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Introduction: The lateral parabrachial nucleus (LPBN), a crucial hub for integrating and modulating diverse sensory information, is known to express both D1 and D2 dopamine receptors and receive dopaminergic inputs. However, the role of the LPBN's dopaminergic system in somatosensory processing remains largely unexplored. In this study, we investigated whether mechanical sensory stimulation triggers dopamine release in the LPBN and how D1- and D2-like receptor signaling in the LPBN influences mechanosensitivity in mice., Methods: We used a G-protein-coupled receptor-based dopamine sensor to monitor dopamine release in the LPBN and a von Frey filament assay to measure the mechanical threshold for nocifensive withdrawal in mouse hind paws after unilateral microinjection of D1- or D2-like receptor antagonist into the LPBN., Results: Noxious mechanical stimulation increased the dopamine sensor signal in the LPBN. Thresholds of nocifensive withdrawal from mechanical stimulation were decreased by the D1-like receptor antagonist SCH-23390 (0.1 µg) but increased by the D2-like receptor antagonist eticlopride (1 µg). In the intraplantar capsaicin injection model that develops mechanical hypersensitivity in the injected paw, the dopamine sensor signal in the LPBN was increased, and eticlopride (1 µg) in the LPBN significantly inhibited the capsaicin-induced mechanical hypersensitivity., Conclusions: These results suggest that endogenous dopaminergic signaling occurs in the LPBN upon noxious mechanical stimulation, inhibiting mechanosensitivity through D1-like receptors while enhancing it through D2-like receptors. D2-like receptor signaling in the LPBN may contribute to an injury-induced increase in mechanical nociception, indicating that inhibiting the receptor within the LPBN could offer potential as a novel analgesic strategy., Competing Interests: The authors have no conflict of interest to declare.Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The International Association for the Study of Pain.)

Chronic abdominal pain in the absence of ongoing disease is the hallmark of disorders of gut-brain interaction (DGBIs), including irritable bowel syndrome (IBS). While the etiology of DGBIs remains poorly understood, there is evidence that both genetic and environmental factors play a role. In this study, we report the identification and validation of arginine-vasopressin receptor 1A (Avpr1a) as a novel candidate gene for visceral hypersensitivity (VH), a primary peripheral mechanism underlying abdominal pain in DGBI/IBS. Comparing 2 C57BL/6 (BL/6) substrains (C57BL/6NTac and C57BL/6J) revealed differential susceptibility to the development of chronic VH following intrarectal zymosan instillation, a validated preclinical model for postinflammatory IBS. Using whole-genome sequencing, we identified a single-nucleotide polymorphism differentiating the 2 strains in the 5' intergenic region upstream of Avpr1a, encoding the protein Avpr1a. We used behavioral, histological, and molecular approaches to identify distal colon-specific gene expression and neuronal hyperresponsiveness covarying with Avpr1a genotype and VH susceptibility. While the 2 BL/6 substrains did not differ across other gastrointestinal phenotypes (eg, fecal water retention), VH-susceptible BL/6NTac mice had higher colonic Avpr1a mRNA and protein expression. These results parallel findings that patients' colonic Avpr1a mRNA expression corresponded to higher pain ratings. Moreover, neurons of the enteric nervous system were hyperresponsive to the Avpr1a agonist arginine-vasopressin, suggesting a role for enteric neurons in the pathology underlying VH. Taken together, these findings implicate differential regulation of Avpr1a as a novel mechanism of VH susceptibility as well as a potential therapeutic target specific to VH. PERSPECTIVE: This article presents evidence of Avpr1a as a novel candidate gene for VH in a mouse model of IBS. Avpr1a genotype and/or tissue-specific expression represents a potential biomarker for chronic abdominal pain susceptibility., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

G protein-coupled receptors (GPCRs) are successful druggable targets, making up around 35% of all FDA-approved medications. However, a large number of receptors remain orphaned, with no known endogenous ligand, representing a challenging but untapped area to discover new therapeutic targets. Among orphan GPCRs (oGPCRs) of interest, G protein-coupled receptor 37 (GPR37) is highly expressed in the central nervous system (CNS), particularly in the spinal cord and oligodendrocytes. While its cellular signaling mechanisms and endogenous receptor ligands remain elusive, GPR37 has been implicated in several important neurological conditions, including Parkinson's disease (PD), inflammation, pain, autism, and brain tumors. GPR37 structure, signaling, emerging physiology, and pharmacology are reviewed while integrating a discussion on potential therapeutic indications and opportunities.

Study Design: Double-blinded, prospective laboratory animal study., Objective: To examine whether intraoperative spinal cord stimulation (SCS) inhibits the development of spine surgery-induced hypersensitivity., Summary of Background Data: Managing postoperative pain after spine surgery is challenging, and as many as 40% of patients may develop failed back surgery syndrome. Although SCS has been shown to effectively reduce chronic pain symptoms, it is unknown whether intraoperative SCS can mitigate the development of central sensitization that causes postoperative pain hypersensitivity and potentially leads to failed back surgery syndrome after spine surgery., Materials and Methods: Mice were randomly stratified into three experimental groups: (1) sham surgery, (2) laminectomy alone, and (3) laminectomy plus SCS. Secondary mechanical hypersensitivity was measured in hind paws using von Frey assay one day before and at predetermined times after surgery. In addition, we also performed a conflict avoidance test to capture the affective-motivational domain of pain at selected time points postlaminectomy., Results: Mice that underwent unilateral T13 laminectomy developed mechanical hypersensitivity in both hind paws. Intraoperative SCS applied to the exposed side of the dorsal spinal cord significantly inhibited the development of hind paw mechanical hypersensitivity on the SCS-applied side. Sham surgery did not produce any obvious secondary mechanical hypersensitivity in the hind paws., Conclusions: These results demonstrate that spine surgery for unilateral laminectomy induces central sensitization that results in postoperative pain hypersensitivity. Intraoperative SCS after laminectomy may be able to mitigate the development of this hypersensitivity in appropriately selected cases., Competing Interests: The authors report no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Abstract: Nociplastic pain conditions develop predominantly in women. We recently established a murine nociplastic pain model by applying postinjury thermal (40°C) stimulation to an injured (capsaicin-injected) area, triggering a transition to a nociplastic pain state manifesting as persistent mechanical hypersensitivity outside of the previously injured area. The nociplastic pain state was centrally maintained by spinal microglia in males but peripherally by ongoing afferent activity at the previously injured area in females. Here, we investigated whether gonadal hormones are critical for the development of this peripherally maintained nociplastic pain state in females. Although the transition to a nociplastic pain state still occurred in ovariectomized females, the pain state was maintained neither by ongoing afferent activity at the previously injured area nor by spinal microglia. Estradiol reconstitution a week before the injury plus postinjury stimulation, but not after the transition had already occurred, restored the development of peripherally maintained nociplastic mechanical hypersensitivity in ovariectomized females. G protein-coupled estrogen receptor antagonism during the transition phase mimicked ovariectomy in gonad-intact females, whereas the receptor antagonism after the transition gradually alleviated the nociplastic mechanical hypersensitivity. At the previously injured area, afferents responsive to allyl isothiocyanate (AITC), a TRPA1 agonist, contributed to the maintenance of nociplastic mechanical hypersensitivity in gonad-intact females. In ex vivo skin-nerve preparations, only AITC-responsive afferents from the nociplastic pain model in gonad-intact females showed ongoing activities greater than control. These results suggest that gonadal hormones are critical for peripherally maintained nociplastic pain state in females by sensitizing AITC-responsive afferents to be persistently active., (Copyright © 2022 International Association for the Study of Pain.)

