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Ashley C Archer,1 Jennifer J DeBerry,2 Cary DeWitte,2 Timothy J Ness2 1University of Alabama at Birmingham School of Medicine, Birmingham, AL, USA; 2Department of Anesthesiology and Perioperative Medicine, University of Alabama at Birmingham, Birmingham, AL, USACorrespondence: Timothy J Ness, Department of Anesthesiology and Perioperative Medicine, University of Alabama at Birmingham, PBMR-208, 901 19th St. S, Birmingham, AL, 35205, USA, Tel +1-205-975-9643, Email tness@uabmc.eduPurpose: Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic pain disorder. Patients with IC/BPS often experience “flares” of symptom exacerbation throughout their lifetime, initiated by triggers, such as urinary tract infections. This study sought to determine whether neonatal bladder inflammation (NBI) alters the sensitivity of adult rat bladders to microbial antigens.Methods: Female NBI rats received intravesical zymosan treatments on postnatal days P14-P16 while anesthetized; Neonatal Control Treatment (NCT) rats were anesthetized. In adults, bladder and spinal cord Toll-like receptor type 2 and 4 (TLR2, TLR4) contents were determined using ELISAs. Other rats were injected intravesically with lipopolysaccharide (LPS; mimics an E. coli infection; 25, 50, 100, or 200 μg/mL) or Zymosan (mimics yeast infection; 0.01, 0.1, 1, and 10 mg/mL) solutions on the following day. Visceromotor responses (VMRs; abdominal contractions) to graded urinary bladder distention (UBD, 10– 60 mm Hg, 20s) were quantified as abdominal electromyograms (EMGs).Results: Bladder TLR2 and TLR4 protein levels increased in NBI rats. These rats displayed statistically significant, dose-dependent, robustly augmented VMRs following all but the lowest doses of LPS and Zymosan tested, when compared with their adult treatment control groups. The NCT groups showed minimal responses to LPS in adults and minimally increased EMG measurements following the highest dose of Zymosan.Conclusion: The microbial antigens LPS and Zymosan augmented nociceptive VMRs to UBD in rats that experienced NBI but had little effect on NCT rats at the doses tested. The greater content of bladder TLR2 and TLR4 proteins in the NBI group was consistent with increased responsiveness to their agonists, Zymosan and LPS, respectively. Given that patients with IC/BPS have a higher incidence of childhood urinary tract infections, this increased responsiveness to microbial antigens may explain the flares in symptoms following “subclinical” tract infections.Plain Language Summary: In a rat model of interstitial cystitis, bladder inflammation during childhood resulted in increased bladder sensitivity (ie, pain) to low-level infections in adults.Keywords: lipopolysaccharide, zymosan, interstitial cystitis, visceromotor responses, TLR2, TLR4, urinary tract infection

Georg Deutsch,1 Hrishikesh Deshpande,1 H Henry Lai,2 Jason J Kutch,3 Timothy J Ness4 1Department of Radiology, University of Alabama at Birmingham, Birmingham, AL, USA; 2Division of Urologic Surgery, Department of Surgery, Washington University School of Medicine, St. Louis, MO, USA; 3Division of Biokinesiology and Physical Therapy, University of Southern California, Los Angeles, CA, USA; 4Department of Anesthesiology and Perioperative Medicine, University of Alabama at Birmingham, Birmingham, AL, USACorrespondence: Timothy J NessDepartment of Anesthesiology, University of Alabama at Birmingham, BMR2-208; 901 19th St. S, Birmingham, AL, 35205, USATel +1 205-975-9643Fax +1 205-934-7437Email tness@uabmc.eduPurpose: Fibromyalgia is a common co-morbidity in patients with interstitial cystitis/bladder pain syndrome. Quantitative sensory testing measures and regional cerebral blood flow measures have been noted to differ from healthy controls in both subjects with fibromyalgia and those with interstitial cystitis when studied independently. The present study examined such measures in subjects with the diagnosis of interstitial cystitis both with and without the co-diagnosis of fibromyalgia to determine whether differences in these measures may be associated with co-morbidity.Patients and Methods: Female subjects with the diagnosis of interstitial cystitis with (n = 15) and without (n = 19) the co-diagnosis of fibromyalgia as well as healthy control subjects (n = 41) underwent quantitative sensory testing. A subset of these patients (9 with and 9 without fibromyalgia) underwent brain perfusion studies using arterial spin labeled functional magnetic resonance imaging. An analysis was performed of absolute regional cerebral blood flow of regions-of-interest when experiencing a full bladder compared with an empty bladder.Results: Subjects with both interstitial cystitis and fibromyalgia were more hypersensitive than those without fibromyalgia as well as healthy controls in most sensory measures except heat. Subjects with interstitial cystitis, but no fibromyalgia, differed from healthy controls only in toleration of the ischemic forearm task. Other co-morbidities were more common in those subjects with both interstitial cystitis and fibromyalgia. Bladder fullness was associated with significantly greater whole brain gray matter blood flow in subjects with interstitial cystitis and fibromyalgia when compared with that of subjects with interstitial cystitis without fibromyalgia. Examination of regional cerebral blood flow in individual regions-of-interest demonstrated statistically significant differences between the subjects with interstitial cystitis with and those without fibromyalgia bilaterally in the thalamus, amygdala and hippocampus, as well as the right prefrontal cortex and greater responsiveness to changes in bladder fullness in the insula.Conclusion: Quantitative sensory testing and brain perfusion data support that there are two phenotypes of interstitial cystitis patients, which can be differentiated by a co-diagnosis of fibromyalgia. This may affect responsiveness to treatment and suggest the utility of stratifying interstitial cystitis patients according to their co-morbidities.Keywords: arterial spin labelling, fMRI, interstitial cystitis, QST

Christina Mueller,1 Timothy J Ness,2 Jarred W Younger1 1Department of Psychology, University of Alabama at Birmingham, Birmingham, AL, USA; 2Department of Anesthesiology and Perioperative Medicine, University of Alabama at Birmingham, Birmingham, AL, USACorrespondence: Jarred W YoungerDepartment of Psychology, University of Alabama at Birmingham, CH233, 1300 University Blvd, Birmingham, AL 35233, USATel +1 (205) 975-5907Fax +1 (205) 934-6440Email younger@uab.eduObjective: Fibromyalgia (FM) is a debilitating chronic pain condition with few treatment options. Central sensitization and neuroinflammation have been forwarded as models of FM pathophysiology, both of which indicate dextromethorphan (DXM) as a potential treatment. DXM is an NMDA-receptor antagonist and microglial modulator with anti-neuroinflammatory properties at low doses. It is available for clinical use but has not been tested as a treatment for FM at low dosages. This study evaluated the effectiveness of DXM in treating FM-associated symptoms.Methods: In a single-blind, placebo-controlled trial, 14 women meeting the 2010 American College of Rheumatology criteria for FM received a placebo for five weeks, followed by 20 mg DXM for ten weeks, while providing daily symptom reports on a 0– 100 scale. Pain and physical activity were the primary and secondary outcomes, respectively. Daily symptom ratings during the last four weeks of placebo were contrasted with ratings during the last four weeks of the active treatment using generalized estimating equations (GEE).Results: DXM was well tolerated, and treatment adherence was high. Baseline pain was reduced by at least 20% in six participants. Self-reported daily pain and physical activity in the entire cohort were not significantly different between the placebo and DXM conditions, and the primary hypotheses were not supported. Exploratory analyses using the entire placebo and DXM data showed that pain was significantly lower in the DXM condition than in the placebo condition (b=− 9.933, p=0.013).Discussion: A strong clinical effect of DXM was not observed at the 20mg/day dosage.Keywords: fibromyalgia, dextromethorphan, clinical trial, neuroinflammation, analgesia

Gabriel Gallegos,1 Charity J Morgan,2 Garrett Scott,1 David Benz,1 Timothy J Ness1 1Department of Anesthesiology and Perioperative Medicine, University of Alabama at Birmingham, Birmingham, AL 35205, USA; 2Department of Biostatistics, University of Alabama at Birmingham, Birmingham, AL 35205, USACorrespondence: Timothy J NessDepartment of Anesthesiology and Perioperative Medicine, University of Alabama at Birmingham, BMR2-208, 901 19 th St. S, Birmingham, AL 35205 Tel +1 205-975-9643Fax +1 205-934-7437Email tness@uabmc.eduBackground: The purpose of this study was to determine whether neuraxial analgesic procedures affect intraoperative hemodynamics and/or postoperative outcomes. Previous studies have examined effects in small samples of patients in highly controlled research environments. This study examined “real-world” data from a large sample of subjects receiving routine clinical cares.Methods: A matched case–control analysis of electronic medical records from a large, academic hospital was performed. Patients who underwent neuraxial procedures preoperatively for postoperative analgesia for abdominal surgery (n=1570) were compared with control patients matched according to age, sex, ASA class and type of surgical procedure. Intraoperative hemodynamic measures, fluids and pressor utilization were quantified. Postoperative outcomes were determined based on the changes in laboratory values, the ordering of imaging studies and admission to an intensive care unit during the seven days following surgery as well as 30-day mortality.Results: Medical records of 1082 patients who received an epidural catheter placement and 488 patients who received a lumbar intrathecal morphine injection were compared with an equal number of matched control patients. Preoperative placement of an epidural catheter for the management of postoperative pain was demonstrated to be associated with significant reductions in mean arterial pressure intraoperatively and poorer postoperative outcomes (more intensive care unit [ICU] admissions, more myocardial injuries) when compared with controls. A similar analysis of preoperatively administered intrathecal morphine injections was not associated with intraoperative alterations in blood pressure and had improved outcomes (less ICU admissions) in comparison with controls.Conclusion: In a “real-world” sample, intrathecal morphine administration proved to be highly beneficial as a neuraxial analgesic procedure as it was not associated with intraoperative hypotension and was associated with improved clinical outcomes, in contrast to opposite findings associated with epidural catheter placement. There should be a careful consideration of elective neuraxial method utilized for postoperative pain control, with the present study raising significant concerns related to the use of epidural analgesia and its potential effect on clinical outcomes.Keywords: neuraxial analgesia, hypotension, perioperative outcomes

