Abstract Chemotherapy-induced peripheral neuropathy (CIPN) is a painful side effect of treating cancer with cisplatin. Cisplatin is a platinum-based chemotherapeutic that induces mast cell activation. Mast cells reside in close proximity of the epidermal layer, vasculature and nerve fibers in the skin. Tryptase and other noxious substances released by mast cells activate nociceptors via protease activated receptor -2 (PAR2) leading to pain. We hypothesized that cisplatin-induced mast cell activation leads to nerve injury and neuropathic pain. We used C57BL/6, wild-type (WT) and mast cell knockout (MC-KO) mice with a spontaneous c-kit "sash" mutation (KitW-sh) on a C57BL/6 background. Male and female mice were treated with cisplatin (i.p., 2.3 mg/kg/day), for 5 days of treatment followed by 5 days of saline (i.p.) for 2 cycles; or pre-treated with 100 mg/kg/day imatinib, an inhibitor of c-Kit and mast cell activation. Mechanical, cold, and thermal hyperalgesia were assessed at regular intervals. At day 18, cisplatin-treated mice showed a significant increase in mechanical hyperalgesia (p<0.001), cold hyperalgesia (p<0.05), and heat hyperalgesia (p<0.0001) compared to vehicle-treated mice. There were no changes in WT mice treated with imatinib or imatinib with cisplatin or in MC-KO mice treated with cisplatin for mechanical or thermal hyperalgesia, suggesting the involvement of mast cells in CIPN. Dorsal skin sections were co-stained with histone H3 (mast cell traps), FcεR1 (mast cell marker), and NF200 (nerve bundles). Cisplatin-treated mice showed activated mast cells surrounding nerve bundles, causing the expulsion of dense traps of citrullinated histones and podia extending into nerve fibers, causing nerve damage. The vehicle-treated mice showed intact, undisturbed thick nerve bundles without mast cell activation. Compared to vehicle, cisplatin-treated WT mice showed a significant increase in the number of non-degranulating and degranulating mast cells in dorsal (p<0.05) and toe skin (p<0.001, p<0.0001, respectively). We found a significant increase in cutaneous chymase (p<0.01), and tryptase (p<0.01), and plasma chymase (p<0.5), and tryptase (p<0.01) levels in cisplatin-treated compared to vehicle-treated WT mice. Human mast cells, HMC1.2, were incubated with 2, 5, and 10 µg/ml cisplatin or vehicle for 10 and 70 min, followed by analysis of chymase and tryptase in the conditioned medium. At the lowest dose of 2 µg/ml, cisplatin significantly stimulated the time-dependent release of chymase and tryptase compared to vehicle (p<0.01 for both). Together, our data show the novel phenomenon of mast cell traps upon cisplatin treatment leading to nerve injury, while tryptase released from mast cells may activate PAR-2 leading to the painful symptoms of CIPN. We speculate that the cotreatment of cisplatin with imatinib or other mast cell stabilizers such as cromolyn may ameliorate the painful symptoms of CIPN. Citation Format: Carolina Mireles, Donovan A. Argueta, Raghda Fouda, Sonal Joshi, Daniela A. Bota, Kalpna Gupta. Mast cell activation leads to cisplatin-induced peripheral neuropathy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5217.

Objective. Low-intensity transcranial focused ultrasound (tFUS) offers high spatial specificity and deep brain penetration, representing a promising non-invasive approach for modulating brain activity and behavior. Although emerging studies indicate that tFUS can modulate pain-related behaviors in rodents and humans, the underlying network-level and cellular mechanisms remain unknown. This study investigates the effects of tFUS neuromodulation on inhibitory neural circuits in a humanized mouse model of chronic pain, integrating electrophysiological, molecular, and histological analyses across a cohort of 50 animals, including wild-type controls. Approach. A 128-element random array transducer was used to precisely target the pain-processing brain circuit, while a non-invasive and flexible 30-channel electroencephalography electrode was applied to record local evoked responses, topographical activity, and global excitation and inhibition dynamics, which were further validated by optogenetics experiments. Cellular-level modulation and safety outcomes were evaluated through blinded histological examination. Main results. We found that tFUS produced robust modulation of local and global brain activity, characterized by suppression of local theta oscillations and enhancement of network-level inhibitory dynamics. These electrophysiological patterns aligned with those observed during optogenetic activation of parvalbumin (PV) interneurons. Immunohistochemistry further showed significant increases in inhibitory neuronal markers, including elevated expressions of glutamate decarboxylase 67 and PV. Blinded histological assessment confirmed the absence of tissue damage, supporting the safety of the stimulation paradigm. Significance. These findings demonstrated that tFUS stimulation non-invasively engages PV GABAergic inhibitory circuits in a chronic pain mouse model, providing mechanistic insight and supporting its development as a precise and safe neuromodulation technology for clinical translation.