Previously we developed a murine model in which postinjury stimulation of an injured area triggers a transition to a nociplastic pain state manifesting as persistent mechanical hypersensitivity outside of the previously injured area. This hypersensitivity was maintained by sex-specific mechanisms; specifically, activated spinal microglia maintained the hypersensitivity only in males. Here we investigated whether spinal microglia drive the transition from acute injury-induced pain to nociplastic pain in males, and if so, how they are activated by normally innocuous stimulation after peripheral injury. Using intraplantar capsaicin injection as an acute peripheral injury and vibration of the injured paw as postinjury stimulation, we found that inhibition of spinal microglia prevents the vibration-induced transition to a nociplastic pain state. The transition was mediated by the ATP-P2X4 pathway, but not BDNF-TrkB signaling. Intrathecally injected GABA receptor agonists after intraplantar capsaicin injection prevented the vibration-induced transition to a nociplastic pain state. Conversely, in the absence of intraplantar capsaicin injection, intrathecally injected GABA receptor antagonists allowed the vibration stimulation of a normal paw to trigger the transition to a spinal microglia-mediated nociplastic pain state only in males. At the spinal level, TNF-α, IL-1β, and IL-6, but not prostaglandins, contributed to the maintenance of the nociplastic pain state in males. These results demonstrate that in males, the transition from acute injury-induced pain to nociplastic pain is driven by spinal microglia causing neuroinflammation and that peripheral injury-induced spinal GABAergic disinhibition is pivotal for normally innocuous stimulation to activate spinal microglia., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Although methadone is effective in the management of acute pain, the complexity of its absorption-distribution-metabolism-excretion profile limits its use as an opioid of choice for perioperative analgesia. Because deuteration is known to improve the pharmacokinetic, pharmacodynamic and toxicological properties of some drugs, here we characterized the single dose pharmacokinetic properties and post-operative analgesic efficacy of d 9 -methadone. The pharmacokinetic profiles of d 9 -methadone and methadone administered intravenously to CD-1 male mice revealed that deuteration leads to a 5.7- and 4.4-fold increase in the area under the time-concentration curve and maximum concentration in plasma, respectively, as well as reduction in clearance (0.9 ± 0.3 L/h/kg vs 4.7 ± 0.8 L/h/kg). The lower brain-to-plasma ratio of d 9 -methadone compared to that of methadone (0.35 ± 0.12 vs 2.05 ± 0.62) suggested that deuteration decreases the transfer of the drug across the blood-brain barrier. The estimated LD 50 value for a single intravenous dose of d 9 -methadone was 2.1-fold higher than that for methadone. Moreover, d 9 -methadone outperformed methadone in the efficacy against postoperative pain by primarily activating peripheral opioid receptors. Collectively, these data suggest that the replacement of three hydrogen atoms in three methyl groups of methadone altered its pharmacokinetic properties, improved safety, and enhanced its analgesic efficacy., 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 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Abstract: Acute injury-induced pain can transition to chronic nociplastic pain, which predominantly affects women. To facilitate studies on the underlying mechanisms of nociplastic pain, we developed a mouse model in which postinjury thermal stimulation (intermittent 40°C water immersion for 10 minutes at 2 hours postcapsaicin) prolongs capsaicin (ie, experimental injury)-induced transient mechanical hypersensitivity outside of the injury area. Although capsaicin injection alone induced mechanical and thermal hypersensitivity that resolved in ∼7 days (slower recovery in females), the postinjury stimulation prolonged capsaicin-induced mechanical, but not thermal, hypersensitivity up to 3 weeks in both sexes. When postinjury stimulation was given at a lower intensity (30°C) or at later time points (40°C at 1-3 days postcapsaicin), chronification of mechanical hypersensitivity occurred only in females. Similar chronification could be induced by a different postinjury stimulation modality (vibration of paw) or with a different injury model (plantar incision). Notably, the 40°C postinjury stimulation did not prolong capsaicin-induced inflammation in the hind paw, indicating that the prolonged mechanical hypersensitivity in these mice arises without clear evidence of ongoing injury, reflecting nociplastic pain. Although morphine and gabapentin effectively alleviated this persistent mechanical hypersensitivity in both sexes, sexually dimorphic mechanisms mediated the hypersensitivity. Specifically, ongoing afferent activity at the previously capsaicin-injected area was critical in females, whereas activated spinal microglia were crucial in males. These results demonstrate that postinjury stimulation of the injured area can trigger the transition from transient pain to nociplastic pain more readily in females, and sex-dependent mechanisms maintain the nociplastic pain state., (Copyright © 2021 International Association for the Study of Pain.)

Opioids are widely used for pain relief; however, chronic opioid use causes a paradoxical state of enhanced pain sensitivity, termed "Opioid-induced hyperalgesia (OIH)." Despite the clinical importance of OIH, the detailed mechanism by which it enhances pain sensitivity remains unclear. In this study, we tested whether repeated morphine induces a neuronal circuit polarization in the mouse spinal dorsal horn (SDH). Transgenic mice expressing GFP to neurokinin 1 receptor-expressing neurons (sNK1Rn) and GABAergic interneurons (sGABAn) that received morphine [20 mg/kg, once daily for four consecutive days (i.p.)] developed mechanical hypersensitivity. Repeated morphine altered synaptic strengths in the SDH as a specific cell-type but not in a gender-dependent manner. In sNK1Rn and non-tonic firing neurons, repeated morphine treatment significantly increased frequency of spontaneous excitatory postsynaptic current (sEPSC) and evoked EPSC (eEPSC). In addition, repeated morphine treatment significantly decreased evoked inhibitory postsynaptic current (eIPSC) in sNK1Rn. Conversely, in sGABAn and tonic firing neurons, repeated morphine treatment significantly decreased sEPSC frequency and eEPSC, but had no change of eIPSC in sGABAn. Interestingly, repeated morphine treatment significantly decreased neuronal rheobase of sNK1Rn but had no effect on sGABAn. These findings suggest that spinal neuronal circuit polarization maybe the mechanism of OIH and identify a potential therapeutic mechanism to prevent or treat opioid-induced pain., 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 Kearns, Jayasi, Liu, Wang, Shi, Chung, La, Tang and Bae.)

Since 1967, spinal cord stimulation (SCS) has been used to manage chronic intractable pain of the trunk and limbs. Compared to traditional high-intensity, low-frequency (<100 Hz) SCS that is thought to produce paresthesia and pain relief by stimulating large myelinated fibers in the dorsal column (DC), low-intensity, high-frequency (10 kHz) SCS has demonstrated long-term pain relief without generation of paresthesia. To understand this paresthesia-free analgesic mechanism of 10 kHz SCS, we examined whether 10 kHz SCS at intensities below sensory thresholds would modulate spinal dorsal horn (DH) neuronal function in a neuron type-dependent manner. By using in vivo and ex vivo electrophysiological approaches, we found that low-intensity (sub-sensory threshold) 10 kHz SCS, but not 1 kHz or 5 kHz SCS, selectively activates inhibitory interneurons in the spinal DH. This study suggests that low-intensity 10 kHz SCS may inhibit pain sensory processing in the spinal DH by activating inhibitory interneurons without activating DC fibers, resulting in paresthesia-free pain relief., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

An important goal of the Zika virus (ZIKV) vaccine is to prevent a congenital syndrome in fetuses of pregnant women, but studies directly evaluating maternal vaccination for ZIKV are lacking. Here we report maternal vaccination using a live-attenuated ZIKV vaccine (3'UTR-∆10-LAV) in a pregnant mouse model. Maternal immunization with 3'UTR-∆10-LAV does not cause any adverse effects on pregnancy, fetal development, or offspring behavior. One maternal immunization fully protects dams against ZIKV infection and in utero transmission. Although neutralizing antibody alone is sufficient to prevent in utero transmission, a higher neutralizing titer is required to protect pregnant mice against in utero transmission than that required to protect non-pregnant mice against viral infection. The immunized dams transfer maternal antibodies to pups, which protect neonates against ZIKV infection. Notably, pregnancy weakens maternal T cell response to 3'UTR-∆10-LAV vaccination. Our results suggest that, besides vaccinating non-pregnant individuals, 3'UTR-∆10-LAV may also be considered for maternal vaccination.

Using a high resolution in situ hybridization technique we have measured PIEZO1 , PIEZO2 , and TRPV1 transcripts in mouse dorsal root ganglion (DRG) neurons. Consistent with previous studies, PIEZO2 transcripts were highly expressed in DRG neurons of all sizes, including most notably the largest diameter neurons implicated in mediating touch and proprioception. In contrast, PIEZO1 transcripts were selectively expressed in smaller DRG neurons, which are implicated in mediating nociception. Moreover, the small neurons expressing PIEZO1 were mostly distinct from those neurons that strongly expressed TRPV1 , one of the channels implicated in heat-nociception. Interestingly, while PIEZO1- and TRPV1- expressing neurons form essentially non-overlapping populations, PIEZO2 showed co-expression in both populations. Using an in vivo functional test for the selective expression, we found that Yoda1, a PIEZO1-specific agonist, induced a mechanical hyperalgesia that displayed a significantly prolonged time course compared with that induced by capsaicin, a TRPV1-specific agonist. Taken together, our results indicate that PIEZO1 should be considered a potential candidate in forming the long sought channel mediating mechano-nociception.

Osteoarthritis (OA) is the most common cause of pain in people aged > 45 years, and the knee is the most commonly affected joint. There is a growing interest in understanding the biological factors that influence pain among older adults, but few studies have examined the relationship between β-endorphin and experimental pain sensitivity in older adults with knee OA pain. The purpose of this study was to investigate the relationship between resting plasma levels of β-endorphin and experimental pain sensitivity. This study was a secondary analysis of data for 40 adults with knee OA pain in whom quantitative sensory testing was used to measure experimental sensitivity to heat- and mechanically induced pain. The mean age of the sample was 60 years ( SD = 9 years), and approximately half were female (53%). Regression analyses indicated that β-endorphin level was negatively related to pressure pain threshold (β = -17.18, p = .02) and positively related to punctate mechanical pain (β = 17.13, p = .04), after controlling for age, gender, and OA severity. We did not find a significant relationship between β-endorphin and heat pain tolerance. The results suggest that higher circulating levels of β-endorphin at rest are associated with increased sensitivity to mechanical pain in older adults with knee OA. These findings add to the literature regarding biological factors associated with pain sensitivity in older adults with chronic pain. Additional studies are needed to identify mediators of the relationship between β-endorphin and pain sensitivity in OA and other musculoskeletal pain conditions.