Hailey W Bulls,1 Emily L Freeman,1 Austen JB Anderson,2 Meredith T Robbins,3 Timothy J Ness,3 Burel R Goodin1,3 1Department of Psychology, University of Alabama at Birmingham, Birmingham, AL, USA; 2Department of Biology, Samford University, Birmingham, AL, USA; 3Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, AL, USA Abstract: It has been suggested that increased pain sensitivity and disruption of endogenous pain inhibitory processes may account, at least in part, for the greater prevalence and severity of chronic pain in women compared to men. However, previous studies addressing this topic have produced mixed findings. This study examined sex differences in pain sensitivity and inhibition using quantitative sensory testing (QST), while also considering the influence of other important factors such as depressive symptoms and sleep quality. Healthy men (n=24) and women (n=24) each completed a QST battery. This battery included an ischemic pain task (IPT) that used a submaximal effort tourniquet procedure as well as a conditioned pain modulation (CPM) procedure for the assessment of endogenous pain inhibition. Prior to QST, participants completed the Center for Epidemiologic Studies Depression Scale and the Pittsburgh Sleep Quality Index. Analyses revealed significant sex differences for the ischemic pain task and the conditioned pain modulation procedure, such that women tolerated the ischemic pain for a shorter amount of time and demonstrated less pain inhibition compared with men. This remained true even when accounting for sex differences in depressive symptoms and sleep quality. The results of this study suggest that women may be more pain sensitive and possess less-efficient endogenous pain inhibitory capacity compared with men. Whether interventions that decrease pain sensitivity and enhance pain inhibition in women ultimately improve their clinical pain outcomes is an area of research that deserves additional attention in the future. Keywords: sex differences, pain sensitivity, inhibition, depressive symptoms, sleep

Introduction:  Baclofen, a clinically available GABA B receptor agonist, produces nonopioid analgesia in multiple models of pain but has had limited studies related to perioperative pain control. The present study seeks to study effects of baclofen on postoperative pain measures and opioid use in adult patients subjected to percutaneous nephrolithotomy (PCNL)., Methods: Using a placebo-controlled, double-blind methodology, a single 10 mg oral dose of baclofen or placebo was given prior to a surgery in 34 patients undergoing PCNL. Standardized intraoperative and postoperative protocols related to opioid use were followed. Use of postoperative opioids in the post-anesthesia care unit and for the first 24 hours following surgery were recorded as were pain scores and other medication use., Results: There was a significant positive correlation in the use of postoperative opioids in patients who had a preceding history of opioid use. However, there were no significant differences in opioid use which could be attributed to baclofen. There were also no differences in postoperative vital signs, side effects or other medication use., Conclusions:  Analgesic benefits of preoperative baclofen were not observed at the dose employed. Safety of the drug was observed., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. University of Alabama at Birmingham Institutional Review Board for Human Studies issued approval IRB-300002466. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Mandabach et al.)

Inflammation is thought to contribute to the etiology of interstitial cystitis/bladder pain syndrome (IC/BPS). It is well-known that disruption in metabolism in immune cells contributes to inflammation in several inflammatory diseases. The purpose of this study was to investigate whether cellular bioenergetics is altered in monocytes and lymphocytes from women with IC/BPS, and if these alterations correlate with systemic inflammatory markers. Age and BMI matched adult healthy women (HS; n = 18) and women with IC/BPS (n = 18) were included in the study. Blood was collected to assess cellular bioenergetics in monocytes and lymphocytes using a Seahorse XF96 Analyzer and plasma cytokine levels were measured using Meso Scale Discovery immunoassays. The correlation between bioenergetic parameters, cytokines, and demographics was determined using Pearson correlation coefficients. Means of the two groups were compared using the two-group t-test. Patients with IC/BPS had reduced monocyte oxygen consumption rates and glycolytic rates compared to healthy subjects. In contrast, lymphocytes from these patients had increased oxygen consumption rates and glycolytic rates. Several cytokines and chemokines including Interferon-gamma (IFN-ɣ), tumor necrosis factor alpha (TNF-ɑ), Interleukin-6 (IL-6), Interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF) levels were significantly elevated in the plasma of patients with IC/BPS. However, Transforming growth factor (TGF-β) and Interleukin-10 (IL-10) levels were significantly decreased in IC/BPS patients compared to HS. In addition, Interferon gamma (IFN-ɣ), TNF-ɑ, IL-8, and TGF-β levels correlated with several bioenergetic parameters in monocytes or lymphocytes from healthy subjects. In contrast, TNF-ɑ and IL-8 correlated with bioenergetic parameters in monocytes from IC/BPS patients. Monocyte and lymphocyte cellular bioenergetics and plasma cytokine levels are different in patients with IC/PBS compared to HS. It appears that systemic inflammation is greater in this cohort which may negatively impact immune cell function. The relationship between cellular bioenergetics and inflammation in monocytes and lymphocytes could be important in understanding the pathogenesis of IC/PBS and warrants further investigation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kumar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

This neuroanatomical study in four, adult, Sprague-Dawley female rats quantified the number of Urothelial (labeled by intravesical DiI dye administration) and Non-Urothelial (labeled by intraparenchymal injection of Fast blue dye) bladder primary afferent neurons (bPANs) located in the T13, L1, L6 and S1 dorsal root ganglia. Additional immunohistochemical labeling using antibodies to detect either Substance P or CGRP further characterized the bPAN samples as peptidergic or non-peptidergic. Cell counts indicated that Urothelial bPANs were more common at the L6/S1 levels and more likely to be identified as peptidergic when compared with bPANs characterized at T13/L1 levels and with Non-Urothelial bPANs. These studies provide additional evidence that at least two distinct neuronal populations, with differing localization of sensory terminals, differing peptide content, and differing projections to the central nervous system, are responsible for bladder sensation., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Corticotropin releasing factor (CRF) receptors have been implicated in stress-induced hyperalgesia. The present study examined the role of CRF receptors Type 1&2 (CRFR1, CRFR2) in stress-induced bladder hyperalgesia in female rats by quantifying changes in receptor and agonist content following chronic (CFS, 7 daily episodes), acute (AFS, single episode) and control (NFS, no episodes) footshock protocols. ELISAs demonstrated that CFS lead to an increase in spinal thoracolumbar and lumbosacral spinal cord CRFR2 content and a decrease in lumbosacral spinal cord CRFR1 content. Content of the endogenous CRFR2 agonist, urocortin 2, was also increased in lumbosacral spinal cord and bladder tissues of CFS-pretreated rats, but urocortin 3 was decreased. Correlative single unit studies of lumbosacral dorsal horn neurons excited by bladder distension, in anesthetized rats that had undergone CFS, AFS or NFS protocols, used a before-after methodology with administration of a CRFR1 antagonist (antalarmin, 24 μg), CRFR2 antagonist (aSVG30, 12 μg) or normal saline topically to the exposed spinal cord following primary characterization. aSVG30 produced a reduction of neuronal responses evoked by bladder distension in CFS-pretreated rats but no statistically significant effects of aSVG30, antalarmin or vehicle were noted in other groups tested with the exception that antalarmin had an inhibitory effect on spontaneous activity in NFS-pretreated rats. The present findings are consistent with previous experiments using reflex responses to bladder distension as endpoints and further support a role for CRFR2-related mechanisms in stress-induced bladder hypersensitivity. This suggests CRFR2 antagonists may have efficacy in the treatment of bladder pain., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The spinal mechanisms of visceral hypersensitivity are poorly understood, particularly when there is an interaction with somatic systems. Recently we demonstrated that rats which were pretreated with neonatal bladder inflammation (NBI) and subsequently pretreated as adults with bladder re-inflammation had augmented reflex and neuronal responses to urinary bladder distension via a corticotropin-releasing factor receptor type II (CRFR2) mechanism. Another insult producing similar augmented responses is somatic inflammation induced by Complete Freund's Adjuvant (CFA) in the hindlimb. Using neurochemical measures and both reflex and neuronal responses to urinary bladder distension as endpoints, the present study probed the role of CRFR2-related mechanisms in bladder hyperalgesia secondary to NBI and CFA-induced hindlimb inflammation. ELISA measures of the lumbosacral spinal cord demonstrated increased CRFR2 protein following pretreatment with NBI+CFA. Intrathecal CRFR2 antagonists blocked the augmentation of visceromotor responses to distension following pretreatment with both NBI+CFA. Lumbosacral dorsal horn neuronal responses to bladder distension in rats pretreated with NBI+CFA were attenuated by the spinal topical administration of a CRFR2 antagonist. These findings are the first demonstration of a somatovisceral interaction working via CRFR2 receptors and support the therapeutic value of these agents in the treatment of painful bladder disorders, particularly when triggered by somatic events. (Word Count 199). PERSPECTIVE: Bladder hypersensitivity occurs following neonatal cystitis and an adult insult such as somatic inflammation. This paper demonstrates that CRFR2-related mechanisms are associated with this hypersensitivity. This supports the therapeutic value of these agents in the treatment of painful bladder disorders, particularly when triggered by somatic events., (Copyright © 2022 United States Association for the Study of Pain, Inc. Published by Elsevier Inc. All rights reserved.)