Abstract Sickle cell disease (SCD) is a hereditary hemoglobinopathy characterized by persistent pain. The mechanisms underlying pain in SCD are poorly understood, and opioids remain the primary treatment, despite their severe side effects. Here, we investigated the contribution of lysophosphatidic acid (LPA), an endogenous pronociceptive lipid mediator, to chronic pain in SCD using humanized transgenic homozygous Berkeley mice that express >99% human sickle hemoglobin (HbSS) and control HbAA mice that express normal human hemoglobin A. Hyperalgesia in HbSS mice was associated with an increase in both plasma level of LPA and expression of LPA receptor 1 (LPA1R) mRNA in L1 to L5 dorsal root ganglion (DRG). Blocking LPA synthesis with BI-2545, or blocking LPA1R function with small interfering RNA (siRNA) or the LPA1R antagonist, AM966, reversed mechanical and heat hyperalgesia in HbSS mice. LPA also produced acute mechanical and heat hyperalgesia in HbAA mice, which resulted from the sensitization of C-fiber nociceptors. In HbSS mice, hyperalgesia was associated with sensitization of nociceptive DRG neurons. Nociceptors from hyperalgesic HbSS mice had lower rheobase, more positive resting membrane potential, and higher frequency of action potential. Although no changes were found in the values of inward currents in nociceptors of HbSS mice compared with HbAA mice, outward-inactivating and noninactivating currents were reduced, indicating the importance of potassium channels to sensitization in SCD. All these parameters were normalized by pretreatment of HbSS mice with LPA1R siRNA. Our results suggest that LPA signaling may be a promising target for treating pain in SCD.

Primary neuronal cultures from the brain are critical for investigating disease-specific cellular and molecular mechanisms in mouse models. Current methods for obtaining primary cultures require embryonic brains that are affected by embryonic lethality and genotypic characterization in severe disease models such as sickle cell disease (SCD). Furthermore, these neuronal cultures require about 14 days in vitro (DIVs) for neurite outgrowth to mature. We adapted and optimized a relatively simplified and reproducible method using brains from postnatal day 1 mouse pups for isolating and culturing hippocampal and cortical neurons. This approach produces viable neurons that attach, extend neurites, and express key synaptic markers by 7 DIV and also minimizes glial outgrowth. We successfully applied this approach to isolating and culturing hippocampal and cortical neurons from the brains of one-day-old (P1) pups of humanized transgenic homozygous BERK sickle cell and control mice. Morphological observations at 3, 7, and 14 DIVs demonstrated robust neuronal attachment, neurite outgrowth, and overall structural development in both male and female hippocampal and cortical neurons. Neurons in culture expressed key markers including neuronal nuclear protein (NeuN/Rbfox3), neurofilament 200 (NF200), microtubule-associated protein 2 (MAP2), vesicular glutamate transporter 1 (VGLUT1), postsynaptic density protein 95 (PSD 95), and glutamate N-methyl-D-aspartate receptor subunit 2B (GluN2B). Notably, male SCD hippocampal neurons evinced a higher density of PSD 95 puncta on dendritic spines compared to controls on 7 as well as 14 DIVs. Incubation of male hippocampal neurons in a sickle cell-like microenvironment with TNF-α and heme further increased the density of PSD 95 puncta and colocalization of GluN2B with PSD 95, supporting the utility of this culture system for examining disease-relevant structural and molecular responses. This optimized culture system provides a simplified and reproducible platform to investigate the mechanisms involving neuronal dysfunction in challenging mouse models of brain disorders.

