Gut microbiota dysbiosis alters chronic pain behaviors in a humanized transgenic mouse model of sickle cell disease.

ABSTRACT: Pain is the most common symptom experienced by patients with sickle cell disease (SCD) throughout their lives and is the main cause of hospitalization. Despite the progress that has been made towards understanding the disease pathophysiology, major gaps remain in the knowledge of SCD pain, the transition to chronic pain, and effective pain management. Recent evidence has demonstrated a vital role of gut microbiota in pathophysiological features of SCD. However, the role of gut microbiota in SCD pain is yet to be explored. We sought to evaluate the compositional differences in the gut microbiota of transgenic mice with SCD and nonsickle control mice and investigate the role of gut microbiota in SCD pain by using antibiotic-mediated gut microbiota depletion and fecal material transplantation (FMT). The antibiotic-mediated gut microbiota depletion did not affect evoked pain but significantly attenuated ongoing spontaneous pain in mice with SCD. Fecal material transplantation from mice with SCD to wild-type mice resulted in tactile allodynia (0.95 ± 0.17 g vs 0.08 ± 0.02 g, von Frey test, P < 0.001), heat hyperalgesia (15.10 ± 0.79 seconds vs 8.68 ± 1.17 seconds, radiant heat, P < 0.01), cold allodynia (2.75 ± 0.26 seconds vs 1.68 ± 0.08 seconds, dry ice test, P < 0.01), and anxiety-like behaviors (Elevated Plus Maze Test, Open Field Test). On the contrary, reshaping gut microbiota of mice with SCD with FMT from WT mice resulted in reduced tactile allodynia (0.05 ± 0.01 g vs 0.25 ± 0.03 g, P < 0.001), heat hyperalgesia (5.89 ± 0.67 seconds vs 12.25 ± 0.76 seconds, P < 0.001), and anxiety-like behaviors. These findings provide insights into the relationship between gut microbiota dysbiosis and pain in SCD, highlighting the importance of gut microbial communities that may serve as potential targets for novel pain interventions.

An alcohol dehydrogenase 7 gene polymorphism associates with both acute and chronic pain in sickle cell disease.

Introduction: As the most distressing complication of sickle cell disease (SCD), pain is marked by considerable heterogenicity. In this study we explored the potential association of alcohol dehydrogenase 7 gene (ADH7) polymorphism rs971074 with sickle cell pain. Methods: We analyzed clinical phenotypes and the rs971074 single-nucleotide polymorphism in ADH7 by MassARRAY-iPlex analysis in a cohort of SCD patients. Results: The synonymous rs971074 was significantly associated with both acute and chronic pain in SCD. Patients with the minor T allele(s) recorded significantly more crisis episodes and severe chronic pain symptoms. Conclusion: Our study has identified the rs971074 minor T allele as a genetic biomarker potentially influencing acute and chronic pain. These findings may ultimately help inform strategies to develop precision pain therapies in SCD.

Candidate gene association study suggests potential role of dopamine beta-hydroxylase in pain heterogeneity in sickle cell disease.

Introduction: Pain is a lifelong companion of individuals with sickle cell disease (SCD) and has a severe impact on their quality of life. Both acute crisis pain and chronic non-crisis pain exhibit high variability between individuals, making it difficult to effectively manage sickle cell-related pain. We investigated the role of dopamine beta-hydroxylase (DBH) gene polymorphisms on pain variability in SCD. DBH is a key enzyme in the catecholamine biosynthesis pathway that catalyzes the conversion of dopamine to norepinephrine, both of which are known mediators of pain and pain-related behaviors. Methods: Acute crisis pain-related utilization and non-crisis chronic pain scores of 131 African Americans with SCD were obtained. Results and discussion: Association analyses revealed that the T allele of upstream variant rs1611115 and downstream variant rs129882 correlated with higher severity of chronic pain in an additive model. On the other hand, the A allele of missense variant rs5324 associated with lower risk of both acute crisis pain and chronic pain. Similarly, the C allele of intronic variant rs2797849 was associated with lower incidence of acute crisis pain in the additive model. In addition, tissue-specific eQTL revealed that the T allele of rs1611115 correlated with decreased expression of DBH in the frontal cortex and anterior cingulate cortex (GTEx), and decreased expression of DBH-AS1 in blood (eQTLGen). Bioinformatic approaches predicted that rs1611115 may be altering a transcription factor binding site, thereby, contributing to its potential effect. Taken together, findings from this study suggest that potential functional polymorphisms of DBH may modulate pain perception in SCD.