Objectives: Spinal cord stimulation (SCS) for the treatment of pelvic visceral pains has been understudied and underused. The goal of the current study was to examine multiple stimulation parameters of SCS to determine optimal settings for the inhibition of responses to urinary bladder distension (UBD) in animal models of bladder pain as a guide for human studies., Materials and Methods: Adult, female isoflurane/urethane-anesthetized rats underwent a T13/L1 mini-laminectomy sufficient to implant an SCS paddle lead for neuromodulation. Silver wire electrodes were inserted into the external oblique musculature. A 22-gauge angiocatheter was placed transurethrally into the bladder and used to deliver phasic, air UBDs at pressures of 10 to 60 mm Hg and visceromotor (abdominal contractile) electromyographic responses to UBD measured in the presence and absence of SCS. Electromyographic activity was quantified using standard differential amplification and rectification. Parameter settings for SCS included both conventional (10, 50, 100 Hz) and high frequency (1,000, 5,000, and 10,000 Hz) biphasic square wave pulses with 50 to 200 μs durations. To create states of hypersensitivity, pretreatment of adult rats included an intravesical zymosan infusion 24 hours before testing with and without a preceding episode of neonatal bladder inflammation., Results: Low frequency (10, 50, and 100 Hz) 200 μs biphasic pulses at submotor thresholds demonstrated inhibition of visceromotor responses (VMRs) to UBD in rats made hypersensitive to UBD by a protocol that included neonatal cystitis. Onset of inhibitory effects occurred within 20 minutes of beginning SCS. Otherwise, SCS at all other parameters studied and in other tested rat models produced either no significant effect or augmentation of VMRs., Conclusions: Demonstration of inhibitory effects of SCS in a clinically relevant model of bladder pain suggests the potential utility of this therapy in patients with painful bladder disorders., (Copyright © 2021 International Neuromodulation Society. All rights reserved.)

Spinal mechanisms associated with visceral hypersensitivity are poorly understood. One model of bladder hypersensitivity with phenotypic features similar to the disorder interstitial cystitis/bladder pain syndrome is the neonatal bladder inflammation (NBI) model. In this model, rat pup bladders are infused with zymosan solutions on post-partum days 14-16 and then rats are retested as adults. Studies of other sites of deep tissue hypersensitivity have suggested a role for corticotropin-releasing factor (CRF) receptors type 1 and 2 (CRFR1 and CRFR2). Using neurochemical measures, pharmacological manipulations and both reflex and neuronal responses to urinary bladder distension as endpoints, the present study probed the role of CRFR2s in bladder hyperalgesia secondary to NBI and acute bladder re-inflammation as an adult (ABI). ELISA measures of the lumbosacral spinal cord demonstrated increased CRFR1s and CRFR2s following pretreatment with both NBI + ABI as well as NBI-related increases in the CRFR2 agonist urocortin 2. Intrathecal CRFR2 antagonists, but not a CRFR1 antagonist, blocked the augmentation of visceromotor responses to distension following pretreatment with both NBI + ABI. Lumbosacral dorsal horn neuronal responses to distension in rats pretreated with NBI + ABI were attenuated by the spinal topical administration of a CRFR2 antagonist. These studies suggest therapeutic value of CRFR2 antagonists in the treatment of painful bladder disorders., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a eukaryotic, post-translational modification catalyzed by GPI transamidase (GPI-T). The Saccharomyces cerevisiae GPI-T is composed of five membrane-bound subunits: Gpi8, Gaa1, Gpi16, Gpi17, and Gab1. GPI-T has been recalcitrant to in vitro structure and function studies because of its complexity and membrane-solubility. Furthermore, a reliable, quantitative, in vitro assay for this important post-translational modification has remained elusive despite its discovery more than three decades ago.Three recent reports describe the structure of GPI-T from S. cerevisiae and humans, shedding critical light on this important enzyme and offering insight into the functions of its different subunits. Here, we present the purification and characterization of a truncated soluble GPI-T heterotrimer complex (Gpi8 23-306 , Gaa1 50-343 , and Gpi16 20-551 ) without transmembrane domains. Using this simplified heterotrimer, we report the first quantitative method to measure GPI-T activity in vitro and demonstrate that this soluble, minimalistic GPI-T retains transamidase activity. These results contribute to our understanding of how this enzyme is organized and functions, and provide a method to screen potential GPI-T inhibitors.

In rodent models, conditioning with acute footshock (AFS) has been demonstrated to produce bladder hypersensitivity which is more robust when rats, tested as adults, had also been pretreated with neonatal bladder inflammation (NBI). The spinal neurochemical mechanisms of pro-nociceptive processes in rats pretreated with NBI are not fully known and so the present study administered intrathecal (IT) opioid (naloxone) and NMDA receptor (MK-801) antagonists to determine whether these receptors' actions had been altered by NBI. Female Sprague-Dawley rat pups were intravesically pretreated on postnatal days P14-P16 with a 1% zymosan solution or with control procedures and then raised to adulthood (12-15 weeks of age). Bladder hypersensitivity was induced through use of an AFS paradigm. Visceromotor responses (VMRs; abdominal muscle contractions) to graded, air pressure-controlled urinary bladder distension were used as nociceptive endpoints. Immediately following AFS pretreatments, rats were anesthetized and surgically prepared. Pharmacological antagonists were administered via an IT catheter onto the lumbosacral spinal cord and VMRs determined 15 min later. Administration of IT naloxone hydrochloride (10 μg) to rats which had been pretreated only with AFS resulted in VMRs that were more robust than VMRs in similarly pretreated rats that received IT normal saline. In contrast, IT naloxone had no significant effect on rats that had been pretreated with both NBI&AFS, although MK-801 was inhibitory. These effects of IT naloxone suggest the presence of inhibitory influences in normal rats that are absent in rats pretreated with NBI. Absence of inhibitory influences produced by AFS was also demonstrated in rats pretreated with NBI&AFS using measures of thermal paw withdrawal latency (PWL): rats pretreated with only AFS had longer PWLs than rats pretreated with both NBI&AFS. Together, a reduction in anti-nociceptive mechanisms coupled with pro-nociceptive NMDA-linked mechanisms results in more robust nociceptive responses to distension in rats which had experienced NBI., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Bladder pain and hypersensitivity to bladder filling are clinically common, but animal models examining syndromes with these features are limited. A rat model of bladder hypersensitivity produced by neonatal bladder inflammation (NBI) has been reported to have many of the clinical features of bladder pain syndromes. The present study sought to determine whether similar hypersensitivity might be induced by NBI in mice. Female C57BL6/J mice had NBI induced on postnatal days P12-14 by the intravesical administration of zymosan. As adults (12-14 weeks of age), the mice were examined for hypersensitivity of their bladders as: spontaneous voiding and evoked cystometrograms at baseline, and visceromotor responses (VMRs) to urinary bladder distension (UBD) following a secondary insult (either repeated bladder inflammation or acute stress induced by footshock). Mice that experienced NBI demonstrated hypersensitivity, when compared with control mice, manifested as increased spontaneous voiding, increased frequency of evoked voids during intravesical saline infusion, and increased vigor of VMRs to UBD following either acute bladder inflammation or acute stress. This recapitulates the hallmark features of clinical painful bladder disorders and suggest utility of this murine model for the study of these disorders while allowing methodological expansion into well-established genetic and immunological models., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Clodfelder-Miller, Ness and DeBerry.)