Chemotherapy is frequently associated with long-term cognitive impairments in cancer survivors that negatively impact their quality of life. Effective mitigation strategies for cancer therapy-related cognitive impairments (CRCI) are still underdeveloped. Our clinical studies on breast cancer patients treated with doxorubicin (Adriamycin®, ADR) and cyclophosphamide (CYP) found significant CRCI associated with neurodegenerative and neuroinflammatory signatures. Current preclinical and clinical studies highlight the potential of cannabidiol (CBD) for alleviating cognitive deficits in neurodegenerative conditions. For example, Epidiolex® is an FDA-approved 99% pure formulation of CBD for treating pediatric epilepsy. CBD, a non-psychoactive component of cannabis, is recognized for its neuroprotective and anti-inflammatory effects. This study, using a mouse model of adjuvant chemotherapy (ADR and CYP)-induced cognitive decline, tested the efficacy of oral administration of 99% pure CBD (20 mg/kg) in sesame oil. ADR + CYP-treated mice receiving CBD for one month showed significant neurocognitive improvements in learning and memory, executive function, and memory consolidation tasks often impaired in cancer survivors. CBD treatment also restored brain endocannabinoid (ECB) levels and reduced ECB-metabolizing enzymes in vivo. Notably, CBD mitigated chemotherapy-induced loss of neurogenesis, neuronal plasticity, synaptic density, and elevated gliosis. In summary, this data provides preclinical evidence for a translationally feasible approach to alleviate CRCI, an unmet medical need.

Abstract Background Acute pain episodes in sickle cell disease (SCD) are unpredictable, frequently require hospitalization, and are associated with reduced survival. While vasoocclusion and hypoxia are major triggers, the neuroimmune mechanisms that amplify nociception during these events remain incompletely defined. Hemolysis and vasoocclusion release damage-associated signals, including heme, that promote oxidative stress and inflammatory activation in the periphery and central nervous system (CNS). Increasing evidence supports a cytokine-driven axis in which interleukin-17A (IL-17A) amplifies tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) signaling and engages stress kinases such as p38 mitogen-activated protein kinase (p38 MAPK), thereby sustaining microglial activation, neuroinflammation, and neuronal dysfunction. Defining how this IL-17A–TNF-α/IL-6–p38 MAPK cascade links systemic inflammation to spinal mechanisms of pain during hypoxic stress is essential for developing preventive, non-opioid strategies for acute SCD pain. Methods Acute vasoocclusive stress was modeled using hypoxia/reoxygenation (H/R; 8% O2 for 3 h followed by reoxygenation at room air) in homozygous HbSS-BERK sickle mice with HbAA-BERK controls. Mechanical sensitivity, cold sensitivity, and grip force were assessed at baseline, immediately after H/R-1, 24 h post-H/R-1, immediately after H/R-2, and 24 h post-H/R-2 (H/R-1 and H/R-2 separated by 48 h). To test the role of IL-17A signaling, anti–IL-17A antibody (0.25 µg/g body weight) was administered intrathecally 4 h prior to each H/R exposure. To evaluate therapeutic modulation, mice received daily transdermal curcumin (TDC; VAS-101, Vascarta Inc.) or vehicle for 2 weeks prior to H/R, which was continued until the end of the study. Plasma cytokines and spinal neuroinflammatory signaling were analyzed by immunoassays and immunofluorescence. Mechanistic studies were performed in primary dorsal root ganglion (DRG) neurons, primary microglia, and HT22 hippocampal neurons exposed to a hypoxic sickle microenvironment (TNF-α + hemin ± CoCl2-induced hypoxia), with or without the p38 MAPK inhibitor neflamapimod. Results Vehicle-treated HbSS-BERK mice displayed increased mechanical and cold hypersensitivity at all post-H/R time points, as predicted, compared with baseline and HbAA controls (P < 0.0001). Grip force declined after H/R-1 (P < 0.05) and further after H/R-2 (P < 0.0001), indicating progressive musculoskeletal hyperalgesia. H/R increased spinal phospho-p38 MAPK and elevated IL-17, TNF-α, and IL-6 compared with normoxia (P < 0.05). Under normoxia, HbSS-BERK mice exhibited higher basal microglial activation, indicated by increased ionized calcium-binding adapter molecule 1 (Iba1) immunoreactivity, and higher phospho-p38 MAPK immunoreactivity in the spinal dorsal horn compared with controls; both were amplified after H/R. Plasma IL-17A was significantly increased in H/R treated compared with normoxic sickle mice (P < 0.05), consistent with a systemic inflammatory response to HR. Intrathecal anti–IL-17A antibody significantly reduced mechanical (P < 0.0001), cold (P < 0.0001), and musculoskeletal hyperalgesia (P < 0.05), attenuated spinal microglial activation, and decreased spinal IL-17 and TNF-α following H/R-2. In primary sickle microglia, TNF-α plus hemin under hypoxic conditions markedly increased IL-17A production, and this response was attenuated by TDC or neflamapimod, supporting microglia as a p38 MAPK-regulated source of IL-17A. Pre-treatment with TDC significantly attenuated H/R-evoked mechanical and cold hypersensitivity at 24 h post-H/R-1, H/R-2, and 24 h post-H/R-2 (P < 0.0001), improved grip force (P < 0.05), suppressed spinal microglial and p38 MAPK activation, reduced systemic and spinal cytokines, and decreased oxidative stress while preserving mitochondrial membrane potential in DRG and HT22 neurons. Conclusions Acute hypoxia reoxygenation in sickle mice engages an IL-17A–TNF-α/p38 MAPK axis that links systemic inflammation to spinal microglial activation and downstream neuronal oxidative stress, culminating in mechanical, cold, and musculoskeletal hyperalgesia. TDC disrupts this multi-level inflammatory cascade and mitigates acute pain behaviors, supporting its potential as a preventive, non-opioid drug candidate for acute pain episodes in SCD.

