR surgery.

BACKGROUND:Heterotopic ossification (HO) can form after amputation and may cause pain and functional impairment. We aimed to describe the prevalence of HO in a civilian population of transtibial amputees. We hypothesized that the decreased rate of symptomatic neuroma following active nerve surgery (Targeted Muscle Reinnervation (TMR) or Regenerative Peripheral Nerve Interface (RPNI)) may subsequently lead to a lower prevalence of HO compared to passive nerve surgery (i.e. traction neurectomy) performed at the time of amputation. METHODS:Adult patients undergoing transtibial amputation at a tertiary care center between 2000 and 2023 were included. Patient data were collected through chart review. The most recent post-amputation X-ray of the residual limb was assessed for HO presence, according to the Walter Reed classification. A random subset of X-rays (10.0 %) was independently assessed by five clinicians and two radiologists, and inter-rater reliability (IRR) was calculated using Cohen's kappa (κ). Multivariable logistic regression was conducted to identify factors associated with HO presence. RESULTS:In total, 665 limbs of 632 patients were included. The median time between amputation and X-ray was 1.7 years (IQR: 0.3-6.2). HO was identified in 326 X-rays (49.0 %) and was commonly present on the distal residual tibia (68.1 %) and fibula (69.0 %). Traditional amputations (i.e. those without TMR or RPNI (OR: 2.0, p = 0.014)), and the presence of a symptomatic neuroma (OR: 2.3, p < 0.001), were independently associated with a higher prevalence of HO. The IRR of the two radiologists was κ = 0.99, the overall IRR of all evaluators was κ = 0.92. CONCLUSIONS:HO is a common finding in transtibial amputees. Peripheral nerve surgerical techniques that actively address amputated nerve endings to reduce symptomatic neuroma formation may decrease the prevalence of HO.

Objective: To characterize the role of neutrophil extracellular traps (NETs) in heterotopic ossification (HO) formation and progression and to use mechanical and pharmacological methods to decrease NETosis and mitigate HO formation. Background: Traumatic HO is the aberrant osteochondral differentiation of mesenchymal progenitor cells after traumatic injury, burns, or surgery. While the innate immune response has been shown to be necessary for HO formation, the specific immune cell phenotype and function remain unknown. Neutrophils, one of the earliest immune cells to respond after HO-inducing injuries, can extrude DNA, forming highly inflammatory NETs. We hypothesized that neutrophils and NETs would be diagnostic biomarkers and therapeutic targets for the detection and mitigation of HO. Methods: C57BL6J mice underwent burn/tenotomy (a well-established mouse model of HO) or a non-HO-forming sham injury. These mice were either (1) ambulated ad libitum, (2) ambulated ad libitum with daily intraperitoneal hydroxychloroquine, ODN-2088 (both known to affect NETosis pathways), or control injections, or (3) had the injured hind limb immobilized. Single-cell analysis was performed to analyze neutrophils, NETosis, and downstream signaling after the HO-forming injury. Immunofluorescence microscopy was used to visualize NETosis at the HO site and neutrophils were identified using flow cytometry. Serum and cell lysates from HO sites were analyzed using enzyme-linked immunosorbent assay for myeloperoxidase-DNA and ELA2-DNA complexes to identify NETosis. Micro-computerized tomography was performed on all groups to analyze the HO volume. Results: Molecular and transcriptional analyses revealed the presence of NETs within the HO injury site, which peaked in the early phases after injury. These NETs were highly restricted to the HO site, with gene signatures derived from both in vitro NET induction and clinical neutrophil characterizations showing a high degree of NET "priming" at the site of injury, but not in neutrophils in the blood or bone marrow. Cell-cell communication analyses revealed that this localized NET formation coincided with high levels of toll-like receptor signaling specific to neutrophils at the injury site. Reducing the overall neutrophil abundance within the injury site, either pharmacologically through treatment with hydroxychloroquine, the toll-like receptor 9 inhibitor OPN-2088, or mechanical treatment with limb offloading, results in the mitigation of HO formation. Conclusions: These data provide a further understanding of the ability of neutrophils to form NETs at the injury site, clarify the role of neutrophils in HO, and identify potential diagnostic and therapeutic targets for HO mitigation.