Psychological stress has been demonstrated to increase reports of pain in humans with pelvic pain of urologic origin. In rodent models, conditioning with acute footshock (AFS) has been demonstrated to increase measures of stress/anxiety as well as bladder hypersensitivity. The spinal neurochemical mechanisms of this pro-nociceptive process are unknown and so the present study administered antagonists for multiple receptors that have been associated with facilitatory mechanisms into the spinal intrathecal space. Bladder hypersensitivity was induced through use of an AFS paradigm in which female Sprague-Dawley rats received a 15-min intermittent shock treatment. Visceromotor responses (VMRs; abdominal muscle contractions) to air pressure-controlled urinary bladder distension (UBD) were used as nociceptive endpoints. Immediately following AFS treatments, rats were anesthetized (inhaled isoflurane, IP urethane) and surgically prepared. Pharmacological antagonists were administered via an intrathecal (IT) catheter onto the lumbosacral spinal cord and VMRs to graded UBD determined 15 min later. Administration of IT naloxone hydrochloride (10 μg) and IT phentolamine hydrochloride (10 μg) resulted in VMRs that were more robust than VMRs in rats that received AFS and IT normal saline whereas there was no significant effect of these drugs on VMRs in rats which underwent non-footshock procedures. In contrast, a low dose of the NMDA-receptor antagonist, MK-801 (30 μg), significantly reduced VMRs in rats made hypersensitive to UBD by AFS, but had no significant effect on rats that underwent non-footshock procedures. This study suggests that pro-nociceptive effects of AFS in otherwise healthy rats involve a spinal NMDA-linked mechanism. The effects of IT naloxone and IT phentolamine suggest the presence of inhibitory influences that are opioidergic and/or alpha-adrenergic and that are masked by the pro-nociceptive mechanisms. Other agents with no statistically significant effect on VMRs include methysergide (30 μg), ondansetron (10 μg), mecamylamine (50 μg), antalarmin (24 μg), aSVG30 (12 μg), and SSR149415 (50 μg)., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Protein-protein interactions play critical roles in biology, but the structures of many eukaryotic protein complexes are unknown, and there are likely many interactions not yet identified. We take advantage of advances in proteome-wide amino acid coevolution analysis and deep-learning–based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes within the Saccharomyces cerevisiae proteome. We use a combination of RoseTTAFold and AlphaFold to screen through paired multiple sequence alignments for 8.3 million pairs of yeast proteins, identify 1505 likely to interact, and build structure models for 106 previously unidentified assemblies and 806 that have not been structurally characterized. These complexes, which have as many as five subunits, play roles in almost all key processes in eukaryotic cells and provide broad insights into biological function.

This report describes methodological and exploratory investigations of the zymosan-induced neonatal bladder inflammation (NBI) model of interstitial cystitis/bladder pain syndrome (IC/BPS) in female rats. These results validate and extend the currently employed model by evaluating critical timepoints for obtaining treatment effects and identified that a second insult as an adult including repeat intravesical zymosan, intravesical lipopolysaccharide, acute footshock stress, neuropathic nociception (facial) or somatic inflammation (hindpaw) all resulted in magnified visceromotor responses to urinary bladder distension (UBD) in rats which had experienced NBI when compared with their controls. NBI also resulted in increased tone and reactivity of pelvic floor musculature to UBD, as well as increased responsiveness to intravesical potassium chloride solutions, abnormal anxiety measures (elevated plus maze) and an increased number of submucosal petechial hemorrhages following 30 min of hydrodistension of the bladder. These phenotypic findings have correlates to the clinical features of IC/BPS in humans and so support use of this model system to examine mechanisms of and treatments for IC/BPS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ness, DeWitte, DeBerry, Hart, Clodfelder-Miller, Gu, Ling and Randich.)

Background: Baclofen, a clinically available GABA B receptor agonist, produces non-opioid analgesia in multiple models of pain but has not been tested for effects on bladder nociception., Methods: A series of experiments examined the effects of systemic and spinally administered baclofen on bladder nociception in female anesthetized rats. Models of bladder nociception included those which employed neonatal and adult bladder inflammation to produce bladder hypersensitivity., Results: Cumulative intraperitoneal dosing (1-8 mg/kg IP) and cumulative intrathecal dosing (10-160 ng IT) of baclofen led to dose-dependent inhibition of visceromotor responses (VMRs) to urinary bladder distension (UBD) in all tested models. There were no differences in the magnitude of the analgesic effects of baclofen as a function of inflammation versus no inflammation treatments. Hemodynamic (pressor) responses to UBD were similarly inhibited by IT baclofen as well as UBD-evoked excitatory responses of spinal dorsal horn neurons. The GABA B receptor antagonist, CGP 35,348, antagonized the antinociceptive effects of IT baclofen on VMRs in all tested models but did not affect the magnitude of the VMRs by itself suggesting no tonic GABA B activity was present in this preparation. Tolerance to a seven day continuous IT infusion of baclofen was not observed., Conclusions: These data provide support for a clinical trial of baclofen as a non-opioid treatment of human bladder pain., (© 2021. The Author(s).)

Non-human primates (NHPs) are a preferred animal model for optimizing adeno-associated virus (AAV)-mediated CNS gene delivery protocols before clinical trials. In spite of its inherent appeal, it is challenging to compare different serotypes, delivery routes, and disease indications in a well-powered, comprehensive, multigroup NHP experiment. Here, a multiplex barcode recombinant AAV (rAAV) vector-tracing strategy has been applied to a systemic analysis of 29 distinct, wild-type (WT), AAV natural isolates and engineered capsids in the CNS of eight macaques. The report describes distribution of each capsid in 15 areas of the macaques' CNS after intraparenchymal (putamen) injection, or cerebrospinal fluid (CSF)-mediated administration routes (intracisternal, intrathecal, or intracerebroventricular). To trace the vector biodistribution (viral DNA) and targeted tissues transduction (viral mRNA) of each capsid in each of the analyzed CNS areas, quantitative next-generation sequencing analysis, assisted by the digital-droplet PCR technology, was used. The report describes the most efficient AAV capsid variants targeting specific CNS areas after each route of administration using the direct side-by-side comparison of WT AAV isolates and a new generation of rationally designed capsids. The newly developed bioinformatics and visualization algorithms, applicable to the comparative analysis of several mammalian brain models, have been developed and made available in the public domain., Competing Interests: Declaration of interests At the time of project design and implementation, R.D.B and J.R. were employees of Pfizer, whereas E.R.-L. is currently an employee of Lacerta Therapeutics Inc. Aspects of this work were funded by Pfizer and Lacerta Therapeutics Inc. R.J.M., S.Z., and E.R.-L. are co-founders and shareholders of Lacerta Therapeutics Inc., (Copyright © 2021 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

This report is third in a three-part clinical trial series screening potential treatments for Gulf War Illness (GWI). The goal of the project was to rapidly identify agents to prioritize for further efficacy research. We used a placebo-controlled, pseudo-randomized, crossover design to test the effects of reishi mushroom ( Ganoderma lucidum ), stinging nettle ( Uritca dioica ), and epimedium ( Epimedium sagittatum ) in 29 men with GWI. Participants completed 30 days of symptom reports for baseline, then a botanical line consisting of 30 days of placebo, followed by 30 days each of lower-dose and higher-dose botanical. After completing a botanical line, participants were randomized to complete the protocol with another botanical, until they completed three botanical trials. GWI symptom severity, pain, and fatigue were contrasted between the four conditions (baseline, placebo, lower-dose, higher dose) using linear mixed models. GWI symptom severity was unchanged from placebo in the reishi lower-dose condition ( p = 0.603), and was higher in the higher-dose condition ( p = 0.012). Symptom severity was not decreased from placebo with lower-dose stinging nettle ( p = 0.604), but was significantly decreased with higher-dose stinging nettle ( p = 0.048). Epimedium showed no significant decreases of GWI symptoms in the lower ( p = 0.936) or higher ( p = 0.183) dose conditions. Stinging nettle, especially at higher daily dosages, may help reduce the symptoms of GWI. Epimedium does not appear to beneficially affect GWI symptom severity, and reishi may exaggerate symptoms in some GWI sufferers. These results are in a small sample and are preliminary. Further research is required to determine if stinging nettle is indeed helpful for the treatment of GWI, and what dosage is optimal. This trial was registered on ClinicalTrials.gov (NCT02909686).

A chronic multi-symptom illness of unknown etiology, Gulf War Illness (GWI) affects 175,000 to 250,000 veterans of the Gulf War. Because inflammation has suspected involvement in the pathophysiology of GWI, botanical treatments that target inflammation may be beneficial in reducing symptoms. No FDA-approved treatments currently exist for GWI, and rapid prioritization of agents for future efficacy testing is important. This study is part of a larger project that screened nine different botanical compounds with purported anti-inflammatory properties for potential treatment of GWI. We tested three botanicals (resveratrol [ Polygonum cuspidatum ], luteolin, and fisetin [ Rhus succedanea ]) on symptom severity of GWI in this placebo-controlled, pseudo-randomized clinical trial. Twenty-one male veterans with GWI completed the study protocol, which consisted of 1 month (30 days ± 3) of baseline symptom reports, 1 month of placebo, 1 month of lower-dose botanical, and 1 month of higher-dose botanical. Participants completed up to 3 different botanicals, repeating the placebo, lower-dose, and higher-dose cycle for each botanical assigned. Linear mixed models were used for analyses. Resveratrol reduced GWI symptom severity significantly more than placebo at both the lower ( p = 0.035) and higher ( p = 0.004) dosages. Luteolin did not decrease symptom severity more than placebo at either the lower ( p = 0.718) or higher dosages ( p = 0.492). Similarly, fisetin did not reduce symptom severity at either the lower ( p = 0.504) or higher ( p = 0.616) dosages. Preliminary findings from this screening study suggest that resveratrol may be beneficial in reducing symptoms of GWI and should be prioritized for future testing. Larger trials are required to determine efficacy, response rates, durability of effects, safety, and optimal dosage. This trial was registered on ClinicalTrials.gov (NCT02909686) on 13 September 2016.