Abstract Cisplatin-induced neuropathy remains a major challenge to treat. Cisplatin disrupts mitochondrial homeostasis and increases reactive oxygen species (ROS) contributing to neuronal injury. We examined the ability of curcumin to prevent CIPN because it has antioxidant and neuroprotective properties. However, a major challenge is the reduced absorption and bioavailability of oral and systemically administered curcumin. To address this challenge, we used a novel transdermal curcumin (TDC) preparation which is bioavailable in the blood and central nervous system after topical application to the abdomen of mice. We used a transgenic mouse model of breast cancer (C3TAg) which shows the evolutionary spectrum of human breast cancer and its isotype control FVB/N mice. At ∼4 months of age female C3TAg mice develop palpable tumors and demonstrate mechanical, thermal and musculoskeletal hyperalgesia (P<0.0001 vs FVB/N). Mice were treated with vehicle or cisplatin (2.3 mg/kg/day i.p.) for two cycles of 5-days and 5 days of rest in the presence or absence of TDC/VAS-101 (0.1 mL) applied daily by rubbing on the abdomen of mice through the endpoint. Similar to cisplatin, TDC alone significantly reduced tumor weight (P<0.05 vs vehicle), and didn’t decrease the anti-tumor efficacy of cisplatin. By day 5, cisplatin induced significant mechanical and cold hyperalgesia in both strains (p<0.001 vs vehicle and BL), and musculoskeletal hyperalgesia at day 16 in C3TAg mice (P<0.001 vs BL; P<0.0001 vs vehicle). TDC co-treatment significantly attenuated cisplatin induced hyperalgesia (mechanical and cold, P<0.0001 vs cisplatin) and prevented musculoskeletal hyperalgesia (P<0.001 vs cisplatin). Notably, in C3TAg mice, TDC alone significantly decreased constitutive mechanical (P<0.001 vs vehicle; P<0.01 vs BL) and cold hyperalgesia (P<0.01 vs vehicle). These changes in hyperalgesia were accompanied by a significant reduction in phospho-p38 mitogen-activated protein kinase (MAPK) in dorsal root ganglion (DRG) neurons in C3TAg mice co-treated with TDC and cisplatin compared to cisplatin treatment (P<0.001) suggesting the activation of pain signaling. Furthermore, in primary DRG neurons and HT22 hippocampal neuronal cell line in culture, cisplatin elevated ROS and caused mitochondrial depolarization (P<0.0001; P<0.001 vs vehicle), which was prevented by TDC (P<0.0001). In HT22 neurons, cisplatin increased calcium release and lowered subsequent metabolic activity and viability (P<0.001 vs. vehicle), which were significantly inhibited by TDC (P<0.001), indicating that TDC targets the pain generating Ca2+ release from neuronal cells. In conclusion, TDC alleviates cancer- and chemotherapy-related hyperalgesia via inhibition of p38 MAPK and oxidative stress, while restoring mitochondrial function and limiting tumor growth. Thus, the novel TDC has a translational potential for preventing CIPN. Citation Format: Yugal Goel, Carolina Mireles, Dahlia Ordaz, Kendall O’Daniel, Kristen A. Peterson, Naomi Lomeli, Reina Lomeli, Daniela A. Bota, Joel Friedman, Kalpna Gupta. Novel transdermal curcumin attenuates cisplatin induced neuropathy in a mouse model of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5216.