Scutellaria baicalensis ( S. baicalensis ) has been used to manage diarrhea, and its anti-inflammatory effects are responsible for anti-diarrheal effects. However, there are no data concerning its direct effect on colonic motility. Therefore, the effects of the major components of S. baicalensis (baicalin, baicalein and wogonin) on colonic motility were investigated. A segment of the distal colon of rats was placed in Krebs solution to monitor spontaneous giant contractions (GCs). Changes in GCs were recorded after applying baicalin, baicalein or wogonin. After pretreatment with N ω -nitro-L-arginine methyl ester hydrochloride (L-NAME), 1H-(1,2,4)-oxadiazolo (4,2-a) quinoxalin-1-one (ODQ), tetradotoxin, w-conotoxin, apamin, and iberiotoxin, changes in GCs by wogonin were recorded and analyzed. The segment of the distal colon showed spontaneous GCs at a mean amplitude of 3.7±0.3 g with a frequency of 0.8±0.1/min. Baicalin, baicalein, and wogonin reduced both the amplitude and the frequency of GCs in a dose-dependent manner. Wogonin had the most potent inhibitory effect on GCs (IC 50 was 14.6 µM in amplitude and 14.2 µM in frequency). Wogonin-induced GC reduction was not significantly affected by the inhibition of nitric oxide/cGMP pathways with L-NAME and ODQ. Blocking the enteric neurotransmission with tetradotoxin and ω-conotoxin was ineffective on the wogonin-induced reduction of GCs. Ca 2+ -activated K + (K Ca ) channel blockers (apamin and iberiotoxin) significantly attenuated the inhibitory effects of wogonin on GCs (P<0.01). Wogonin was effective in inhibiting colonic motility, probably through the opening of K Ca channels located in the smooth muscle apparatus. These findings suggest that wogonin may be a candidate drug for the management of dysmotility-related diarrhea.

Chemotherapy-induced peripheral neuropathy (CIPN) is an adverse side effect of many anti-cancer chemotherapeutic treatments. CIPN often causes neuropathic pain in extremities, and oxidative stress has been shown to be a major contributing factor to this pain. In this study, we determined the site of oxidative stress associated with pain (specifically, mechanical hypersensitivity) in cisplatin- and paclitaxel-treated mouse models of CIPN and investigated the neurophysiological mechanisms accounting for the pain. C57BL/6N mice that received either cisplatin or paclitaxel (2 mg/kg, once daily on four alternate days) developed mechanical hypersensitivity to von Frey filament stimulations of their hindpaws. Cisplatin-induced mechanical hypersensitivity was inhibited by silencing of Transient Receptor Potential channels V1 (TRPV1)- or TRPA1-expressing afferents, whereas paclitaxel-induced mechanical hypersensitivity was attenuated by silencing of Aβ fibers. Although systemic delivery of phenyl N-tert-butylnitrone, a reactive oxygen species scavenger, alleviated mechanical hypersensitivity in both cisplatin- and paclitaxel-treated mice, intraplantar phenyl N-tert-butylnitrone was effective only in cisplatin-treated mice, and intrathecal phenyl N-tert-butylnitrone, only in paclitaxel-treated mice. In a reactive oxygen species-dependent manner, the mechanosensitivity of Aδ/C fiber endings in the hindpaw skin was increased in cisplatin-treated mice, and the excitatory synaptic strength in the spinal dorsal horn was potentiated in paclitaxel-treated mice. Collectively, these results suggest that cisplatin-induced mechanical hypersensitivity is attributed to peripheral oxidative stress sensitizing mechanical nociceptors, whereas paclitaxel-induced mechanical hypersensitivity is due to central (spinal) oxidative stress maintaining central sensitization that abnormally produces pain in response to Aβ fiber inputs.

In this work, a switched-capacitor-based stimulator circuit that enables efficient energy harvesting for neurostimulation applications is presented, followed by the discussion on the optimization of the inductive coupling front-end through a codesign approach. The stimulator salvages input energy and stores it in a storage capacitor, and, when the voltage reaches a threshold, releases the energy as an output stimulus. The dynamics of the circuit are automatically enabled by a positive feedback, eliminating any stimulation control circuit blocks. The IC is fabricated in a 180 nm CMOS process and achieves a quiescent current consumption of 1.8 μA. The inductive coupling front-end is optimized as a loaded resonator, in which the input impedance of the custom rectifier directly loads the inductive loop antenna. The loaded quality factor and the rectifier's efficiency determine the reception sensitivity of the stimulator, while a balance should be achieved for the robustness of the system against dielectric medium variations by increasing the reception bandwidth. The finalized stimulator adopts a 4.9 mm × 4.9 mm inductive loop antenna and achieves an overall assembly dimension of 5 mm × 7.5 mm. Operating at the resonant frequency of 198 MHz, the stimulator works at a 14 cm distance from the transmitter in the air. An animal experiment was performed, in which a fully implanted stimulator excited the sciatic nerve of a rat that consequently triggered the movement of the limb.

Reactive oxygen species has been suggested as a key player in neuropathic pain, causing central sensitization by changing synaptic strengths in spinal dorsal horn neurons. However, it remains unclear as to what type of reactive oxygen species changes what aspect of synaptic strengths for central sensitization in neuropathic pain conditions. In this study, we investigated whether mitochondrial superoxide affects both excitatory and inhibitory synaptic strengths in spinal dorsal horn neurons after peripheral nerve injury. Upregulation of mitochondrial superoxide level by knockout of superoxide dismutase-2 exacerbated neuropathic mechanical hypersensitivity caused by L5 spinal nerve ligation, whereas downregulation of mitochondrial superoxide level by overexpression of superoxide dismutase-2 alleviated the hypersensitivity. In spinal nerve ligation condition, the frequency of miniature excitatory postsynaptic currents increased, while that of miniature inhibitory postsynaptic currents decreased in spinal dorsal horn neurons. Superoxide dismutase-2-knockout augmented, whereas superoxide dismutase-2-overexpression prevented, the spinal nerve ligation-increased miniature excitatory postsynaptic currents frequency. However, superoxide dismutase-2-knockout had no effect on the spinal nerve ligation-decreased miniature inhibitory postsynaptic current frequency, and superoxide dismutase-2-overexpression unexpectedly decreased miniature inhibitory postsynaptic current frequency in the normal condition. When applied to the spinal cord slice during in vitro recordings, mitoTEMPO, a specific scavenger of mitochondrial superoxide, reduced the spinal nerve ligation-increased miniature excitatory postsynaptic currents frequency but failed to normalize the spinal nerve ligation-decreased miniature inhibitory postsynaptic current frequency. These results suggest that in spinal dorsal horn neurons, high levels of mitochondrial superoxide increase excitatory synaptic strength after peripheral nerve injury and contribute to neuropathic mechanical hypersensitivity. However, mitochondrial superoxide does not seem to be involved in the decreased inhibitory synaptic strength in this neuropathic pain condition.

Spinal synaptic plasticity is believed to drive central sensitization that underlies the persistent nature of neuropathic pain. Our recent data showed that synaptic plasticity in the dorsal horn is cell type specific: intense afferent stimulation produced long-term potentiation (LTP) in excitatory spinothalamic tract neurons (STTn), whereas it produced long-term depression (LTD) in inhibitory GABAergic interneurons (GABAn). In addition, reactive oxygen species (ROS) were shown to be involved in LTP in STTn (STTn-LTP) and in LTD in GABAn (GABAn-LTD). This study examined the roles of 2 biologically important ROS--superoxide [·O2] and hydroxyl radicals [·OH]--in neuropathic mechanical hyperalgesia and cell type-specific spinal synaptic plasticity. The [·O2] donor induced stronger mechanical hyperalgesia than the [·OH] donor in naive mice. The [·O2] scavenger showed greater antihyperalgesic effect than [·OH] scavengers in the spinal nerve ligation (SNL) mouse model of neuropathic pain. In addition, the [·O2] donor induced both STTn-LTP and GABAn-LTD, but the [·OH] donor induced only GABAn-LTD. On the other hand, the [·O2] scavenger inhibited STTn-LTP and GABAn-LTD induction in naive mice and alleviated SNL-induced potentiation in STTn and depression in GABAn. The [·OH] scavenger, however, inhibited depression in GABAn but did not interfere with potentiation in STTn. These results indicate that mechanical hyperalgesia in SNL mice is the result of the combination of STTn-LTP and GABAn-LTD. Behavioral outcomes compliment electrophysiological results which suggest that [·O2] mediates both STTn-LTP and GABAn-LTD, whereas [·OH] is involved primarily in GABAn-LTD.