Modified Akuamma Alkaloids with Increased Potency at the Mu-opioid Receptor.

Akuammine (1) and pseudoakuammigine (2) are indole alkaloids found in the seeds of the akuamma tree (Picralima nitida). Both alkaloids are weak agonists of the mu opioid receptor (µOR); however, they produce minimal effects in animal models of antinociception. To probe the interactions of 1 and 2 at the opioid receptors, we have prepared a collection of 22 semisynthetic derivatives. Evaluation of this collection at the µOR and kappa opioid receptor (κOR) revealed structural-activity relationship trends and derivatives with improved potency at the µOR. Most notably, the introduction of a phenethyl moiety to the N1 of 2 produces a 70-fold increase in potency and a 7-fold increase in selectivity for the µOR. The in vitro potency of this compound resulted in increased efficacy in the tail-flick and hot-plate assays of antinociception. The improved potency of these derivatives highlights the promise of exploring natural product scaffolds to probe the opioid receptors.

Correction: The gut microbiota mediates the protective effects of anserine supplementation on hyperuricaemia and associated renal inflammation.

Correction for 'The gut microbiota mediates the protective effects of anserine supplementation on hyperuricaemia and associated renal inflammation' by Jiaojiao Han et al., Food Funct., 2021, 12, 9030-9042, DOI: 10.1039/D1FO01884A.

Analysis of AVPR1A, thermal and pressure pain thresholds, and stress in sickle cell disease.

Aim: In patients with sickle cell disease (SCD), negative physical and emotional experiences result from intense chronic and acute pain episodes, but factors underlying these, and their interactions, are not well understood. The arginine vasopressin receptor 1a gene (AVPR1A) single nucleotide polymorphism rs10877969 has been previously associated with aspects of acute pain and stress related pain. In this study, we tested for associations between this SNP, thermal and pressure pain thresholds, clinical pain, and stress in people with SCD. Methods: 150 adults enrolled with SCD completed pain intensity measures (Average Pain Intensity, API) and the Perceived Stress Questionnaire (PSQ). Thermal and pressure pain threshold data were available from quantitative sensory testing (QST), and rs10877969 genotypes were obtained. Results: In models adjusted for age and gender, between rs10877969 genotypes, we observed no significant differences in thermal (cold, p = 0.66; heat, p = 0.91) and mechanical (pressure, p = 0.33) pain thresholds. The association of rs10877969 with API (p = 0.09) was borderline, but non-significant with PSQ (p = 0.51). The correlation between clinical pain and environmental stress was significant, r = 0.18, p = 0.024, however, the interaction of genotype and PSQ was not significant (p = 0.63). Conclusion: Clinical and experimental pain were not significantly associated with the rs10877969 genotype. The rs10877969 genotype did not moderate the correlation between environmental stress and clinical pain in this population. However, a trend toward a protective T allele effect on average pain rating in SCD warrants future exploration of this SNP/gene in SCD.

The gut microbiota mediates the protective effects of anserine supplementation on hyperuricaemia and associated renal inflammation.

Hyperuricaemia is a disease associated with elevated serum uric acid content, which has emerged rapidly in recent decades. The drugs used to treat clinical hyperuricaemia have side effects, and their safety is poor. However, anserine is a natural carnosine derivative that shows an anti-hyperuricaemic effect. A previous study demonstrated that anserine inhibits uric acid synthesis and promotes uric acid excretion, but there is no evidence regarding the effect of anserine from the perspective of the gut microbiota. In this study, the anti-hyperuricaemic and anti-inflammatory effects of anserine were explored in a diet-induced hyperuricaemic mouse model. Anserine alleviated hyperuricaemia and renal inflammation phenotypes, inhibited uric acid biosynthesis, promoted uric acid excretion, and inhibited NLRP3 inflammasome and TLR4/MyD88/NF-κB signalling pathway activation. The results showed that the anti-hyperuricaemic effect of anserine was dependent on the gut microbiota in the germ-free mice experiment. Furthermore, anserine treatment reversed gut microbiota dysbiosis, repaired the intestinal epithelial barrier and increased short-chain fatty acid production. Moreover, the anti-hyperuricaemic effect of anserine was transmissible by transplanting the faecal microbiota from anserine-treated mice, indicating that the protective effects were at least partially mediated by the gut microbiota. Thus, we identified a new and safe prebiotic material to alleviate hyperuricaemia and provided ideas for the development of oligopeptides.