Background: Mu opioid receptors (MORs) in peripheral tissues mediate adverse effects of opioids that impair health-related quality of life (HRQoL) and may stimulate cancer progression via mitogenic signaling. Naloxegol, a peripherally acting MOR antagonist (PAMORA), is approved for opioid-induced constipation. Safety and efficacy of naloxegol have not been evaluated concurrently with systemic cancer therapy. Methods: We conducted a randomized, double-blind, placebo-controlled trial of naloxegol in patients with advanced lung adenocarcinoma starting first-line systemic therapy. Results: Only 50 patients were enrolled; the trial was terminated early due to slow accrual. Two of the three components of the feasibility primary endpoint were not met (accrual and PRO completion). At 6 months, FACT-L emotional well-being was better with naloxegol (p = 0.0113). There were trends towards better Trial Outcome Index (p = 0.0505) and physical well-being (p = 0.0628) with naloxegol. Bowel function favored naloxegol for constipation (p = 0.0223), rectal pain during defecation (p = 0.0075), and abdominal pain from constipation (p = 0.0113). Adverse event frequency and severity, PRO-CTCAE, urinary hesitancy, pain scores, and progression-free and overall survival were comparable between naloxegol and placebo. Conclusions: Naloxegol appears to be safe and tolerable, with a signal of improved HRQoL and previously unappreciated benefit for emotional well-being, without adverse clinical outcomes. Our findings should be confirmed in larger studies. ClinicalTrials.gov ID: NCT03087708.

Pain is a debilitating feature of hemophilia A (HA), yet it remains understudied. We developed models of chronic and acute pain in factor VIII knock-out (FVIIIKO; B6;129S-FVIIItm1Kaz/J), demonstrating characteristic features of hemarthrosis pain and gait. Using these models, we examined the mechanisms involving inflammation and vascular changes triggered by bleeding to produce pain. To recapitulate hemarthrosis, we utilized an acute knee-injury model to assess acute pain. We observed an increase in nocifensive behaviors, mechanical and deep tissue hyperalgesia, impaired weight bearing and gait changes 4 days post-subpatellar injury which persisted through 8 weeks. The uninjured FVIIIKO mice showed similar changes in behaviors and gait at 10 weeks of age, suggestive of chronic pain. Dynamic gait changes indicated compensatory behavior. Hemarthropathy induced a significant elevation in circulating serum amyloid P (SAP) and interleukin (IL)-6 and the injured knee joint showed a significant increase in neutrophil elastase, myeloperoxidase, mast cell degranulation, substance P (SP) and calcitonin gene related peptide (CGRP) suggestive of global inflammation. SP and CGRP stimulate vascular permeability and arteriolar dilatation, respectively, but are also involved in the generation and maintenance of pain. Mast cell tryptase and neutrophil elastase activate protease-activated receptor 2, which causes nociceptor activation leading to pain. Recombinant FVIII treatment led to partial improvement in pain behaviors. Thus, pain may persist following current HA treatments. Our study shows that a mouse model of HA can be used to study the mechanisms of pain and gait, which will enable us to develop treatable targets for pain in HA.

Background: Sickle cell disease (SCD) is a systemic disorder marked by chronic pain and neurocognitive deficits, yet the molecular drivers of these neurocognitive features remain poorly defined. Platelets, central to inflammation and vascular homeostasis, may reflect broad pathophysiologic processes in SCD. Methods: We performed high-resolution mass spectrometry on ultra-purified platelets from 16 adults with SCD and moderate to severe pain (self-reported ≥ 3/10 in the past year), identifying 4196 proteins, of which 1046 were significant (FDR < 0.05). Unsupervised clustering was used to stratify individuals into high- and low-pain phenotypes. Results: Contrary to expectations, canonical pain pathways were not enriched. Instead, significant alterations were observed in neurodegeneration, mitochondrial metabolism, ATP regulation, mitophagy, and tRNA aminoacylation pathways between high- and low-pain phenotypes. High-pain individuals exhibited elevated levels of proteins involved in proteostasis and neurodegenerative disease processes, whereas low-pain individuals showed increased expression of proteins linked to mitochondrial integrity, neuroprotection, and reduced oxidative stress. Protein-protein interaction networks revealed tightly connected clusters within neurodegenerative and central nervous system-related pathways. Disease association analysis ranked neurodegenerative and mitochondrial pathways above traditional hematologic and nociceptive mechanisms. Conclusions: These findings suggest that platelet proteomics may serve as a peripheral window into PNS or CNS vulnerability and cognitive risk in SCD. The enrichment of tRNA aminoacylation and mitochondrial regulation pathways underscores the metabolic complexity of SCD and highlights novel targets for biomarker development and therapeutic intervention.