This report is part of a larger study designed to rapidly and efficiently screen potential treatments for Gulf War Illness (GWI) by testing nine different botanicals. In this placebo-controlled, pseudo-randomized, crossover clinical trial of 20 men with GWI, we tested three botanical agents with putative peripheral and central anti-inflammatory actions: curcumin ( Curcuma longa ), boswellia ( Boswellia serrata ), and French maritime pine bark extract ( Pinus pinaster ). Participants completed 30 +/- 3 days of baseline symptom reports, followed by 30 +/- 3 days of placebo, 30 +/- 3 days of lower-dose botanical, and 30 +/- 3 days of higher-dose botanical. Participants then repeated the process with a new botanical until completing up to three botanical cycles. Data were analyzed using linear mixed models. Curcumin reduced GWI symptom severity significantly more than placebo at both the lower ( p < 0.0001) and higher ( p = 0.0003) dosages. Boswellia was not more effective than placebo at reducing GWI symptoms at either the lower ( p = 0.726) or higher ( p = 0.869) dosages. Maritime pine was not more effective than placebo at the lower dosage ( p = 0.954) but was more effective than placebo at the higher dosage ( p = 0.006). This study provides preliminary evidence that curcumin and maritime pine may help alleviate symptoms of GWI. As a screening study, a final determination of the efficacy of these compounds for all individuals with GWI cannot be made, and further studies will need to be conducted to determine strength and durability of effects, as well as optimal dosage. These results suggest that GWI may, at least in part, involve systemic inflammatory processes. This trial was registered on ClinicalTrials.gov (NCT02909686) on 13 September 2016.

Objective To evaluate the post-operative outcomes of patients with obstructive sleep apnea (OSA) given intraoperative ketamine. Design: case-control study A total of 574 patients (287 received ketamine and 287 were matched controls) diagnosed with OSA and body mass index (BMI) > 30 who received general anesthesia were included in this study. Patients given intraoperative ketamine were matched (1:1) with those who did not receive ketamine for age, gender, BMI, ethnicity, anesthesia time, intraoperative fentanyl dose, ketamine dose, and surgery type. A sub-analysis was performed based on the dose of ketamine administered and also on the surgery type. Measured outcomes include post-operative pain scores, post-operative opioid requirements, respiratory status, oxygen use, and duration post-operatively. Results Intraoperative ketamine use did not decrease pain scores or post-operative opioid use when compared with the control (no intraoperative ketamine) group. Patients who received high-dose ketamine had significantly higher post-operative pain scores (p=0.048) while in the post-anesthesia care unit (PACU) and required supplemental oxygen for a longer period of time (p = 0.030), pain scores were not significant for patients who underwent orthopedic/spine procedures (p = 0.074), and high-dose ketamine group patients who underwent orthopedic/spine surgery required significantly more opioids in the PACU (p = 0.031). Among patients who received low-dose ketamine, those who underwent head, ear, nose, and throat surgery required significantly more opioids in PACU (p = 0.022). Conclusions Low-dose intraoperative ketamine did not decrease pain scores or post-operative opioid use significantly and did not improve standard respiratory recovery parameters for OSA patients after surgery. Neither low- nor high-dose ketamine demonstrated the anticipated benefits of low pain scores and reduced post-operative opioid use. These outcomes will differ depending on the surgery type and dose of ketamine used., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2020, Schreiner et al.)

Aims: Neuromodulation (nerve stimulation) can produce analgesia. One form, bilateral pudendal nerve stimulation (bPNS), suppresses responses to urinary bladder distension (UBD) in hypersensitive rats. Drugs can modify this effect (eg, benzodiazepines, but not opioids, suppress bPNS effects). Prior to a clinical trial of bPNS effects on bladder pain, we felt it was prudent to survey the effects of medications commonly used in patients with bladder disorders., Methods: Bladder hypersensitivity was produced by neonatal bladder inflammation in rat pups coupled with a second inflammatory insult as an adult. Antimuscarinic (oxybutynin), β 3 -adrenoceptor agonist (mirabegron, CL316243), α 1 -adrenoceptor antagonist (tamsulosin), antidepressant (amitriptyline), muscle relaxing (baclofen), and sedative (propofol) agents were administered and effects of bPNS on responses to UBD assessed. bPNS consisted of bilateral biphasic electrical stimulation of the mixed motor/sensory component of the pudendal nerves. Visceromotor responses (VMRs; abdominal muscle contractile responses) were used as nociceptive endpoints., Results: Many of these drugs directly inhibited the VMRs to UBD, but only mirabegron, at the doses employed, significantly reduced inhibitory effects of bPNS. In the presence of the other drugs, bPNS continued to produce statistically significant inhibition of VMRs to UBD., Conclusions: This study suggests that concurrent therapy with drugs used to treat bladder disorders could affect assessment of the effects of bPNS on bladder hypersensitivity. This study gives guidance to clinical trials using bPNS for the treatment of painful bladder syndromes and suggests potential clinical use of some of these medications in the treatment of these same disorders., (© 2020 Wiley Periodicals, Inc.)

Introduction: Low back pain (LBP) is a complex and growing global health problem in need of more effective pain management strategies. Spinal mobilization (SM) is a non-pharmacological approach recommended by most clinical guidelines for LBP, but greater utilization and treatment optimization are hampered by a lack of mechanistic knowledge underlying its hypoalgesic clinical effects., Methods: Groups of female Sprague-Dawley rats received unilateral trunk (L5 vertebral level) injections (50 μl) of either vehicle (phosphate-buffer solution, PBS; VEH) or nerve growth factor (NGF; 0.8 μM) on Days 0 and 5 with or without daily L5 SM (VEH, NGF, VEH + SM, VEH + SM). Daily passive SM (10 min) was delivered by a feedback motor (1.2 Hz, 0.9N) from Days 1 to 12. Changes in pain assays were determined for mechanical and thermal reflexive behavior, exploratory behavior (open field events) and spontaneous pain behavior (rat grimace scale). On Day 12, lumbar (L1-L6) dorsal root ganglia (DRG) were harvested bilaterally and calcitonin gene-related peptide (CGRP) positive immunoreactive neurons were quantified from 3 animals (1 DRG tissue section per segmental level) per experimental group., Results: NGF induced bilateral trunk (left P = 0.006, right P = 0.001) mechanical hyperalgesia and unilateral hindpaw allodynia ( P = 0.006) compared to the vehicle group by Day 12. Additionally, we found for the first time that NGF animals demonstrated decreased exploratory behaviors (total distance traveled) and increased grimace scale scoring compared to the VEH group. Passive SM prevented this development of local (trunk) mechanical hyperalgesia and distant (hindpaw) allodynia, and normalized grimace scale scores. NGF increased CGRP positive immunoreactive neurons in ipsilateral lumbar DRGs compared to the VEH group ([L1] P = 0.02; [L2] P = 0.007) and SM effectively negated this increase in pain-related neuropeptide CGRP expression., Conclusion: SM prevents the development of local (trunk) NGF-induced mechanical hyperalgesia and distant (hindpaw) allodynia, in part, through attenuation of CGRP expression in lumbar DRG sensory neurons. NGF decreases rat exploratory behavior and increases spontaneous pain for which passive SM acts to mitigate these pain-related behavioral changes. These initial study findings suggest that beginning daily SM soon after injury onset might act to minimize or prevent the development of LBP by reducing production of pain-related neuropeptides., (Copyright © 2020 Reed, Little, Lima, Sorge, Yarar-Fisher, Eraslan, Hurt, Ness, Gu, Martins and Li.)

Background: Neuromodulation, as a therapeutic modality for pain treatment, is an alternative to opioid therapies and therefore receiving increased interest and use. Neuromodulation at a peripheral nerve target, in the form of bilateral electrical pudendal nerve stimulation (bPNS), has been shown to reduce bladder hypersensitivity in rats and anecdotally reduces pain in humans with pelvic pain of urological origin. Recent studies have identified a role for spinal γ-aminobutyric acid (GABA) receptors in this effect. Concomitant medication use, such as benzodiazepines, could alter responses to neuromodulation, and so before the development of a clinical trial to confirm translation of this potential therapy, the potential interactions between acute and chronic use of benzodiazepines and bPNS were examined in a preclinical model., Methods: Bladder hypersensitivity was produced by neonatal bladder inflammation in rat pups coupled with a second inflammatory insult as an adult. Diazepam (1-5 mg/kg intraperitoneal [i.p.]) or vehicle was administered acutely (with or without bPNS) and chronically (5 mg/kg subcutaneous [s.c.] daily for 2 weeks before the final experiment). bPNS was delivered as bilateral biphasic electrical stimulation of the mixed motor/sensory component of the pudendal nerves. Visceromotor responses (VMRs; abdominal muscle contractile responses to urinary bladder distension [UBD]) were used as nociceptive end points. Due to the profound effects of diazepam, the effect of midazolam (0.5-1.0 mg/kg i.p.) on VMRs and bPNS effects was also studied., Results: Diazepam and midazolam both produced a dose-dependent, flumazenil-reversible inhibition of VMRs to UBD. bPNS resulted in statistically significant inhibition of VMRs to UBD in hypersensitive rats that had received vehicle injections. Select doses of diazepam and midazolam suppressed the inhibitory effect of bPNS on VMRs., Conclusions: This study suggests that inhibitory effects of bPNS on bladder pain could be suppressed in subjects receiving benzodiazepine therapy, suggesting that potential clinical testing of pudendal nerve stimulation for the treatment of painful bladder syndromes may be confounded by the use of benzodiazepines. Clinical assessment of other forms of neuromodulation should also be screened for impacts of benzodiazepines.