Protein kinase Cδ as a neuronal mechanism for headache in a chronic intermittent nitroglycerin model of migraine in mice.

ABSTRACT: Migraine is one of the most common neurological disorders characterized by recurrent attacks of typically throbbing and unilateral headaches, affecting up to 20% of the population worldwide. Despite the high prevalence and severity of this primary headache disorder, it remains to be a challenge to fully understand and treat migraine headaches. By characterizing and validating a mouse migraine model, this study aimed to investigate the functional contribution of protein kinase C (PKC) isoforms in migraine. In this study, we identified the presence of migraine-like ongoing pain in mice after chronic intermittent treatment with nitroglycerin (NTG). The peptide antagonist of calcitonin gene-related peptide α-CGRP (8-37), but not topiramate nor sumatriptan, effectively blocked ongoing pain and elicited pain relief-induced conditioned place preference in NTG-treated mice. Prominent activation of PKCδ was observed in chronic NTG-treated mice. Functional inhibition of PKCδ significantly attenuated ongoing spontaneous pain in chronic NTG-treated mice. Furthermore, we recapitulated the NTG-triggered migraine behavior in wild-type mice, but not in PKCδ-null mice. In response to repeated administration of NTG, ongoing spontaneous pain was not developed in mice lacking the specific PKC isoform. This study identified the presence of ongoing pain in mice treated with NTG, a known human migraine trigger that closely resembles the common manifestation of spontaneous migraine attacks in humans. These findings demonstrated a critical regulatory role of PKCδ in migraine pathophysiology, which may offer new pharmacological targets for antimigraine treatment.

S100B single nucleotide polymorphisms exhibit sex-specific associations with chronic pain in sickle cell disease in a largely African-American cohort.

BACKGROUND: Pain in sickle cell disease (SCD) is severe and multifaceted resulting in significant differences in its frequency and intensity among individuals. In this study, we examined the influence of S100B gene single nucleotide polymorphisms (SNP) on acute and chronic pain variability in SCD. METHODS: Composite pain index (CPI) scores captured chronic pain. Painful crisis related emergency care utilization recorded acute pain incidence. Genotyping was performed using MassARRAY iPLEX platform. RESULTS: Regression analysis revealed associations of increased CPI with rs9722 A allele in additive (p = 0.005) and dominant (p = 0.005) models. Rs1051169 G allele on the other hand was associated with decreased CPI in additive (p = 0.001), and dominant (p = 0.005) models. Sex-specific analysis found that these associations were significant in females but not males in this cohort. Linkage analysis identified two haploblocks. Block 1 (rs9983698-rs9722) haplotype T-A was associated with increased CPI (p = 0.002) while block 2 (rs1051169-rs11911834) haplotype G-G was associated with decreased CPI (p = 0.001). Both haplotypic associations were only significant in females. No association of S100B SNPs with utilization reached statistical significance. CONCLUSIONS: S100B SNPs and haplotypes are associated with chronic pain in female, but not male, patients with SCD, implicating a potential role of S100B polymorphism in SCD pain heterogeneity in a sex-dependent manner.

Protective effects of tuna meat oligopeptides (TMOP) supplementation on hyperuricemia and associated renal inflammation mediated by gut microbiota.

Recently, interest in using whole food-derived mixtures to alleviate chronic metabolic syndrome through potential synergistic interactions among different components is increasing. In this study, the effects and mechanisms of tuna meat oligopeptides (TMOP) on hyperuricemia and associated renal inflammation were investigated in mice. Dietary administration of TMOP alleviated hyperuricemia and renal inflammation phenotypes, reprogramed uric acid metabolism pathways, inhibited the activation of NLRP3 inflammasome and TLR4/MyD88/NF-κB signaling pathways, and suppressed the phosphorylation of p65-NF-κB. In addition, TMOP treatments repaired the intestinal epithelial barrier, reversed the gut microbiota dysbiosis and increased the production of short-chain fatty acids. Moreover, the antihyperuricemia effects of TMOP were transmissible by transplanting the fecal microbiota from TMOP-treated mice, indicating that the protective effects were at least partially mediated by the gut microbiota. Thus, for the first time, we clarify the potential effects of TMOP as a whole food derived ingredient on alleviating hyperuricemia and renal inflammation in mice, and additional efforts are needed to confirm the beneficial effects of TMOP on humans.