Hypoxia signaling has been previously demonstrated to be a critical regulator in age-dependent skeletal muscle regeneration. While animal models provide a crucial scientific substrate for medical innovation and translation, discordant findings have been commonly described in existing literature between animals and humans. The nature and extent of this divergence in the context of aging and its effect on skeletal muscle regeneration remains incompletely understood. Quadriceps muscles from young (Y, 12 weeks, n=3) and old (O, 25 months, n=3) C57BL/6 mice were harvested and analyzed via RNA bulk-sequencing. These results were compared to healthy human vastus lateralis muscles (Y, 22-30 years, n=8; O, 80-83 years, n=5) from the GESTALT study (NIA/NIH) analyses as found in the Gene Expression Omnibus (NIH) database. Gross examination of the most differentially expressed genes demonstrated low variance between samples within each mouse cohort (Disp = 0.04467, BCV = 0.2114) relative to young/old human (Disp = 0.61261, BCV = 0.7827) specimens. Enrichment analyses were agnostically performed on filtered differentially expressed human genes via edgeR (Bioconductor) resulting in significant overlap with the HIF-1 signaling pathway KEGG term (Eno1, Tfrc, Ldha, Eno3, and Pdha1) with significant upregulation in young human muscles. However, these same genes demonstrated no significant differences within mice. Interestingly, when examining angiogenesis gene lists determined a priori, mice demonstrated several significantly upregulated genes in the young cohort (Anxa2, Lox, Mmp9, Angptl4, Adora2b, and Pgf) that thematically correspond to modulators of the extracellular matrix in the process of neoangiogenesis. Conversely, none of these genes were found to be significantly different between old and young humans. To further compare the muscle transcriptomes between humans and mice in the context of aging, genes with the largest magnitude fold changes and most significant p-values were identified. The most significant differentially expressed genes in mice demonstrated robust upregulation of Mss51, Igkv3-2, Ighg2c, Rasd2, Igkc, Eda2r, and Gdf11 in old mice. These same genes in human specimens demonstrated no comparable segregation. Similarly, the most upregulated genes in old human specimens (Gda, Mtcybp32, Fgf7, Fam83b, Lyve1, Plag1, Eda2r, Dkk2, and Col21a1) showed no similar segregations in mice. The most differentially expressed genes for hypoxia and angiogenesis in both mice and humans were not found to be concordantly upregulated when cross-examined across the other species. Thus, skeletal muscle in inbred mice exhibits differential transcriptome expression and decreased variance as compared to humans in aging. Given these findings, great care should be taken in making inferences between findings between mice and humans. As NextGen sequencing becomes routine, incorporation of genetically diverse mice to better approximate human genetic variation may provide further insights in translation from bench to bedside.

The Interdisciplinary Cardiac Arrest Research Review (ICARE) group was formed in 2018 to conduct an annual search of peer-reviewed literature relevant to cardiac arrest. Now in its fourth year, the goals of this review are to highlight annual updates on clinically relevant and impactful clinical and population-level studies in the interdisciplinary world of cardiac arrest research from 2021. To achieve these goals, a search of PubMed using keywords related to clinical research in cardiac arrest was conducted. Titles and abstracts were screened for relevance and sorted into seven categories: Epidemiology & Public Health; Prehospital Resuscitation; In-Hospital Resuscitation & Post-Arrest Care; Prognostication & Outcomes; Pediatrics; Interdisciplinary Guidelines; and Coronavirus disease 2019. Screened manuscripts underwent standardized scoring of methodological quality and impact by reviewer teams lead by a subject matter expert editor. Articles scoring higher than 99(th) percentile by category were selected for full critique. Systematic differences between editors' and reviewers' scores were assessed usingWilcoxon signed-rank test. A total of 4,730 articles were identified on initial search; of these, 1,677 were scored after screening for relevance and deduplication. Compared to the 2020 ICARE review, this represents a relative increase of 32% and 63%, respectively. Ultimately, 44 articles underwent full critique. The leading category was In-Hospital Resuscitation, representing 41% of fully reviewed articles, followed by Prehospital Resuscitation (20%) and Interdisciplinary Guidelines (16%). In conclusion, several clinically relevant studies in 2021 have added to the evidence base for the management of cardiac arrest patients including implementation and incorporation of resuscitation systems, technology, and quality improvement programs to improve resuscitation.