Spinal GABAergic disinhibition allows microglial activation mediating the development of nociplastic pain in male mice.

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-1ß, 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.

Peripheral and central oxidative stress in chemotherapy-induced neuropathic pain.

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 Aß 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 Aß fiber inputs.

Mitochondrial superoxide increases excitatory synaptic strength in spinal dorsal horn neurons of neuropathic mice.

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.

Differential involvement of reactive oxygen species in a mouse model of capsaicin-induced secondary mechanical hyperalgesia and allodynia.

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.

A novel role for follistatin in hypersensitivity following cystitis.

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.

Chronic Prostatitis Induces Bladder Hypersensitivity and Sensitizes Bladder Afferents in the Mouse.

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.

Experimental and computational evidence for an essential role of NaV1.6 in spike initiation at stretch-sensitive colorectal afferent endings.

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.

Activation of guanylate cyclase-C attenuates stretch responses and sensitization of mouse colorectal afferents.

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.

TRPV1 and TRPA1 antagonists prevent the transition of acute to chronic inflammation and pain in chronic pancreatitis.

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.

Identification of Arginine-Vasopressin Receptor 1a (Avpr1a/Avpr1a) as a Novel Candidate Gene for Chronic Visceral Pain Sheds Light on the Potential Role of Enteric Neurons in the Development of Visceral Hypersensitivity.

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.

Orphan G Protein-Coupled Receptor GPR37 as an Emerging Therapeutic Target.

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.

Gonadal hormone-dependent nociceptor sensitization maintains nociplastic pain state in female mice.

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.

Intraoperative Spinal Cord Stimulation Mitigates Central Sensitization After Spine Surgery in Mice.

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.

Identification of arginine-vasopressin receptor 1a (Avpr1a/AVPR1A) as a novel candidate gene for chronic visceral 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 Avpr1a as a novel candidate gene for visceral hypersensitivity (VH), a primary peripheral mechanism underlying abdominal pain in DGBI/IBS. Comparing two C57BL/6 (BL/6) substrains (C57BL/6NTac and C57BL/6J) revealed differential susceptibility to the development of chronic VH following intrarectal zymosan (ZYM) instillation, a validated preclinical model for post-inflammatory IBS. Using whole genome sequencing, we identified a SNP differentiating the two strains in the 5' intergenic region upstream of Avpr1a, encoding the protein arginine-vasopressin receptor 1A (AVPR1A). We used behavioral, histological, and molecular approaches to identify distal colon-specific gene expression differences and neuronal hyperresponsiveness covarying with Avpr1a genotype and VH susceptibility. While the two BL/6 substrains did not differ across other gastrointestinal (GI) phenotypes (e.g., GI motility), VH-susceptible BL/6NTac mice had higher colonic Avpr1a mRNA and protein expression. Moreover, neurons of the enteric nervous system were hyperresponsive to the AVPR1A agonist AVP, suggesting a role for enteric neurons in the pathology underlying VH. These results parallel our findings that patients' colonic Avpr1a mRNA expression was higher in patients with higher pain ratings. 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.

Irritable bowel syndrome: methods, mechanisms, and pathophysiology. Neural and neuro-immune mechanisms of visceral hypersensitivity in irritable bowel syndrome.

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.

Luminal hypertonicity and acidity modulate colorectal afferents and induce persistent visceral hypersensitivity.

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.

Altered colorectal afferent function associated with TNBS-induced visceral hypersensitivity in mice.

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.

Long-term sensitization of mechanosensitive and -insensitive afferents in mice with persistent colorectal 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.

Synergistic role of TRPV1 and TRPA1 in pancreatic pain and inflammation.

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.

Postinjury stimulation triggers a transition to nociplastic pain in mice.

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.