Aims: Previous studies have shown that the activin-binding protein follistatin reduces inflammation in several mouse models of colitis. To determine whether follistatin also has a beneficial effect following bladder inflammation, we induced cystitis in mice using cyclophosphamide (CYP) and examined the relationship between bladder hypersensitivity and bladder follistatin expression., Methods: Adult female C57BL/6 mice were treated with CYP (100 mg/kg) or vehicle (saline) three times over 5 days. Bladder hypersensitivity was assessed by recording the visceromotor response (VMR) to urinary bladder distension and in vitro single-fiber bladder afferent recording. Follistatin gene expression was measured using qRT-PCR. Immunohistochemistry was employed for further characterization., Results: Bladder hypersensitivity was established by day 6 and persisted to day 14 in CYP-treated mice. On day 14, hypersensitivity was accompanied by increases in follistatin gene expression in the bladder. Follistatin-like immunoreactivity colocalized with laminin, and the percentage of structures in the lamina propria that were follistatin-positive was increased in CYP-treated mice. Exogenous follistatin increased VMR and afferent responses to bladder distension in CYP- but not vehicle-treated mice., Conclusions: Chronic bladder pain following CYP treatment is associated with increased follistatin expression in the bladder. These results suggest a novel, pro-nociceptive role for follistatin in cystitis, in contrast with its proposed therapeutic role in colitis. This protein has exciting potential as a biomarker and therapeutic target for bladder hypersensitivity. Neurourol. Urodynam. 36:286-292, 2017. © 2015 Wiley Periodicals, Inc., (© 2015 Wiley Periodicals, Inc.)

Abstract: Intradermally injected capsaicin induces secondary mechanical hyperalgesia and allodynia outside the primary (i.e., capsaicininjected) site. This secondary mechanical hypersensitivity is attributed to central sensitization in which reactive oxygen species (ROS) play a key role. We examined whether ROS would be differentially involved in secondary mechanical hyperalgesia and allodynia using a mouse intraplantar capsaicin injection model. In mice, capsaicin-induced secondary mechanical hyperalgesia outlasted its allodynia counterpart. Unlike the hyperalgesia, the allodynia was temporarily abolished by an anesthetic given at the capsaicin-injected site. The ROS scavenger phenyl-N-tert-butylnitrone slowed the development of both secondary mechanical hyperalgesia and allodynia when administered before intraplantar capsaicin injection, whereas it inhibited only the allodynia when administered after capsaicin had already induced secondary mechanical hyperalgesia and allodynia. Intrathecal injection of the ROS donor KO2 induced both mechanical hyperalgesia and allodynia with the former outlasting the latter. Metformin, an activator of redox-sensitive adenosine monophosphate-activated protein kinase, selectively inhibited capsaicin-induced secondary mechanical allodynia and intrathecal KO2-induced mechanical allodynia. These results suggest that ROS is required for rapid activation of central sensitization mechanisms for both secondary mechanical hyperalgesia and allodynia after intraplantar capsaicin injection. Once activated, the mechanism for the hyperalgesia is longlasting without being critically dependent on ongoing afferent activities arising from the capsaicin-injected site and the continuous presence of ROS. On the contrary, the ongoing afferent activities, ROS presence and adenosine monophosphate-activated protein kinase inhibition are indispensable for the maintenance mechanism for capsaicin-induced secondary mechanical allodynia.

Purpose: Chronic prostatitis/chronic pelvic pain syndrome causes symptoms that include the frequent and urgent need to urinate, pain or burning during urination and pain radiating to the back, abdomen and/or colorectum. These bladder symptoms suggest that chronic prostatitis/chronic pelvic pain syndrome is associated with sensitization of adjacent organs, termed cross-organ sensitization. The objective of this study was to determine the extent of 1) changes in immunomodulatory mediators in the prostate and bladder after inflammation of the prostate and 2) bladder function and bladder afferent sensitization., Materials and Methods: Prostate and bladder histology, immunohistochemistry and expression of immunomodulatory targets were examined weekly after zymosan or vehicle was injected in the dorsal lobe of the mouse prostate. Cystometry, bladder and bladder afferent sensitivity were also assessed weekly., Results: Prostate inflammation induced significant up-regulation in proinflammatory and anti-inflammatory cytokines TNF-α (tumor necrosis factor-α) and IL-10 (interleukin-10), growth factor NGF (nerve growth factor), and T-lymphocyte markers FoxP3, CD4 and CD8 in the prostate and the bladder. Notably, prostatitis significantly increased urinary voiding frequency, induced hypersensitivity to bladder distension and sensitized bladder afferents. We also examined sensory (afferent) co-innervation by injecting retrograde tracers DiI and Fast Blue in the bladder wall and the prostate, respectively. This showed that a significant proportion (approximately 17%) of dorsal root ganglion afferent somata contained tracers from the bladder and the prostate., Conclusions: These observations support an afferent contribution to chronic prostatitis/chronic pelvic pain syndrome and cross-organ sensitization from prostate to bladder., (Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Isolectin B4-binding (IB4+) dorsal root ganglion (DRG) neurons are distinct from peptidergic DRG neurons in their terminal location in the spinal cord and respective contributions to various classes and modalities of nociception. In DRG neurons innervating the mouse colon (c-DRG neurons), the reported proportion of IB4+ population is inconsistent across studies, and little is known regarding their role in colorectal mechanonociception. To address these issues, in C57BL/6J mice, we quantified IB4+ binding after labeling c-DRG neurons with Fast Blue and examined functional consequences of ablating these neurons by IB4-conjugated saporin. Sixty-one percent of Fast Blue-labeled neurons in the L6 DRG were IB4+, and 95% of these IB4+ c-DRG neurons were peptidergic. Intrathecal administration of IB4-conjugated saporin reduced the proportion of IB4+ c-DRG neurons to 37%, which was due to the loss of c-DRG neurons showing strong to medium IB4+ intensity; c-DRG neurons with weak IB4+ intensity were spared. However, this loss altered neither nociceptive behaviors to colorectal distension nor the relative proportions of stretch-sensitive colorectal afferent classes characterized by single-fiber recordings. These findings demonstrate that more than 1 half of viscerosensory L6 c-DRG neurons in C57BL/6J mouse are IB4+ and suggest, in contrast to the reported roles of IB4+/nonpeptidergic neurons in cutaneous mechanonociception, c-DRG neurons with strong-to-medium IB4+ intensity do not play a significant role in colorectal mechanonociception.

Background/aims: Abnormal visceral sensitivity and disordered motility are common in patients with diabetes mellitus. The purpose of the present study was to investigate whether visceral sensation and bowel motility were altered in a rat model of type 2 diabetes mellitus accompanied by weight loss., Methods: A type 2 diabetic rat model in adulthood was developed by administrating streptozotocin (STZ; 90 mg/kg, i.p.) to neonatal rats. Eight weeks after STZ administration, rats with blood glucose level of 200 mg/dL or higher were selected and used as diabetic group (n = 35) in this study. Abdominal withdrawal reflex and arterial pulse rate were measured to examine visceral nociception induced by colorectal distension (0.1-1.0 mL). The amplitude, frequency, and area under the curve (AUC) of spontaneous phasic contractions of colonic circular muscles were recorded in vitro to examine colonic motility., Results: STZ-treated diabetic rats gained significantly less weight for 8 weeks than control (P < 0.01). Forty-eight percent of the diabetic rats showed enhanced visceral nociceptive response to colorectal distension. Diabetic rats did not differ from control rats in colorectal compliance. However, the frequency and AUC, not the amplitude, of colonic spontaneous contraction in vitro was significantly decreased in diabetic rats compared to control rats (P < 0.01 in frequency and P < 0.05 in AUC)., Conclusions: These results demonstrate visceral hypersensitivity and colonic dysmotility in a rat model of type 2 diabetes mellitus accompanied by weight loss.

Chronic nonbacterial prostatitis, characterized by genitourinary pain in the pelvic region in the absence of an identifiable cause, is common in adult males. Surprisingly, the sensory innervation of the prostate and mediators that sensitize its innervation have received little attention. We thus characterized a mouse model of chronic prostatitis, focusing on the prostate innervation and how organ inflammation affects gene expression of putative nociceptive markers in prostate afferent somata in dorsal root ganglia (DRG) and mediators in the prostate. Retrograde tracing (fast blue) from the prostate revealed that thoracolumbar and lumbosacral DRG are the principal sources of somata of prostate afferents. Nociceptive markers (eg, transient receptor potential, TREK, and P2X channels) were upregulated in fast blue-labeled thoracolumbar and lumbosacral somata for up to four weeks after inflaming the prostate (intraprostate injection of zymosan). Prostatic inflammation was evident histologically, by monocyte infiltration and a significant increase in mast cell tryptase activity 14, 21, and 28 days after zymosan injection. Interleukin 10 and NGF were also significantly upregulated in the prostate throughout the 4 weeks of inflammation. Open-field pain-related behaviors (eg, rearing) were unchanged in prostate-inflamed mice, suggesting the absence of ongoing nociception, but withdrawal thresholds to lower abdominal pressure were significantly reduced. The increases in IL-10, mast cell tryptase, and NGF in the inflamed prostate were cotemporaneous with reduced thresholds to probing of the abdomen and upregulation of nociceptive markers in DRG somata innervating the prostate. The results provide insight and direction for the study of mechanisms underlying pain in chronic prostatitis., Competing Interests: There were no financial or other relationships that led to a conflict of interest.