The classification of chronic visceral pain is complex, resulting from persistent inflammation, vascular (ischemic) mechanisms, cancer, obstruction or distension, traction or compression, and combined mechanisms, as well as unexplained functional mechanisms. Despite the prevalence, treatment options for chronic visceral pain are limited. Given this unmet clinical need, the development of novel analgesic agents, with defined targets derived from preclinical studies, is urgently needed. While various animal models have played an important role in our understanding of visceral pain, our knowledge is far from complete. Due to the complexity of visceral pain, this document will focus on chronic abdominal pain, which is the major complaint in patients with disorders of the gut-brain interaction, also referred to as functional gastrointestinal disorders, such as irritable bowel syndrome (IBS). Models for IBS are faced with challenges including a complex clinical phenotype, which is comorbid with other conditions including anxiety, depression, painful bladder syndrome, and chronic pelvic pain. Based upon the multifactorial nature of IBS with complicated interactions between biological, psychological, and sociological variables, no single experimental model recapitulates all the symptoms of IBS. This position paper will contextualize chronic visceral pain using the example of IBS and focus on its pathophysiology while providing a critical review of current animal models that are most relevant, robust, and reliable in which to screen promising therapeutics to alleviate visceral pain and delineate the gaps and challenges with these models. We will also highlight, prioritize, and come to a consensus on the models with the highest face/construct validity., (© 2019 John Wiley & Sons Ltd.)

Background and Objectives: Bilateral electrical pudendal nerve stimulation (bPNS) reduces bladder hypersensitivity in rat models and anecdotally reduces pain in humans with pelvic pain of urologic origin. Concomitant opioids are known to alter responses to neuromodulation in some systems. So prior to the development of a clinical trial for purposes of regulatory approval, the preclinical interaction between opioids and stimulation effectiveness was examined., Methods: Bladder hypersensitivity was produced by neonatal bladder inflammation in rat pups coupled with a second inflammatory insult as an adult. Morphine was administered acutely (1-4 mg/kg intraperitoneal) or chronically (5 mg/kg subcutaneously daily for 2 weeks prior to the terminal experiment). bPNS consisted of bilateral biphasic electrical stimulation of the mixed motor/sensory component of the pudendal nerves. Visceromotor responses (VMR; abdominal muscle contractile responses to urinary bladder distension (UBD)) were used as nociceptive endpoints., Results: Morphine produced a dose-dependent inhibition of VMRs to UBD that was naloxone reversible. bPNS resulted in statistically significant inhibition of VMRs to UBD in hypersensitive rats that had received acute or chronic subcutaneous morphine injections., Conclusions: This study suggests that inhibitory effects of bPNS can still be evoked in subjects who are receiving opioid therapy, thus giving guidance to potential clinical trials seeking regulatory approval for the treatment of chronic bladder pain., Competing Interests: Competing interests: XS and DEN were employees of Medtronic which funded this study., (© American Society of Regional Anesthesia & Pain Medicine 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Altered Toll-like receptor (TLR)4 activation has been identified in several chronic pain conditions but has not been well studied in interstitial cystitis/bladder pain syndrome (IC/BPS). Our previously published human studies indicated that patients with IC/BPS present altered systemic TLR4-mediated inflammatory responses, which were significantly correlated with reported pain severity. In the present study, we sought to determine whether altered TLR4 activation plays a role in pelvic/bladder pain seen in patients with IC/BPS using our validated IC/BPS-like transgenic autoimmune cystitis model (URO-OVA). URO-OVA mice developed responses consistent with pelvic and bladder pain after cystitis induction, which was associated with increased splenocyte production of TLR4-mediated proinflammatory cytokines IL-1β, IL-6, and TNF-α. Increased spinal expression of mRNAs for proinflammatory cytokines IL-6 and TNF-α, glial activation markers CD11b and glial fibrillary acidic protein, and endogenous TLR4 ligand high mobility group box 1 was also observed after cystitis induction. Compared with URO-OVA mice, TLR4-deficient URO-OVA mice developed significantly reduced nociceptive responses, although similar bladder inflammation and voiding dysfunction, after cystitis induction. Intravenous administration of TAK-242 (a TLR4-selective antagonist) significantly attenuated nociceptive responses in cystitis-induced URO-OVA mice, which was associated with reduced splenocyte production of TLR4-mediated IL-1β, IL-6, and TNF-α as well as reduced spinal expression of mRNAs for IL-6, TNF-α, CD11b, glial fibrillary acidic protein, and high mobility group box 1. Our results indicate that altered TLR4 activation plays a critical role in bladder nociception independent of inflammation and voiding dysfunction in the URO-OVA model, providing a potential mechanistic insight and therapeutic target for IC/BPS pain.

Small open reading frames (smORFs) encoding polypeptides of less than 100 amino acids in eukaryotes (50 amino acids in prokaryotes) were historically excluded from genome annotation. However, recent advances in genomics, ribosome footprinting, and proteomics have revealed thousands of translated smORFs in genomes spanning evolutionary space. These smORFs can encode functional polypeptides, or act as cis-translational regulators. Herein we review evidence that some smORF-encoded polypeptides (SEPs) participate in stress responses in both prokaryotes and eukaryotes, and that some upstream ORFs (uORFs) regulate stress-responsive translation of downstream cistrons in eukaryotic cells. These studies provide insight into a regulated subclass of smORFs and suggest that at least some SEPs may participate in maintenance of cellular homeostasis under stress.

Bilateral electrical pudendal nerve stimulation (bPNS) reduces bladder hypersensitivity in rat models of bladder pain and anecdotally reduces pain in humans with pelvic pain of urologic origin. The spinal neurochemical mechanisms of this antinociception are unknown. In the present study, bladder hypersensitivity was produced by neonatal bladder inflammation in rat pups coupled with a second inflammatory insult as an adult. Visceromotor responses (VMRs; abdominal muscle contractions) to urinary bladder distension (UBD) were used as a nociceptive endpoint under urethane-isoflurane anesthesia. bPNS consisted of bilateral biphasic electrical stimulation of the mixed motor/sensory component of the pudendal nerves. Following determination of the inhibitory effect of bPNS on VMRs, pharmacological antagonists were administered via an intrathecal catheter onto the lumbosacral spinal cord and bPNS effects on VMRs redetermined. bPNS resulted in statistically significant inhibition of VMRs to UBD in hypersensitive rats that was statistically reduced by the intrathecal administration of methysergide, WAY100636, CGP35348 and strychnine but was unaffected by naloxone, bicuculline, phentolamine, ondansetron and normal saline. This study suggests that inhibitory effects of bPNS may include serotonergic, GABA-B-ergic and glycinergic mechanisms suggesting the potential for interaction of the neuromodulatory effect with concommitant drug therapies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

There is increasing evidence that chronic pain may be associated with events that occur during critical periods of development. Recent studies have identified behavioral, spinal neurophysiological and spinal/peripheral neurochemical differences in rats that have experienced neonatal bladder inflammation (NBI): a putative model of the chronically painful bladder disorder, interstitial cystitis. Stress has been shown to exacerbate symptoms of interstitial cystitis and produces bladder hypersensitivity in animal models. We recently reported that Acute Footshock-induced bladder hypersensitivity was eliminated in otherwise normal rats by prior bilateral lesions of the central nucleus of the amygdala. Since the spinal and peripheral nervous systems of NBI-treated rats are known to differ from normal rats, the present experiments sought to determine whether a supraspinal nervous system structure, the central amygdala, is still necessary for the induction of Acute Footshock-induced hypersensitivity. The effect of bilateral amygdala electrolytic lesions on Acute Footshock-induced bladder hypersensitivity in adult female rats was tested in Control rats which underwent a control protocol as neonates and in experimental rats which experienced NBI. Consistent with our previous report, in Control rats, Acute Footshock-induced bladder hypersensitivity was eliminated by bilateral Amygdala Lesions. In contrast, Acute Footshock-induced bladder hypersensitivity in NBI-treated rats was unaffected by bilateral Amygdala Lesions. These findings provide evidence that NBI results in the recruitment of substrates of bladder hypersensitivity that may differ from those of normal rats. This, in turn, suggests that unique therapeutics may be needed for painful bladder disorders like interstitial cystitis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Known complications of endoscopic retrograde cholangiopancreatography (ERCP) include pancreatitis, bleeding, duodenal perforation, and venous air embolism (VAE). The aim of this study was to determine the incidence of VAE during ERCP and be able to differentiate high-risk versus low-risk ERCP procedures., Methods: This is a prospective cohort study consisting of patients who underwent ERCP and were monitored with a precordial Doppler ultrasound (PDU) for VAE. PDU monitoring was digitally recorded and analyzed to confirm the suspected VAE. Demographic and clinical data related to the anesthetic care, endoscopic procedure, and intraoperative hemodynamics were analyzed., Results: A total of 843 ERCP procedures were performed over a 15-month period. The incidence of VAE was 2.4% (20 patients). All VAE's occurred during procedures in which stent placement, sphincterotomy, biopsy, duct dilation, gallstone retrieval, cholangioscopy, or necrosectomy occurred. Ten of 20 (50%) of VAEs were associated with hemodynamic alterations. None occurred if the procedure was only diagnostic or for stent removal. Subanalysis for the type of procedure showed that VAE was statistically more frequent when stents were removed and then replaced or if a cholangioscopy was performed., Conclusions: The high incidence of VAE highlights the need for practitioners to be aware of this potentially serious event. Use of PDU can aid in the detection of VAE during ERCP and should be considered especially during high-risk therapeutic procedures. Detection may allow appropriate interventions before serious adverse events such as cardiovascular collapse occur.