This Viewpoint calls for a reconsideration of chronic pain as a symptom of nervous system dysfunction and recommends several neuromodulation therapies for refractory clinical pain.

Objectives:Stroke in sickle cell disease (SCD) is often attributed to large vessel involvement in the disorder, whereas the contribution of cerebral microvascular disease has been less explored. In this study, we investigated the formation of cerebral microvascular lesions and the involvement of mast cells in a humanized SCD mouse model. Methods:We studied hemorrhagic microvascular disease in a well-characterized mouse model of humanized transgenic sickle (HbSS-BERK) expressing >99% human sickle hemoglobin (HbS) and a control (HbAA-BERK) mouse model expressing normal human hemoglobin A (HbA). Mouse brains were analyzed by Prussian blue staining to detect cerebral microhemorrhage (CMH) formation. Mast cell identification was performed by toluidine blue staining. Results:SCD brain sections exhibited approximately 86% more CMH than controls (mean ± SE of 1.17 ± 0.22 vs. 0.63 ± 0.13 number/cm2, P = .02). Mast cells were positively correlated with CMH number in SCD mice (Spearman r = 0.42, P < .05), but not in control mice. Conclusion:SCD mice demonstrated significantly increased CMH load compared with control mice, and SCD microhemorrhages were associated with the number of mast cells. These findings highlight the significance of cerebral microvascular disease in SCD and imply that cerebral mast cells may be a novel therapeutic target in SCD.

Abstract Background Approximately 5% of pregnant women in the United States and 20% of pregnant women aged 18-24 years in California reported using cannabis (Young-Wolff et al JAMA 2017). Cannabinoid (CB) use in persons with sickle cell disease (SCD) has been reported more than other conditions. Hydroxyurea (HU) improves survival in SCD, however, HU is a teratogen that is withdrawn during pregnancy, thus increasing the probability of seeking CBs to control pain. This may have devastating consequences; a single in utero dose of synthetic- (CP55,940) or phyto-CBs (Δ9-tetrahydrocannabinol) produced developmental changes in the offspring of pregnant C57BL/6 mice (Fish et al Sci. Rep. 2019). We examined if CB use during pregnancy in SCD will have an effect on the development and long-term generational behavioral outcomes related to pain in the offspring. Methods We utilized the well-established humanized sickle, transgenic HbSS-BERK mouse model of SCD which expresses >99% human α- and ßS- sickle hemoglobin (Hb) and complete knockout of murine α- and β-globins. Sickle mice recapitulate many of the clinical features of human SCD, which include spontaneous chronic pain, hypoxia/reoxygenation-incited acute pain, and end organ damage. Homozygous (HbSS/SS) sickle BERK mice breed poorly, thus we bred hemizygous sickle (HbAS/AS) and normal human Hb expressing control mice (HbAA/AA) females with SS and AA males, respectively. Females were treated with HU (i.p., 50 mg/kg/day), CP55,940 (CP; i.p., 0.3 mg/kg/day), or vehicle (Veh; 2% DMSO in sterile saline) for 2-weeks prior to breeding. During gestation, mice receiving HU were switched to Veh or CP (to recapitulate HU cessation and CB replacement, respectively); mice receiving Veh or CP continued their exposure. Placenta were evaluated at embryonic day (ED) 18. Offspring were assessed for teratogenicity (physical malformations) at postnatal day 1 (PD1) using high-resolution digital images; a subset of offspring were harvested at PD21 to evaluate brain weights; lastly, offspring were evaluated for hyperalgesia at 2- and 3-months (mos) of age. Results Replacing HU with CP55,940 (HU+CP) during pregnancy significantly reduced head circumference on PD1 in male and female sickle and control offspring compared to Veh (P < 0.05); HU+CP significantly reduced eye size in male and female sickle offspring compared to Veh (P < 0.05). CP caused limb lengthening in female sickle offspring compared to Veh and HU+CP (P < 0.01). Brains from male PD21 sickle offspring exposed to CP showed significant size reduction compared to Veh and HU+CP (P < 0.05). Further, placenta weights at ED18 were significantly reduced by HU+CP compared to Veh or HU (P < 0.05), which was further exacerbated by CP compared to HU+CP (P < 0.05). Placental histology revealed that CP led to a marked reduction in the junctional zone (JZ), with decreased cellularity, disrupted labyrinth architecture, diminished maternal blood spaces, and focal areas suggestive of hemorrhage and infarction, consistent with ischemic injury. This contrasts with Veh, which showed mildly dilated vascular channels and sinusoidal congestion, likely reflecting SCD-related pathology. HU preserved placental structure, maintaining organized vasculature and intact JZ. HU+CP displayed intermediate features, suggesting partial protection. The observed JZ thinning in CP, may reflect CB-induced vasoconstriction or hypoxic remodeling. Placental dysfunction gives rise to poor outcomes for cognition and development, which may contribute to an overt, early onset hyperalgesia phenotype. SCD presents with chronic hyperalgesia, indicated by greater sensitivity to mechanical and cold stimuli, and reduced grip strength, which typically arises at 3 mos in sickle mice. CP and HU+CP significantly reduced grip strength, indicating greater musculoskeletal hyperalgesia, in male and female sickle offspring starting at 2 mos. CP or HU+CP exacerbated features of chronic hyperalgesia compared to Veh or HU in 3 mos male and female sickle mice, indicated by greater paw withdrawal frequency to mechanical and cold stimuli (P < 0.05). No differences in hyperalgesia were observed in control offspring. Conclusions Thus, prenatal CB exposure, with or without HU, may have a profound generational effect on pain in offspring, specific to SCD. The offspring with in utero CB exposure may be at risk of multi-organ developmental defects, and early onset of hyperalgesia. Our data highlight the risks associated with CB use during pregnancy in SCD, which requires investigation to understand the mechanisms conferring risk to offspring health.