Stretch-sensitive afferents comprise ∼33% of the pelvic nerve innervation of mouse colorectum, which are activated by colorectal distension and encode visceral nociception. Stretch-sensitive colorectal afferent endings respond tonically to stepped or ramped colorectal stretch, whereas dissociated colorectal dorsal root ganglion neurons generally fail to spike repetitively upon stepped current stimulation. The present study investigated this difference in the neural encoding characteristics between the soma and afferent ending using pharmacological approaches in an in vitro mouse colon-nerve preparation and complementary computational simulations. Immunohistological staining and Western blots revealed the presence of voltage-gated sodium channel (NaV) 1.6 and NaV1.7 at sensory neuronal endings in mouse colorectal tissue. Responses of stretch-sensitive colorectal afferent endings were significantly reduced by targeting NaV1.6 using selective antagonists (μ-conotoxin GIIIa and μ-conotoxin PIIIa) or tetrodotoxin. In contrast, neither selective NaV1.8 (A803467) nor NaV1.7 (ProTX-II) antagonists attenuated afferent responses to stretch. Computational simulation of a colorectal afferent ending that incorporated independent Markov models for NaV1.6 and NaV1.7, respectively, recapitulated the experimental findings, suggesting a necessary role for NaV1.6 in encoding tonic spiking by stretch-sensitive afferents. In addition, computational simulation of a dorsal root ganglion soma showed that, by adding a NaV1.6 conductance, a single-spiking neuron was converted into a tonic spiking one. These results suggest a mechanism/channel to explain the difference in neural encoding characteristics between afferent somata and sensory endings, likely caused by differential expression of ion channels (e.g., NaV1.6) at different parts of the neuron., (Copyright © 2015 the American Physiological Society.)

Background: A low-level inflammation has been hypothesized to mediate visceral hypersensitivity in functional bowel disorders that persist after or even in the absence of gut inflammation. We aimed to test the efficacy of a steroidal anti-inflammatory treatment, and identify local inflammatory molecules mediating post- and non-inflammatory colorectal hypersensitivity using two mouse models., Methods: Visceromotor responses to colorectal distension were quantified as a measure of colorectal sensitivity. On day 1, mice received intracolonic saline (control), trinitrobenzenesulfonic acid (postinflammatory on day 15), or acidified hypertonic saline (non-inflammatory). Colorectal sensitivity before (day 10) and after (day 15) 4-day dexamethasone (Dex) treatment was compared, and colonic gene expression of inflammatory molecules was quantified., Key Results: Dexamethasone effectively inhibited gene expression of inflammatory molecules such as interleukin (IL)-1β and mast cell protease-1 in the colon, but did not attenuate colorectal hypersensitivity in either model. Gene expression of inflammatory molecules in the colon did not differ between control and the non-inflammatory model, but the postinflammatory model showed increased IL-10 and tight junction protein 2, and decreased IL-6, transforming growth factor (TGF)-β, a precursor of β-endorphin, occludin, and mucin 2. While no common molecule explained colorectal hypersensitivity in these models, hypersensitivity was positively correlated with TGF-β2 mRNA in control, and with IL-1β, inhibin βA, and prostaglandin E2 synthase in the Dex-treated postinflammatory model. In the non-inflammatory model, cyclooxygenase-2 mRNA was negatively correlated with colorectal sensitivity., Conclusions & Inferences: These results suggest that persistent functional colorectal hypersensitivity is mediated by condition-specific mediators whose gene expression in the colon is not inevitably sensitive to steroidal anti-inflammatory treatment., (© 2014 John Wiley & Sons Ltd.)

Background: Anterograde in vitro tracing of the pelvic nerve (PN) and visualization in the horizontal plane in whole mount preparations has been fundamental in the analysis of distribution of peripheral nerves innervating the colorectum. Here, we performed a similar analysis, but in cryostat sections of the mouse colorectum, allowing for a more direct visualization of nerve distribution in all tissue layers., Methods: Colorectum with attached PNs was dissected from adult male BalbC mice. Presence of active afferents was certified by single fiber recording of fine PN fibers. This was followed by 'bulk' (all fibers) anterograde tracing using biotinamide (BTA). Histo- and immunohistochemical techniques were used for visualization of BTA-positive nerves, and evaluation of co-localization with calcitonin gene-related peptide (CGRP), respectively. Tissue was analyzed using confocal microscopy on transverse or longitudinal colorectum sections., Key Results: Abundant BTA-positive nerves spanning all layers of the mouse colorectum and contacting myenteric plexus neurons, distributing within the muscle layer, penetrating deeper into the organ and contacting blood vessels, submucosal plexus neurons or even penetrating the mucosa, were regularly detected. Several traced axons co-localized CGRP, supporting their afferent nature. Finally, anterograde tracing of the PN also exposed abundant BTA-positive nerves in the major pelvic ganglion., Conclusions & Inferences: We present the patterns of innervation of extrinsic axons across layers in the mouse colorectum, including the labile mucosal layer. The proposed approach could also be useful in the analysis of associations between morphology and physiology of peripheral nerves targeting the different layers of the colorectum., (© 2014 John Wiley & Sons Ltd.)

Irritable bowel syndrome (IBS) is characterized by altered bowel habits, persistent pain and discomfort, and typically colorectal hypersensitivity. Linaclotide, a peripherally restricted 14 aa peptide approved for the treatment of IBS with constipation, relieves constipation and reduces IBS-associated pain in these patients presumably by activation of guanylate cyclase-C (GC-C), which stimulates production and release of cyclic guanosine monophosphate (cGMP) from intestinal epithelial cells. We investigated whether activation of GC-C by the endogenous agonist uroguanylin or the primary downstream effector of that activation, cGMP, directly modulates responses and sensitization of mechanosensitive colorectal primary afferents. The distal 2 cm of mouse colorectum with attached pelvic nerve was harvested and pinned flat mucosal side up for in vitro single-fiber recordings, and the encoding properties of mechanosensitive afferents (serosal, mucosal, muscular, and muscular-mucosal; M/M) to probing and circumferential stretch studied. Both cGMP (10-300 μM) and uroguanylin (1-1000 nM) applied directly to colorectal receptive endings significantly reduced responses of muscular and M/M afferents to stretch; serosal and mucosal afferents were not affected. Sensitized responses (i.e., increased responses to stretch) of muscular and M/M afferents were reversed by cGMP, returning responses to stretch to control. Blocking the transport of cGMP from colorectal epithelia by probenecid, a mechanism validated by studies in cultured intestinal T84 cells, abolished the inhibitory effect of uroguanylin on M/M afferents. These results suggest that GC-C agonists like linaclotide alleviate colorectal pain and hypersensitivity by dampening stretch-sensitive afferent mechanosensitivity and normalizing afferent sensitization.

Visceral afferents expressing transient receptor potential (TRP) channels TRPV1 and TRPA1 are thought to be required for neurogenic inflammation and development of inflammatory hyperalgesia. Using a mouse model of chronic pancreatitis (CP) produced by repeated episodes (twice weekly) of caerulein-induced AP (AP), we studied the involvement of these TRP channels in pancreatic inflammation and pain-related behaviors. Antagonists of the two TRP channels were administered at different times to block the neurogenic component of AP. Six bouts of AP (over 3 wks) increased pancreatic inflammation and pain-related behaviors, produced fibrosis and sprouting of pancreatic nerve fibers, and increased TRPV1 and TRPA1 gene transcripts and a nociceptive marker, pERK, in pancreas afferent somata. Treatment with TRP antagonists, when initiated before week 3, decreased pancreatic inflammation and pain-related behaviors and also blocked the development of histopathological changes in the pancreas and upregulation of TRPV1, TRPA1, and pERK in pancreatic afferents. Continued treatment with TRP antagonists blocked the development of CP and pain behaviors even when mice were challenged with seven more weeks of twice weekly caerulein. When started after week 3, however, treatment with TRP antagonists was ineffective in blocking the transition from AP to CP and the emergence of pain behaviors. These results suggest: (1) an important role for neurogenic inflammation in pancreatitis and pain-related behaviors, (2) that there is a transition from AP to CP, after which TRP channel antagonism is ineffective, and thus (3) that early intervention with TRP channel antagonists may attenuate the transition to and development of CP effectively.