There is mounting evidence underscoring a role for the urothelium in urinary bladder sensation. Previous functional studies have identified bladder primary afferents with mechanosensitive properties suggesting urothelial innervation and/or communication. The current study identifies a group of urothelium-innervating afferent neurons in rat, and characterizes and compares the properties of these and non-urothelial afferent neuron populations. Lumbosacral (LS) primary afferent neurons were retrogradely labeled using intraparenchymal (IPar) microinjection or intravesical (IVes) infusion of tracer into the bladder. Using these techniques, separate populations of neurons were differentiated by dorsal root ganglion (DRG) somata labeling and dye distribution within the bladder. IPar- and IVes-labeled neurons accounted for 85.0% and 14.4% of labeled L6-S1 neurons (P < .001), respectively, with only 0.6% of neurons labeled by both techniques. Following IVes labeling, dye was contained only within the periurothelial bladder region in contrast to non-urothelial distribution of dye after IPar labeling. Electrophysiological characterization by in situ patch-clamp recordings from whole-mount DRG preparations indicated no significant difference in passive or active membrane properties of IPar and IVes DRG neurons. However, calcium imaging of isolated neurons indicates that a greater proportion of IPar- than IVes-labeled neurons express functional TRPA1 (45.7% versus 25.6%, respectively; P < .05). This study demonstrates that two anatomically distinct groups of LS bladder afferents can be identified in rat. Further studies of urothelial afferents and the phenotypic differences between non-/urothelial afferents may have important implications for normal and pathophysiological bladder sensory processing., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background and Objectives: The Pain Sensitivity Questionnaire, English version (PSQ-E), is predictive of pain-related responses to experimental stimuli. Ethnic differences have been noted in experimental measures of pain sensation using quantitative sensory testing. The present study sought to determine if the PSQ-E also identified similar ethnic differences., Methods: Fifty-seven subjects who self-identified as African Americans (AAs) and who were scheduled to undergo a low-back interventional procedure completed the PSQ-E and other questionnaires. Their data were compared with an age-, sex-, and opioid usage-matched sample of 57 self-identified non-Hispanic white (NHW) subjects. Pain ratings on a visual analog scale (VAS) were obtained following 2 standardized injections of subcutaneous lidocaine (VAS1-infiltration in hand, VAS2-infiltration of procedural site). Correlations between PSQ-E scores, VAS measures, and other inventories were tested., Results: The PSQ-E scores and clinical and experimental pain scores were all significantly elevated in AA compared with NHW patients (P < 0.05 for experimental pain scores, P < 0.001 for PSQ and clinical pain scores). Measures of pain interference, depression, anxiety, and pain catastrophizing were not different between groups. Similar to our previous study, PSQ-E scores significantly correlated with both experimental and clinical pain scores (eg, PSQ-E with Brief Pain Inventory pain score: r = 0.39, P < 0.001)., Conclusions: The study demonstrated significantly elevated pain sensitivity in AA compared with NHW patients as measured by the PSQ-E and experimental and clinical pain intensity scores. This shows that the PSQ reflects the known elevation of pain sensitivity in AA subjects and suggests that it may be useful in assessing pain treatment disparities by identifying and standardizing differences in pain sensitivity.

Both acute and chronic stress has been shown to exacerbate symptoms of chronic visceral pain conditions such as interstitial cystitis. Studies using animal models support these findings in that both acute and chronic exposure to foot shock-induced stress (FS) augment nociceptive reflex responses to urinary bladder distension (UBD). Only a few studies have examined the neural substrates mediating these phenomena and it is not clear whether acute and chronic stress engage the same or different substrates to produce bladder hypersensitivity. The present studies examined the role of two important central nervous system structures - the amygdala (AMG) and the ventromedial medulla (VMM) - in mediating/modulating hypersensitivity evoked by acute versus chronic FS using responses to graded UBD in adult, female Sprague-Dawley rats. Bladder hypersensitivity produced by acute FS was significantly reduced by either bilateral central AMG or VMM lesions using measures generated by graded UBD, but these lesions had no significant effects using the same measures on bladder hyperalgesia produced by chronic FS. Our findings provide evidence that neural substrates underlying bladder hypersensitivity produced by chronic stress differ from those produced by acute stress. These findings suggest that while the AMG and VMM participate in pain processing during periods of limited exposure to stress, prolonged stress may recruit a new set of neural substrates not initially activated by acute exposure to stress., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Glycosylphosphatidylinositol transamidase (GPI-T) catalyzes the post-translational addition of the GPI anchor to the C-terminus of some proteins. In most eukaryotes, Gpi8, the active site subunit of GPI-T, is part of a hetero-pentameric complex containing Gpi16, Gaa1, Gpi17, and Gab1. Gpi8, Gaa1, and Gpi16 co-purify as a heterotrimer from Saccharomyces cerevisiae, suggesting that they form the core of the GPI-T. Details about the assembly and organization of these subunits have been slow to emerge. We have previously shown that the soluble domain of S. cerevisiae Gpi8 (Gpi8 23-306 ) assembles as a homodimer, similar to the caspases with which it shares weak sequence homology (Meitzler, J. L. et al., 2007). Here we present the characterization of a complex between the soluble domains of Gpi8 and Gaa1. The complex between GST-Gpi8 23-306 (α) and His 6 -Gaa1 50-343 (β) was characterized by native gel analysis and size exclusion chromatography (SEC) and results are most consistent with an α 2 β 2 stoichiometry. These results demonstrate that Gpi8 and Gaa1 interact specifically without a requirement for other subunits, bring us closer to determining the stoichiometry of the core subunits of GPI-T, and lend further credence to the hypothesis that these three subunits assemble into a dimer of a trimer., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Chronic pain is often measured with a severity score that overlooks its spatial distribution across the body. This widespread pain is believed to be a marker of centralization, a central nervous system process that decouples pain perception from nociceptive input. Here, we investigated whether centralization is manifested at the level of the brain using data from 1079 participants in the Multidisciplinary Approach to the Study of Chronic Pelvic Pain Research Network (MAPP) study. Participants with a clinical diagnosis of urological chronic pelvic pain syndrome (UCPPS) were compared to pain-free controls and patients with fibromyalgia, the prototypical centralized pain disorder. Participants completed questionnaires capturing pain severity, function, and a body map of pain. A subset (UCPPS N = 110; fibromyalgia N = 23; healthy control N = 49) underwent functional and structural magnetic resonance imaging. Patients with UCPPS reported pain ranging from localized (pelvic) to widespread (throughout the body). Patients with widespread UCPPS displayed increased brain gray matter volume and functional connectivity involving sensorimotor and insular cortices (P < 0.05 corrected). These changes translated across disease diagnoses as identical outcomes were present in patients with fibromyalgia but not pain-free controls. Widespread pain was also associated with reduced physical and mental function independent of pain severity. Brain pathology in patients with centralized pain is related to pain distribution throughout the body. These patients may benefit from interventions targeting the central nervous system.

Background: Every day, millions of people undergo surgical procedures facilitated by anesthesia. Yet, there is no clinically accepted measure to predict the effects of sedation or anesthesia on the central nervous system. Auditory brain activation may provide an objective and quantifiable method to measure of the effects of sedation on neuronal processing., Methods: This is a randomized clinical trial. Forty-eight healthy volunteers were randomly assigned to receive 1 of 3 sedative drugs (midazolam [n = 11], propofol [n = 12], or dexmedetomidine [n = 12]) at a concentration adjusted to achieve mild sedation by self-rating, or to a no-drug control group (n = 13). Participants underwent functional magnetic resonance imaging while listening to music in a 5-minute block design experiment. We tested the hypothesis that mild sedation changes the magnitude or extent of cortical activation of an auditory stimulus., Results: We observed a significant reduction in auditory activation in both the dexmedetomidine (P = .001) and midazolam (P = .029) but not the propofol group (P = .619) when compared with saline control., Conclusions: Our findings indicate that, compared with saline control, there is a significant reduction of brain activation in the auditory cortex in response to midazolam and dexmedetomidine but not propofol when given at mildly sedative doses. This method serves as a novel approach to quantify the effects of sedative agents in an objective fashion.