Tuna Bone Powder Alleviates Glucocorticoid-Induced Osteoporosis via Coregulation of the NF-κB and Wnt/ß-Catenin Signaling Pathways and Modulation of Gut Microbiota Composition and Metabolism.

SCOPE: The effects and mechanism of tuna bone powder (TBP) on glucocorticoid-induced osteoporosis (GIOP) alleviation in terms of signaling pathway coregulation and gut microbiota modulation are investigated. METHODS AND RESULTS: The powder size distribution and composition of TBP are measured. The GIOP female mice induced by dexamethasone intramuscular injection are used to examine the anti-osteoporosis effects of TBP in a 10 week experiment, and improved bone mineral density and bone microarchitecture are observed via micro-CT. In addition, qRT-PCR results show that the NF-κB pathway is inhibited to reduce bone resorption, whereas the Wnt/ß-catenin pathway is activated to enhance bone formation after treatment. Moreover, TBP treatments suppress the release of pro-inflammatory cytokines, repair dysfunction of the intestinal epithelial barrier, and prevent aggravated systemic inflammation in mRNA levels. Additionally, 16S rRNA gene sequencing indicate that TBP treatments enhance the abundance of anti-inflammatory bacteria and short-chain fatty acid (SCFA) producers, which is consistent with increased SCFA contents in feces measured via GC-MS. CONCLUSION: These data show that TBP ameliorates GIOP in mice through four aspects, including coregulating signaling pathways, blocking proinflammatory cytokines, repairing the intestinal epithelial barrier, and modulating gut microbiota. Therefore, TBP may be a potential prebiotic agent to alleviate osteoporosis in humans.

Ketamine reduces remifentanil-induced postoperative hyperalgesia mediated by CaMKII-NMDAR in the primary somatosensory cerebral cortex region in mice.

Remifentanil is commonly used clinically for perioperative pain relief, but it may induce postoperative hyperalgesia. Low doses of ketamine have remained a common choice in clinical practice, but the mechanisms of ketamine have not yet been fully elucidated. In this study, we examined the possible effects of ketamine on calcium/calmodulin-dependent protein kinase II α (CaMKIIα) and N-methyl-d-aspartate receptor (NMDAR) subunit NR2B in a mouse model of remifentanil-induced postoperative hyperalgesia (RIPH) in the primary somatosensory cerebral cortex (SI) region. The paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were used to assess mechanical allodynia and thermal hyperalgesia, respectively, before and after intraoperative remifentanil administration. Before surgery, mice received intrathecal injections of the following drugs: ketamine, NMDA, BayK8644 (CaMKII activator), and KN93 (CaMKII inhibitor). Immunofluorescence was performed to determine the anatomical location and expression of activated CaMKIIα, phosphorylated CaMKIIα (p-CaMKIIα). Additionally, western blotting was performed to assess p-CaMKIIα and NMDAR expression levels in the SI region. Remifentanil decreased the PWMT and PWTL at 0.5 h, 2 h, and 5 h and increased p-CaMKIIα expression in the SI region. Ketamine increased the PWMT and PWTL and reversed the p-CaMKIIα upregulation. Both BayK8644 and NMDA reversed the effect of ketamine, decreased the PWMT and PWTL, and upregulated p-CaMKIIα expression. In contrast, KN93 enhanced the effect of ketamine by reducing hyperalgesia and downregulating p-CaMKIIα expression. These results suggested that ketamine reversed RIPH by inhibiting the phosphorylation of CaMKIIα and the NMDA receptor in the SI region in mice.

Neuropathic Pain Screening: Construct Validity in Patients With Sickle Cell Disease.