Importance:The interplay between sickle cell disease (SCD) and reproductive health remains understudied. Menstruation is particularly complex in individuals with SCD, who may have delayed puberty, early menopause, abnormal uterine bleeding, and overlapping SCD-related and menstruation-related pain. Objective:To assess menstrual patterns in female patients with SCD, these patients' access to and use of contraceptive therapy, and clinician practices associated with reproductive health. Design, Setting, and Participants:This cross-sectional survey study was conducted in 13 outpatient SCD centers across the US. Female patients with SCD who had reached menarche 12 or more months prior to enrollment between March 1, 2022, and May 31, 2024, and were not pregnant or post menopausal were included. Clinicians involved in the care of enrolled participants were also surveyed on a voluntary basis. Main Outcomes and Measures:Age-appropriate menstrual bleeding questionnaires were used to assess menstrual patterns, and global health was evaluated using the Patient-Reported Outcomes Measurement Information System mental and physical health scales. Newly designed patient-facing surveys were used to assessed SCD health and contraceptive use. Clinician-facing surveys assessed participants' clinical history and care plans after reviewing the patient surveys. Results:A total of 211 participants (mean [SD] age 23.7 [10.1] years) were enrolled. All patient-facing surveys with a corresponding clinician survey were completed for 183 participants (86.7%). Abnormal uterine bleeding was noted in 52 participants (24.6%) and was associated with a higher rate of hospitalizations (59.1% hospitalized in this group vs 34.5% in the non-abnormal uterine bleeding group). Most participants (134 of 208 [64.4%]) endorsed sickle cell pain with menses. Among these participants, 42 (36.5%) had no emergency department (ED) visits, 51 (44.3%) had 1 to 3 ED visits, and 22 (19.1%) had 4 or more ED visits in the 6 months prior to enrollment compared with those who did not endorse sickle cell pain associated with menstruation (38 [58.5%], 24 [36.9%], and 3 [4.6%], respectively). In addition, among the participants who endorsed sickle cell pain, 57 (49.6%) had 0 hospitalizations, 48 (41.7%) had 1 to 3 hospitalizations, and 10 (8.7%) had 4 or more hospitalizations compared with those who did not endorse sickle cell pain associated with menstruation (50 [76.9%], 14 [21.5%], and 1 [1.5%], respectively). There was minimal use of hormonal contraception (40 of 208 participants [19.2%]), and 46 participants (22.1%) reported that they had never heard of these medications. After reviewing the patient survey, clinicians reported several intended steps to improve reproductive health for their patients, including reproductive health education (131 participants [72.4%]), referral to a reproductive health specialist (90 participants [49.7%]), or workup for iron deficiency (29 participants [16.0%]) or an underlying bleeding disorder (21 participants [11.6%]). Three participating sites established new multidisciplinary clinics for reproductive health and hematology during the enrollment period. Conclusions and Relevance:In this multicenter survey study of female individuals with SCD, sickle cell pain with menses was prevalent, and hormonal contraceptive use was low. The findings suggest that brief assessments may facilitate dialogue between female patients with SCD and their sickle cell clinicians and lay the groundwork for improved reproductive health and medical management.