Background and Objectives: Neuromodulation has been reported to reliably improve symptoms of bladder overactivity and sometimes pain. The effect of electrical stimulation of several nerve pathways demonstrated to alter cystometric responses to bladder distension was examined on nociceptive responses in models of bladder hypersensitivity., Methods: Bladder hypersensitivity was produced by several published methods including neonatal inflammation, acute inflammation, and chronic stress. Effects of different sites of stimulation (L6 and T13 nerve roots, proximal and distal pudendal nerves [PNs]) on nociceptive reflex responses to urinary bladder distension in urethane-anesthetized female rats were assessed and a parametric analysis of parameters of stimulation was performed., Results: Bilateral biphasic stimulation of the proximal PNs resulted in statistically significant inhibition of visceromotor and cardiovascular responses to bladder distension in rats made hypersensitive by neonatal bladder inflammation. We found a range of optimal stimulation frequencies (5-10 Hz) which produced robust inhibitory effects when using short pulse widths (100-240 μs). Onset of inhibition was within minutes and persisted for several minutes after the stimulus was discontinued. Use of bilateral PN stimulation in acute inflammation and stress-induced hypersensitivity models as well as unilateral stimulation, very distal PN cutaneous branch stimulation, and stimulation of the T13 and L6 nerve roots all proved ineffective with the parameters used., Conclusions: This study suggests that inhibitory effects of bilateral PN stimulation can be evoked in a rodent hypersensitivity model at relatively low frequencies with short pulse widths. The onset of effect is rapid, which suggests the potential for treating episodic pain in painful bladder disorders.

Pelvic nerve (PN) bladder primary afferent neurons were retrogradely labeled by intraparenchymal (IPar) microinjection of fluorescent tracer or intravesical (IVes) infusion of tracer into the bladder lumen. IPar and IVes techniques labeled two distinct populations of PN bladder neurons differentiated on the basis of dorsal root ganglion (DRG) soma labeling, dye distribution within the bladder, and intrinsic electrophysiological properties. IPar (Fast blue)- and IVes (DiI)-labeled neurons accounted for 91.5% (378.3±32.3) and 8% (33.0±26.0) of all labeled neurons, respectively (p<0.01), with only 2.0±1.2 neurons labeled by both techniques. When dyes were switched, IPar (DiI)- and IVes (Fast blue) labeled neurons accounted for 77.6% (103.0±25.8) and 22.4% (29.8±10.5), respectively (P<0.05), with 6.0±1.5 double-labeled neurons. Following IPar labeling, DiI was distributed throughout non-urothelial layers of the bladder. In contrast, dye was contained within the urothelium and occasionally the submucosa after IVes labeling. Electrophysiological properties of DiI-labeled IPar and IVes DRG neurons were characterized by whole-mount, in situ patch-clamp recordings. IPar- and IVes-labeled neurons differed significantly with respect to rheobase, input resistance, membrane capacitance, amplitude of inactivating and sustained K(+) currents, and rebound action potential firing, suggesting that the IVes population is more excitable. This study is the first to demonstrate that IVes labeling is a minimally invasive approach for retrograde labeling of PN bladder afferent neurons, to selectively identify urothelial versus non-urothelial bladder DRG neurons, and to elucidate electrophysiological properties of urothelial and non-urothelial afferents in an intact DRG soma preparation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Purpose: In healthy control subjects certain brain regions of interest demonstrate increased regional cerebral blood flow in response to painful stimuli. We examined the effect of bladder distension on arterial spin label functional magnetic resonance imaging measures of regional cerebral blood flow in regions of interest in subjects with interstitial cystitis., Materials and Methods: A total of 11 female subjects with interstitial cystitis and 11 healthy controls underwent 3 brain perfusion scan studies using arterial spin label functional magnetic resonance imaging, including 1) with a full bladder, 2) with an empty bladder and 3) while experiencing heat pain. Regional cerebral blood flow was calculated using custom software and individual scans were spatially normalized to the MNI (Montreal Neurological Institute) template. Region of interest based, absolute regional cerebral blood flow was determined for each condition and for the within group/within subject regional cerebral blood flow distribution changes induced by each condition., Results: Bladder distension was associated with robust increases in regional cerebral blood flow in subjects with interstitial cystitis. The increases were greater than those in healthy controls in multiple regions of interest, including the supplemental motor area (mainly Brodmann area 6), the motor and sensory cortex, the insula bilaterally, the hippocampal structures bilaterally, and the middle and posterior cingulate areas bilaterally. During heat pain healthy controls had more robust regional cerebral blood flow increases in the amygdala bilaterally. At baseline with an empty bladder there was lower regional cerebral blood flow in the insula, and the mid and posterior cingulate cortex bilaterally in subjects with interstitial cystitis., Conclusions: Compared to healthy controls, subjects with interstitial cystitis have limited differences in regional cerebral blood flow in baseline (empty bladder) conditions as well as during heat pain. However, they had robust regional cerebral blood flow increases in the full bladder state in regions of interest typically associated with pain, emotion and/or motor control, indicating altered processing of bladder related sensations., (Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

The Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network is an ongoing multi-center collaborative research group established to conduct integrated studies in participants with urologic chronic pelvic pain syndrome (UCPPS). The goal of these investigations is to provide new insights into the etiology, natural history, clinical, demographic and behavioral characteristics, search for new and evaluate candidate biomarkers, systematically test for contributions of infectious agents to symptoms, and conduct animal studies to understand underlying mechanisms for UCPPS. Study participants were enrolled in a one-year observational study and evaluated through a multisite, collaborative neuroimaging study to evaluate the association between UCPPS and brain structure and function. 3D T1-weighted structural images, resting-state fMRI, and high angular resolution diffusion MRI were acquired in five participating MAPP Network sites using 8 separate MRI hardware and software configurations. We describe the neuroimaging methods and procedures used to scan participants, the challenges encountered in obtaining data from multiple sites with different equipment/software, and our efforts to minimize site-to-site variation.

Studies have suggested chronic pain syndromes are associated with neural reorganization in specific regions associated with perception, processing, and integration of pain. Urological chronic pelvic pain syndrome (UCPPS) represents a collection of pain syndromes characterized by pelvic pain, namely Chronic Prostatitis/Chronic Pelvic Pain Syndrome (CP/CPPS) and Interstitial Cystitis/Painful Bladder Syndrome (IC/PBS), that are both poorly understood in their pathophysiology, and treated ineffectively. We hypothesized patients with UCPPS may have microstructural differences in the brain compared with healthy control subjects (HCs), as well as patients with irritable bowel syndrome (IBS), a common gastrointestinal pain disorder. In the current study we performed population-based voxel-wise DTI and super-resolution track density imaging (TDI) in a large, two-center sample of phenotyped patients from the multicenter cohort with UCPPS (N = 45), IBS (N = 39), and HCs (N = 56) as part of the MAPP Research Network. Compared with HCs, UCPPS patients had lower fractional anisotropy (FA), lower generalized anisotropy (GA), lower track density, and higher mean diffusivity (MD) in brain regions commonly associated with perception and integration of pain information. Results also showed significant differences in specific anatomical regions in UCPPS patients when compared with IBS patients, consistent with microstructural alterations specific to UCPPS. While IBS patients showed clear sex related differences in FA, MD, GA, and track density consistent with previous reports, few such differences were observed in UCPPS patients. Heat maps illustrating the correlation between specific regions of interest and various pain and urinary symptom scores showed clustering of significant associations along the cortico-basal ganglia-thalamic-cortical loop associated with pain integration, modulation, and perception. Together, results suggest patients with UCPPS have extensive microstructural differences within the brain, many specific to syndrome UCPPS versus IBS, that appear to be localized to regions associated with perception and integration of sensory information and pain modulation, and seem to be a consequence of longstanding pain.

Serotonin from the descending pain modulatory pathway is critical to nociceptive processing. Its effects on pain modulation may either be inhibitory or facilitatory, depending on the type of pain and which receptors are involved. Little is known about the role of serotonergic systems in bladder nociceptive processing. These studies examined the effect of systemic administration of the serotonin precursor, 5-hydroxytryptophan (5-HTP), on normal bladder and somatic sensation in rats. ELISA was used to quantify peripheral and central changes in serotonin and its major metabolite following 5-HTP administration, and the potential role of the 5-HT3 receptor on changes in bladder sensation elicited by 5-HTP was investigated. 5-HTP produced bladder hypersensitivity and somatic analgesia. The pro-nociceptive effect of 5-HTP was attenuated by intrathecal, but not systemic, ondansetron. Peripheral increases in serotonin, its metabolism and rate of turnover were detectable within 30min of 5-HTP administration. Significant enhancement of serotonin metabolism was observed centrally. These findings suggest that 5-HTP increases serotonin, which may then affect descending facilitatory systems to produce bladder hypersensitivity via activation of spinal 5-HT3 receptors., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)