Individuals with pain from sickle cell disease (SCD) are often treated for nociceptive pain, but recent findings indicate they may also have neuropathic pain. PAINReportIt, a computerized version of the McGill Pain Questionnaire, provides a potential subscale that is the summed number of selected neuropathic pain quality words (PR-NNP), but it lacks construct validity. The study purpose was to ascertain PR-NNP construct validity in adults with SCD and chronic pain. In an outpatient setting, 186 participants completed the PAINReportIt, Neuropathic Pain Symptom Inventory (NPSI), and Leeds Assessment for Neuropathic Symptoms and Signs (S-LANSS). PR-NNP was moderately correlated with NPSI (r = .33, p < .001) and S-LANSS (r = .40, p < .001). Regression analysis indicated that PR-NNP and pain intensity, but not a nociceptive pain subscale, were significant predictors of NPSI and S-LANSS. Findings support construct validity of PR-NNP, which may be useful as a screening tool for neuropathic pain in patients with SCD.

Keys to Recruiting and Retaining Seriously Ill African Americans With Sickle Cell Disease in Longitudinal Studies: Respectful Engagement and Persistence.

OBJECTIVES: Sickle cell disease (SCD) is a serious illness with disabling acute and chronic pain that needs better therapies, but insufficient patient participation in research is a major impediment to advancing SCD pain management. The purpose of this article is to discuss the challenges of conducting an SCD study and approaches to successfully overcoming those challenges. DESIGN: In a repeated-measures, longitudinal study designed to characterize SCD pain phenotypes, we recruited 311 adults of African ancestry. Adults with SCD completed 4 study visits 6 months apart, and age- and gender-matched healthy controls completed 1 visit. RESULTS: We recruited and completed measures on 186 patients with SCD and 125 healthy controls. We retained 151 patients with SCD with data at 4 time points over 18 months and 125 healthy controls (1 time point) but encountered many challenges in recruitment and study visit completion. Enrollment delays often arose from patients' difficulty in taking time from their complicated lives and frequent pain episodes. Once scheduled, participants with SCD cancelled 49% of visits often because of pain; controls canceled 30% of their scheduled visits. To facilitate recruitment and retention, we implemented a number of strategies that were invaluable in our success. CONCLUSION: Patients' struggles with illness, chronic pain, and their life situations resulted in many challenges to recruitment and completion of study visits. Important to overcoming challenges was gaining the trust of patients with SCD and a participant-centered approach. Early identification of potential problems allowed strategies to be instituted proactively, leading to success.

A Stepped-Wedge Randomized Controlled Trial: Effects of eHealth Interventions for Pain Control Among Adults With Cancer in Hospice.

CONTEXT: Unrelieved cancer pain at the end of life interferes with achieving patient-centered goals. OBJECTIVE: To compare effects of usual hospice care and PAINRelieveIt® on pain outcomes in patients and their lay caregivers. METHODS: In a five-step, stepped-wedge randomized, controlled study, 234 patients (49% male, 18% Hispanic, 51% racial minorities) and 231 lay caregivers (26% male, 20% Hispanic, 54% racial minorities) completed pre-pain/post-pain measures. They received usual hospice care with intervention components that included a summary of the patient's pain data, decision support for hospice nurses, and multimedia education tailored to the patient's and lay caregiver's misconceptions about pain. RESULTS: The intervention effect on analgesic adherence (primary outcome) was not significant. Post-test worst pain intensity was significantly higher for the experimental group, but the difference (0.70; CI = [0.12, 1.27]) was not clinically meaningful. There was nearly universal availability of prescriptions for strong opioids and adjuvant analgesics for neuropathic pain in both groups. Lay caregivers' pain misconceptions (0-5 scale) were significantly lower in the experimental group than the usual care group (mean difference controlling for baseline is 0.38; CI = [0.08, 0.67]; P = 0.01). CONCLUSION: This randomized controlled trial was a negative trial for the primary study outcomes but positive for a secondary outcome. The trial is important for clearly demonstrating the feasibility of implementing the innovative set of interventions.

Dezocine attenuates the remifentanil-induced postoperative hyperalgesia by inhibition of phosphorylation of CaMKâ ¡α.