Numerous studies have identified mitochondria as critical players in the pathophysiology of sickle cell disease (SCD). Here, we investigated the bioenergetic impairment in cardiac mitochondria as well as the putative benefits of a transdermal (TD) curcumin (VAS-101) treatment in a Berkeley SCD (BERK-SS) mouse model. Low oxygen consumption rate observed in cardiac mitochondria was indicative of impaired electron transport chain (ETC) activities in these animals. Furthermore, there was a loss of enzymatic activity primarily in complex I (reduced NAD dehydrogenase) and to a lesser degree in complex V (ATP synthase) observed in BERK-SS cardiac mitochondrial fractions. Proteomic analysis of cardiac mitochondria revealed changes in the relative abundance of proteins related to ETC complexes, especially in multiple subunits of complex I and complex V. Changes were also seen in proteins involved in several other pathways (eg, β-oxidation, glycolysis, mitochondrial protein transport, cytoskeleton, Ca+2 regulation, and mitophagy). Moreover, TD curcumin treatment caused improvements in various oxidative stress parameters in heart tissues of BERK-SS mice. Together, our results suggest that novel TD curcumin may affect oxygen homeostasis at cellular and subcellular levels by improving mitochondrial respiration in the heart muscle of BERK-SS mice.

Several comorbidities of sickle cell disease (SCD) originate from red blood cell (RBC) instability, chronic inflammation, and oxidative stress. Development of scalable, cost-effective therapeutics suitable for chronic administration to prevent, attenuate, and perhaps reverse the consequences of RBC instability is needed. Curcumin has many of these attributes as a safe compound with antisickling, antiinflammatory, and antioxidant properties, but its translational potential has been constrained due to limited bioavailability from oral administration. The present study demonstrates the rapid and high bioavailability of a novel topical/transdermal (TD) curcumin gel formulation in the plasma and blood cells and its effectiveness in humanized sickle cell mice in: (i) ameliorating features of sickle cell pain hypersensitivity and axonal injury; (ii) reducing multiple manifestations of RBC instability including evidence of decreased hemolysis (reduced lactate dehydrogenase levels), enhanced RBC ATP levels along with decreased oxidative damage; (iii) decreasing multiple proinflammatory cytokines including interleukin-6, monocyte chemoattractant protein-1, granulocyte-macrophage colony-stimulating factor, and activation, normal T cell expressed and secreted protein in skin secretome; and (iv) reducing mast cell degranulation and activation. Our data suggest that an easy-to-use novel TD curcumin gel formulation has the potential to ameliorate chronic pain, improve RBC stability, and reduce inflammatory consequences of SCD.

Sickle cell disease (SCD) is characterized by impaired red blood cell (RBC) rheology, including increased hemoglobin S (HbS) polymerization, reduced sickle RBC deformability, and elevated blood viscosity, all features that promote lung vasocongestion and contribute to acute and chronic lung disease. Currently, there is an urgent need for new therapies to improve RBC rheology. Here, we investigate whether N-acetyl-lysyltyrosylcysteine amide (KYC), a redox-modulating end-capped tripeptide, improves RBC rheology by reducing oxidative stress. Steady-state and vaso-occlusive crisis (VOC) were modeled in SS mice using normoxic (NOX) and hypoxia/ reoxygenation (H/R) protocols. KYC inhibition of HbS oxidation was assessed in vitro. The effects of KYC on plasma advanced oxidation protein products (AOPP), hematocrit (%Hct), blood viscosity, RBC sickling, lung vasocongestion, and RBC reactive oxygen species (ROS) in the mice were determined ex vivo. In vitro, KYC reduced H2O2-mediated HbS oxidation. KYC decreased plasma AOPP levels in NOX and H/R SS mice. Although KYC did not improve %Hct in NOX mice, it reduced blood viscosity and the percentage of sickled RBCs. After H/R injury, KYC partially restored %Hct, increased blood viscosity, reduced RBC sickling, ablated lung vasocongestion, and reduced RBC ROS production in H/R mice. These findings support the hypothesis that RBCderived ROS causally impair rheology and promote lung vasocongestion in murine SCD. The H/R protocol models lung pathology associated with both VOC and acute chest syndrome (ACS) in SCD. Collectively, these data support a redox-mediated systems pathology model of SCD, where RBC oxidative stress is a critical driver of disease severity.