Previous studies in rat and mouse documented that a subpopulation of dorsal root ganglion (DRG) neurons innervating non-visceral tissues express tyrosine hydroxylase (TH). Here we studied whether or not mouse DRG neurons retrogradely traced with Fast Blue (FB) from colorectum or urinary bladder also express immunohistochemically detectable TH. The lumbar sympathetic chain (LSC) and major pelvic ganglion (MPG) were included in the analysis. Previously characterized antibodies against TH, norepinephrine transporter type 1 (NET-1) and calcitonin gene-related peptide (CGRP) were used. On average, ∼14% of colorectal and ∼17% of urinary bladder DRG neurons expressed TH and spanned virtually all neuronal sizes, although more often in the medium-sized to small ranges. Also, they were more abundant in lumbosacral than thoracolumbar DRGs, and often coexpressed CGRP. We also detected several TH-immunoreactive (IR) colorectal and urinary bladder neurons in the LSC and the MPG, more frequently in the former. No NET-1-IR neurons were detected in DRGs, whereas the majority of FB-labeled, TH-IR neurons in the LSC and MPG coexpressed this marker (as did most other TH-IR neurons not labeled from the target organs). TH-IR nerve fibers were detected in all layers of the colorectum and the urinary bladder, with some also reaching the basal mucosal cells. Most TH-IR fibers in these organs lacked CGRP. Taken together, we show: (1) that a previously undescribed population of colorectal and urinary bladder DRG neurons expresses TH, often CGRP but not NET-1, suggesting the absence of a noradrenergic phenotype; and (2) that TH-IR axons/terminals in the colon or urinary bladder, naturally expected to derive from autonomic sources, could also originate from sensory neurons., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Carbohydrate malabsorption such as in lactose intolerance or enteric infection causes symptoms that include abdominal pain. Because this digestive disorder increases intracolonic osmolarity and acidity by accumulation of undigested carbohydrates and fermented products, we tested whether these two factors (hypertonicity and acidity) would modulate colorectal afferents in association with colorectal nociception and hypersensitivity. In mouse colorectum-pelvic nerve preparations in vitro, afferent activities were monitored after application of acidic hypertonic saline (AHS; pH 6.0, 800 mosM). In other experiments, AHS was instilled intracolonically to mice and behavioral responses to colorectal distension (CRD) measured. Application of AHS in vitro excited 80% of serosal and 42% of mechanically-insensitive colorectal afferents (MIAs), sensitizing a proportion of MIAs to become mechanically sensitive and reversibly inhibiting stretch-sensitive afferents. Acute intracolonic AHS significantly increased expression of the neuronal activation marker pERK in colon sensory neurons and augmented noxious CRD-induced behavioral responses. After three consecutive daily intracolonic AHS treatments, mice were hypersensitive to CRD 4-15 days after the first treatment. In complementary single fiber recordings in vitro, the proportion of serosal class afferents increased at day 4; the proportion of MIAs decreased, and muscular class stretch-sensitive afferents were sensitized at days 11-15 in mice receiving AHS. These results indicate that luminal hypertonicity and acidity, two outcomes of carbohydrate malabsorption, can induce colorectal hypersensitivity to distension by altering the excitability and relative proportions of colorectal afferents, suggesting the potential involvement of these factors in the development of abdominal pain.

Inflammation of the distal bowel is often associated with abdominal pain and hypersensitivity, but whether and which colorectal afferents contribute to the hypersensitivity is unknown. Using a mouse model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis, we investigated colorectal hypersensitivity following intracolonic TNBS and associated changes in colorectum and afferent functions. C57BL/6 mice were treated intracolonically with TNBS or saline. Visceromotor responses to colorectal distension (15-60 mmHg) were recorded over 8 wk in TNBS- and saline-treated (control) mice. In other mice treated with TNBS or saline, colorectal inflammation was assessed by myeloperoxidase assay and immunohistological staining. In vitro single-fiber recordings were conducted on both TNBS and saline-treated mice to assess colorectal afferent function. Mice exhibited significant colorectal hypersensitivity through day 14 after TNBS treatment that resolved by day 28 with no resensitization through day 56. TNBS induced a neutrophil- and macrophage-based colorectal inflammation as well as loss of nerve fibers, all of which resolved by days 14-28. Single-fiber recordings revealed a net increase in afferent drive from stretch-sensitive colorectal afferents at day 14 post-TNBS and reduced proportions of mechanically insensitive afferents (MIAs) at days 14-28. Intracolonic TNBS-induced colorectal inflammation was associated with the development and recovery of hypersensitivity in mice, which correlated with a transient increase and recovery of sensitization of stretch-sensitive colorectal afferents and MIAs. These results indicate that the development and maintenance of colorectal hypersensitivity following inflammation are mediated by peripheral drive from stretch-sensitive colorectal afferents and a potential contribution from MIAs.

Irritable bowel syndrome (IBS) is characterized as functional because a pathobiological cause is not readily apparent. Considerable evidence, however, documents that sensitizing proinflammatory and lipotoxic lipids, mast cells and their products, tryptases, enteroendocrine cells, and mononuclear phagocytes and their receptors are increased in tissues of IBS patients with colorectal hypersensitivity. It is also clear from recordings in animals of the colorectal afferent innervation that afferents exhibit long-term changes in models of persistent colorectal hypersensitivity. Such changes in afferent excitability and responses to mechanical stimuli are consistent with relief of discomfort and pain in IBS patients, including relief of referred abdominal hypersensitivity, upon intra-rectal instillation of local anesthetic. In the aggregate, these experimental outcomes establish the importance of afferent drive in IBS, consistent with a larger literature with respect to other chronic conditions in which pain is a principal complaint (e.g., neuropathic pain, painful bladder syndrome, fibromyalgia). Accordingly, colorectal afferents and the environment in which these receptive endings reside constitute the focus of this review. That environment includes understudied and incompletely understood contributions from immune-competent cells resident in and recruited into the colorectum. We close this review by highlighting deficiencies in existing knowledge and identifying several areas for further investigation, resolution of which we anticipate would significantly advance our understanding of neural and neuro-immune contributions to IBS pain and hypersensitivity.

Afferent input contributes significantly to the pain and colorectal hypersensitivity that characterize irritable bowel syndrome. In the present study, we investigated the contributions of mechanically sensitive and mechanically insensitive afferents (MIAs; or silent afferents) to colorectal hypersensitivity. The visceromotor response to colorectal distension (CRD; 15-60 mmHg) was recorded in mice before and for weeks after intracolonic treatment with zymosan or saline. After CRD tests, the distal colorectum with the pelvic nerve attached was removed for single-fiber electrophysiological recordings. Colorectal afferent endings were located by electrical stimulation and characterized as mechanosensitive or not by blunt probing, mucosal stroking, and circumferential stretch. Intracolonic zymosan produced persistent colorectal hypersensitivity (>24 days) associated with brief colorectal inflammation. Pelvic nerve muscular-mucosal but not muscular mechanosensitive afferents recorded from mice with colorectal hypersensitivity exhibited persistent sensitization. In addition, the proportion of MIAs (relative to control) was significantly reduced from 27% to 13%, whereas the proportion of serosal afferents was significantly increased from 34% to 53%, suggesting that MIAs acquired mechanosensitivity. PGP9.5 immunostaining revealed no significant loss of colorectal nerve fiber density, suggesting that the reduction in MIAs is not due to peripheral fiber loss after intracolonic zymosan. These results indicate that colorectal MIAs and sensitized muscular-mucosal afferents that respond to stretch contribute significantly to the afferent input that sustains hypersensitivity to CRD, suggesting that targeted management of colorectal afferent input could significantly reduce patients' complaints of pain and hypersensitivity.

Vesicular glutamate transporters (VGLUTs) have been extensively studied in various neuronal systems, but their expression in visceral sensory and autonomic neurons remains to be analyzed in detail. Here we studied VGLUTs type 1 and 2 (VGLUT(1) and VGLUT(2) , respectively) in neurons innervating the mouse colorectum. Lumbosacral and thoracolumbar dorsal root ganglion (DRG), lumbar sympathetic chain (LSC), and major pelvic ganglion (MPG) neurons innervating the colorectum of BALB/C mice were retrogradely traced with Fast Blue, dissected, and processed for immunohistochemistry. Tissue from additional naïve mice was included. Previously characterized antibodies against VGLUT(1) , VGLUT(2) , and calcitonin gene-related peptide (CGRP) were used. Riboprobe in situ hybridization, using probes against VGLUT(1) and VGLUT(2) , was also performed. Most colorectal DRG neurons expressed VGLUT(2) and often colocalized with CGRP. A smaller percentage of neurons expressed VGLUT(1) . VGLUT(2) -immunoreactive (IR) neurons in the MPG were rare. Abundant VGLUT(2) -IR nerves were detected in all layers of the colorectum; VGLUT(1) -IR nerves were sparse. A subpopulation of myenteric plexus neurons expressed VGLUT2 protein and mRNA, but VGLUT1 mRNA was undetectable. In conclusion, we show 1) that most colorectal DRG neurons express VGLUT(2) , and to a lesser extent, VGLUT(1) ; 2) abundance of VGLUT2-IR fibers innervating colorectum; and 3) a subpopulation of myenteric plexus neurons expressing VGLUT(2). Altogether, our data suggests a role for VGLUT(2) in colorectal glutamatergic neurotransmission, potentially influencing colorectal sensitivity and motility., (Copyright © 2011 Wiley-Liss, Inc.)