Remifentanil, ultra-short-acting µ-opioid receptor agonist, has the greatest advantage in analgesia but could increase postoperative pain scores and induces postoperative hyperalgesia. Dezocine is a mixed opioid receptor partial agonist/antagonist and has been used for postoperative hyperalgesia management in clinical patients,but the potential molecular mechanism is still unclear. Ca2+/calmodulin-dependent protein kinase Ⅱ(CaMKⅡ) has been reported involved in remifentanil-induced hyperalgesia (RIH) in previous studies, but the relationship between CaMKⅡ and dezocine in RIH is still unclear. To investigate the mechanism of dezocine in RIH, we used a remifentanil induced postoperative hyperalgesia (RIPH) in incisional pain model of mouse. We subcutaneously infused remifentanil (40 µg/kg) to induce postoperative hyperalgesia. Dezocine (1.5 mg/kg, 3.0 mg/kg, and 6.0 mg/kg) was infused subcutaneously with remifentanil using the apparatus pump for 30 min. Paw withdrawal thermal latency (PWTL) and paw withdrawal mechanical threshold (PWMT) were used to assess thermal hyperalgesia and mechanical allodynia. Western blotting analysis and immunohistochemistry analysis were used to assess the expression of phosphorylated CaMKⅡα (p-CaMKⅡα) in somatosensory cortex, hippocampus and spinal cord. Subcutaneous infusion of remifentanil enhanced postoperative pain induced by surgical incision and increased PWTL and PWMT. Dezocine dose-dependently decreased the PWTL and PWMT in RIPH model. Correlating with behavioral effects, dezocine inhibited remifentanil-induced up-regulation of p-CaMKⅡα expression in somatosensory cortex, hippocampus and spinal cord. Dezocine could attenuate RIPH by suppressing p-CaMKⅡα.

Vasopressin SNP pain factors and stress in sickle cell disease.

PURPOSE: Frequencies of single nucleotide polymorphisms (SNPs) from pain related candidate genes are available for individuals with sickle cell disease (SCD). One of those genes, the arginine vasopressin receptor 1A gene (AVPR1A) and one of its SNPs, rs10877969, has been associated with pain and disability in other pain populations. In patients with SCD, clinical factors such as pain and stress have been associated with increased health care utilization, but it is not known if the presence of the AVPR1A SNP plays a role in this observation. The study purpose was to explore the relationships between rs10877969 and self-reported pain, stress, and acute care utilization events among individuals with SCD. METHODS: In a cross-sectional investigation of outpatients with SCD, participants completed PAINReportIt®, a computerized pain measure, to describe their pain experience and contributed blood or saliva samples for genetic analysis. We extracted emergency department and acute care utilization from medical records. RESULTS: The SNP genotype frequencies (%) for this sample were CC 30 (28%), CT 44 (41%), TT 33 (31%). Acute care utilization and stress as an aggravator of pain were significantly associated with the rs10877969 genotype (p = .02 and p = .002, respectively). The CT genotype had the highest mean utilization and CC genotype was associated with not citing stress as a pain aggravator. Chronic pain was not associated with rs10877969 (p = .41). CONCLUSION: This study shows that rs10877969 is related to indicators of stress and acute pain. Further research is recommended with other measures of stress and acute pain.

Alterations of the Brain Proteome and Gut Microbiota in d-Galactose-Induced Brain-Aging Mice with Krill Oil Supplementation.

Brain aging is commonly associated with neurodegenerative disorders, but the ameliorative effect of krill oil and the underlying mechanism remain unclear. In this study, the components of krill oil were measured, and the antiaging effects of krill oil were investigated in mice with d-galactose (d-gal)-induced brain aging via proteomics and gut microbiota analysis. Krill oil treatment decreased the expression of truncated dopamine- and cAMP-regulated phosphoproteins and proteins involved in the calcium signaling pathway. In addition, the concentrations of dopamine were increased in the serum (p < 0.05) and brain (p > 0.05) due to the enhanced expressions of tyrosine-3-monooxygenase and aromatic l-amino acid decarboxylase. Moreover, krill oil alleviated gut microbiota dysbiosis, decreased the abundance of bacteria that consume the precursor tyrosine, and increased the abundance of Lactobacillus spp. and short-chain fatty acid producers. This study revealed the beneficial effect of krill oil against d-gal-induced brain aging and clarified the underlying mechanism through proteomics and gut microbiota analysis.