The bladder and distal colon are innervated by lumbar splanchnic (LSN) and pelvic nerves (PN) whose axons arise from dorsal root ganglia (DRG) neurons at thoracolumbar (TL) and lumbosacral (LS) spinal levels, respectively. In an attempt to understand the molecular basis of differences between LSN and PN mechanosensitive afferents, we analyzed the gene expression of two potentially counteracting ion channel groups involved in mechanosensation, transient receptor potential channels (TRPV1 and TRPA1) and mechanosensitive two pore-domain K(+) (K(2P)) channels (TREK-1, TREK-2 and TRAAK), in TL and LS DRG neurons innervating mouse bladder or distal colon. The proportion of TRPV1-expressing cells (41∼61%) did not differ between TL and LS neurons innervating bladder or colon. TRPA1 was seldom detected in bladder LS neurons whereas it was expressed in 64∼66% of bladder TL, colon TL and colon LS neurons. Coexpression of TRPV1 and TRPA1 was frequent. TREK-1-expressing cells were more prevalent in LS than TL ganglia in both bladder- and colon-DRG neurons. All three K(2P) channels were detected more frequently in TRPV1-positive neurons in TL ganglia. More than half of TL neurons expressing only TRPA1 were devoid of any of the three K(2P) channels, whereas all TL neurons expressing both TRPA1 and TRPV1 expressed at least one of the K(2P) channels. These results reveal clear differences between LSN and PN sensory pathways in TRPA1 and TREK-1 gene expression and in the gene expression of K(2P) channels in TRPV1-expressing neurons. This study further documents heterogeneity of visceral afferents based on combinations of the five channels examined., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

TREK-1, TREK-2 and TRAAK are mechanosensitive two-pore domain K(+) (K(2P)) channels thought to be involved in the attenuation of mechanotransduction. Because colon inflammation is associated with colon mechanohypersensitivity, we hypothesized that the role of these channels in colon sensory (dorsal root ganglion, DRG) neurons would be reduced by colon inflammation. Accordingly, we studied the functional expression of mechanosensitive K(2P) channels in colon sensory neurons in both thoracolumbar (TL) and lumbosacral (LS) DRG that represent the splanchnic and pelvic nerve innervations of the colon, respectively. In colon DRG neurons identified by retrograde tracer previously injected into the colon wall, 62% of TL neurons and 83% of LS neurons expressed at least one of three K(2P) channel mRNAs; the proportion of neurons expressing the TREK-1 gene was greater in LS than in TL DRG. In electrophysiological studies, single-channel activities of TREK-1a, TREK-1b, TREK-2, and TRAAK-like channels were detected in cultured colon DRG neuronal membranes. After trinitrobenzene sulfonic acid-induced colon inflammation, we observed significant decreases in the amount of TREK-1 mRNA, in the response of TREK-2-like channels to membrane stretch, and in the whole cell outward current during osmotic stretch in LS colon DRG neurons. These findings document that the majority of DRG neurons innervating the mouse colon express mechanosensitive K(2P) channels and suggest that a decrease in their expression and activities contributes to the increased colon mechanosensitivity that develops in inflammatory bowel conditions.

Background & Aims: The transient receptor potential (TRP) channels TRPV1 and TRPA1 have each been associated with regulation of efferent properties of primary afferent neurons that initiate neurogenic inflammation and are required for the development of inflammatory hyperalgesia. To evaluate the role of these channels in producing pain during pancreatic inflammation, we studied pancreatic nodose ganglion (NG) and dorsal root ganglion (DRG) sensory neurons (identified by content of retrograde tracer) and behavioral outcomes in a mouse model of acute pancreatitis., Methods: Pancreatic inflammation was induced by 8 hourly injections of cerulein (50 μg/kg). The extent of inflammation, pancreatic neuron TRP channel expression and function and excitability, and pain-related behaviors were evaluated over the course of the following week., Results: Histology and myeloperoxidase activity confirmed pancreatic inflammation that was associated with increased excitability and messenger RNA expression of the TRP channels in NG and DRG pancreatic neurons. Calcium imaging of pancreatic NG and DRG neurons from mice given cerulein revealed increased responses to TRP agonists. TRPV1 and TRPA1 antagonists attenuated cerulein-induced pain behaviors and pancreatic inflammation; they had a synergistic effect., Conclusions: Pancreatic inflammation significantly increased the expression and functional properties of TRPV1 and TRPA1, as well as the excitability of pancreatic sensory neurons in vagal and spinal pathways. TRP channel antagonists acted synergistically to reverse pancreatic inflammation and associated pain behaviors; reagents that target interactions between these channels might be developed to reduce pain in patients with acute pancreatitis., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by pain and hypersensitivity in the relative absence of colon inflammation or structural changes. To assess the role of P2X receptors expressed in colorectal dorsal root ganglion (c-DRG) neurons and colon hypersensitivity, we studied excitability and purinergic signaling of retrogradely labeled mouse thoracolumbar (TL) and lumbosacral (LS) c-DRG neurons after intracolonic treatment with saline or zymosan (which reproduces 2 major features of IBS-persistent colorectal hypersensitivity without inflammation) using patch-clamp, immunohistochemical, and RT-PCR techniques. Although whole cell capacitances did not differ between LS and TL c-DRG neurons and were not changed after zymosan treatment, membrane excitability was increased in LS and TL c-DRG neurons from zymosan-treated mice. Purinergic agonist adenosine-5'-triphosphate (ATP) and α,β-methylene ATP [α,β-meATP] produced inward currents in TL c-DRG neurons were predominantly P2X(3)-like fast (∼70% of responsive neurons); P2X(2/3)-like slow currents were more common in LS c-DRG neurons (∼35% of responsive neurons). Transient currents were not produced by either agonist in c-DRG neurons from P2X(3)(-/-) mice. Neither total whole cell Kv current density nor the sustained or transient Kv components was changed in c-DRG neurons after zymosan treatment. The number of cells expressing P2X(3) protein and its mRNA and the kinetic properties of ATP- and α,β-meATP-evoked currents in c-DRG neurons were not changed by zymosan treatment. However, the EC(50) of α,β-meATP for the fast current decreased significantly in TL c-DRG neurons. These findings suggest that colorectal hypersensitivity produced by intracolonic zymosan increases excitability and enhances purinergic signaling in c-DRG neurons.

We conducted a single-center retrospective study to compare the characteristics of Takayasu arteritis (TA) among white, North African, and black patients in a French tertiary care center (Hospital Pitié-Salpêtrière, Paris). Eighty-two patients were studied (82.9% female) during a median follow-up of 5.1 years (range, 1 mo to 30 yr). Among these 82 patients, 39 (47.6%) were white, 20 (24.4%) were North African, and 20 (24.4%) were black patients. Median age at diagnosis was 39.3 years (range, 14-70 yr) in white patients vs. 28.4 years (range, 12-54 yr) in North African (p = 0.02), and 28.0 years (range, 13-60 yr) in black patients (p = 0.08). Patients aged >40 years at TA onset were more frequently white than non-white (40.0% vs. 18.6%, p = 0.03). North African patients had more frequent occurrence of ischemic stroke (p = 0.03) and poorer survival (p = 0.01) than white patients. Type V of the Hata classification was the most frequent type among white (38.5%), North African (65.0%), and black patients (40.0%). Corticosteroids were used in 96.1% of patients. Fifty-three percent of white and North African patients, and 44% of black patients required a second line of immunosuppressive treatment (p = 0.60). Vascular surgical procedures were respectively performed in 46.1%, 50.0%, and 55.0% of white, North African, and black patients, p = 0.81. The 5-year and 10-year survival rates were 100% and 95.0%, respectively, in white patients; 67.4% at both 5 years and 10 years in North African patients; and 100% at both 5 years and 10 years in black patients. This study is one of the first direct comparisons of TA profiles among patients of distinct ethnic backgrounds. Our data support the idea that late-onset TA or an overlap between TA and large-vessel giant cell arteritis may be observed in white patients. North African patients have a higher occurrence of ischemic stroke and poorer survival than white patients.

Visceral hypersensitivity often develops after intestinal inflammation, but the pathogenic mechanism has not been clearly elucidated. We investigated whether this post-inflammatory visceral hypersensitivity is mediated by 5-hydroxytryptamine through activation of the 5-hydroxytryptamine 3 receptor. In male Sprague-Dawley rats recovered from acetic acid-induced colitis, we monitored visceral nociceptive response by scoring the abdominal withdrawal reflex and simultaneously measuring the changes in arterial pulse rate. Seven days after induction of colitis, 52% of the rats showed an increased abdominal withdrawal reflex score and arterial pulse rate changes to colorectal distension, indicating that they had post-inflammatory visceral hypersensitivity. The 5-hydroxytryptamine 3 receptor antagonists, alosetron (20 mg/kg, p.o.) and granisetron (10 microg/kg, s.c.), inhibited post-inflammatory visceral hypersensitivity. Administration of a 5-hydroxytryptamine precursor, 5-hydroxytryptophan; 10 mg/kg, s.c.), induced visceral hypersensitivity in naïve rats, which was antagonized by granisetron. Increase in 5-hydroxytryptamine immunoreactive cells in colonic mucosal layer was found both in the rats with post-inflammatory visceral hypersensitivity and in the 5-hydroxytryptophan-treated rats. These results suggest that increased 5-hydroxytryptamine in colonic mucosa mediates post-inflammatory visceral hypersensitivity through activation of the 5-hydroxytryptamine 3 receptor.

Single-channel recordings of TASK-1 and TASK-3, members of two-pore domain K(+) channel family, have not yet been reported in dorsal root ganglion (DRG) neurons, even though their mRNA and activity in whole-cell currents have been detected in these neurons. Here, we report single-channel kinetics of the TASK-3-like K(+) channel in DRG neurons and up-regulation of TASK-3 mRNA expression in tissues isolated from animals with spinal cord injury (SCI). In DRG neurons, the single-channel conductance of TASK-3-like K(+) channel was 33.0+/-0.1 pS at -60 mV, and TASK-3 activity fell by 65+/-5% when the extracellular pH was changed from 7.3 to 6.3, indicating that the DRG K(+) channel is similar to cloned TASK-3 channel. TASK-3 mRNA and protein levels in brain, spinal cord, and DRG were significantly higher in injured animals than in sham-operated ones. These results indicate that TASK-3 channels are expressed and functional in DRG neurons and the expression level is up-regulated following SCI, and suggest that TASK-3 channel could act as a potential background K(+) channel under SCI-induced acidic condition.

We identified human TRPC3 protein by yeast two-hybrid screening of a human brain cDNA library with human TRPM4b as a bait. Immunoprecipitation and confocal microscopic analyses confirmed the protein-protein interaction between TRPM4b and TRPC3, and these two TRPs were found to be highly colocalized at the plasma membrane of HEK293T cells. Overexpression of TRPM4b suppressed TRPC3-mediated whole cell currents by more than 90% compared to those in TRPC3-expressed HEK293T cells. Furthermore, HEK293T cells stably overexpressing red fluorescent protein (RFP)-TRPM4b exhibited an almost complete abolition of UTP-induced store-operated Ca(2+) entry, which is known to take place via endogenous TRPC channels in HEK293T cells. This study is believed to provide the first clear evidence that TRPM4b interacts physically with TRPC3, a member of a different TRP subfamily, and regulates negatively the channel activity, in turn suppressing store-operated Ca(2+) entry through the TRPC3 channel.

Dorsal root ganglion (DRG) neurons express mRNAs for numerous two-pore domain K(+) (K(2P)) channels and G-protein coupled receptors (GPCR). Recent studies have shown that TRESK is a major background K(+) channel in DRG neurons. Here, we demonstrate the pharmacological properties of TRESK, including GPCR agonist-induced effects on DRG neurons. TRESK mRNA was highly expressed in DRG compared to brain and spinal cord. Similar to cloned TRESK, native TRESK was inhibited by acid and arachidonic acid (AA), but not zinc. Native TRESK was also activated by GPCR agonists such as acetylcholine, glutamate, and histamine. The glutamate-activated TRESK was blocked by lamotrigine in DRG neurons. In COS-7 cells transfected with mouse TRESK, 30 microM lamotrigine inhibited TRESK by approximately 50%. Since TRESK is target of modulation by acid, AA, GPCR agonists, and lamotrigine, it is likely to play an active role in the regulation of excitability in DRG neurons.

Aim: To investigate whether peripheral corticotropin releasing hormone (CRH), which is up-regulated in intestinal inflammation, mediates the post-inflammatory visceral hypersensitivity in a rat model of colitis., Methods: We measured mucosal myeloperoxidase (MPO) activity as a marker of inflammation, plasma CRH level, and abdominal withdrawal reflex (AWR) to colorectal distension as a visceral nociceptive response at 2, 7 and 14 d after the induction of colitis with 4% acetic acid., Results: Colonic inflammation, quantified by MPO activity, significantly increased on d 2 and subsided thereafter, which indicated a resolution of inflammation within 7 d. On the contrary, plasma CRH level and AWR score were increased on d 2, remained high on d 7, and returned to control level on d 14. Intraperitoneal injection of a CRH antagonist, astressin (30 mug/kg), significantly attenuated the post-inflammatory visceral hypersensitivity on d 7. Furthermore, intraperitoneal administration of CRH (3 and 10 mug/kg) mimicked the post-inflammatory visceral hypersensitivity in naive rats., Conclusion: These results suggest that increased peripheral CRH mediates the enhanced visceral nociception in rats recovered from experimental colitis.

Recent studies have suggested that acid-sensitive background K+ channels such as TASK-1 and TASK-3, members of two-pore domain K+ (K2P) channel family, express and contribute to extracellular acidification-induced responses in dorsal root ganglia (DRG) neurons. However, it has remained to address whether other acid-sensitive background K+ channels are functionally expressed in DRG neurons. Here we characterized biophysical and pharmacological properties of a novel acid-sensitive background K+ channel in DRG neurons isolated from neonatal rats. We recorded an 80-pS K+ channel with a weak inward rectification current-voltage relationship in cell-attached patches in 150mM KCl bath solution. The 80-pS K+ channel was inhibited by extracellular low pH (pHo 6.3). Interestingly, the channel was similar to TASK-2 cloned from mouse and rat in biophysical and pharmacological properties. However, extracellular alkaline condition which activates TASK-2 channel, failed to activate the 80-pS K+ channel. Lidocaine and quinine more inhibited the channel activity of 80-pS K+ channel than that of TASK-2 channel. Our results suggest that the acid-sensitive 80-pS K+ channels may regulate resting membrane potential and may play a critical role in various processes such as cell metabolism, pH, and pain sensation in DRG neurons.

Nitric oxide (NO) is a non-adrenergic, non-cholinergic neurotransmitter found in the enteric nervous system that plays a role in a variety of enteropathies, including inflammatory bowel disease. Alteration of nitrergic neurons has been reported to be dependent on the manner by which inflammation is caused. However, this observed alteration has not been reported with acetic acid-induced colitis. Therefore, the purpose of the current study was to investigate changes in nitrergic neuromuscular transmission in experimental colitis in a rat model. Distal colitis was induced by intracolonic administration of 4 % acetic acid in the rat. Animals were sacrificed at 4 h and 48 h postacetic acid treatment. Myeloperoxidase activity was significantly increased in the acetic acid-treated groups. However, the response to 60 mM KCl was not significantly different in the three groups studied. The amplitude of phasic contractions was increased by Nomega-nitro-L-arginine methyl ester (L-NAME) in the normal control group, but not in the acetic acid-treated groups. Spontaneous contractions disappeared during electrical field stimulation (EFS) in normal group. However, for the colitis groups, these contractions initially disappeared, and then reappeared during EFS. Moreover, the observed disappearance was diminished by L-NAME; this suggests that these responses were NO-mediated. In addition, the number of NADPH-diaphorase positive nerve cell bodies, in the myenteric plexus, was not altered in the distal colon; whereas the area of NADPH-diaphorase positive fibers, in the circular muscle layer, was decreased in the acetic acidtreated groups. These results suggest that NO-mediated inhibitory neural input, to the circular muscle, was decreased in the acetic acid-treated groups.

COS-7 cells originally isolated from monkey kidney and used in many transfection studies were found to express a background K+ channel and therefore, its biophysical and pharmacological properties were examined. In cell-attached patches, a 32-pS K+ channel with a linear current-voltage relationship could be recorded. The open probability was highly voltage-dependent, with greater channel activity at depolarized potentials. The channel was markedly sensitive to changes in extracellular pH (pH(o)), showing a 70+/-10% inhibition by changing the pH(o) from 7.3 to 6.3. Arachidonic acid (5 microM) augmented channel activity 12-fold. Applying negative pressure (-40 mmHg) to the membrane patch also increased channel activity by 4-fold. These results show that COS-7 cells express a K+ channel with unique properties that must be considered when using these cells as transfection system.