A Clinically Relevant Animal Model of Temporomandibular Disorder and Irritable Bowel Syndrome Comorbidity

Temporomandibular disorder and irritable bowel syndrome are comorbid functional chronic pain disorders of unknown etiology that are triggered/exacerbated by stress. Here we present baseline phenotypic characterization of a novel animal model to gain insight into the underlying mechanisms that contribute to such comorbid pain conditions. In this model, chronic visceral hypersensitivity, a defining symptom of irritable bowel syndrome, is dependent on 3 factors: estradiol, existing chronic somatic pain, and stress. In ovariectomized rats, estradiol replacement followed by craniofacial muscle injury and stress induced visceral hypersensitivity that persisted for months. Omission of any 1 factor resulted in a transient (1 week) visceral hypersensitivity from stress alone or no hypersensitivity (no inflammation or estradiol). Maintenance of visceral hypersensitivity was estradiol dependent, resolving when estradiol replacement ceased. Referred cutaneous hypersensitivity was concurrent with visceral hypersensitivity. Increased spinal Fos expression suggests induction of central sensitization. These data demonstrate the development and maintenance of visceral hypersensitivity in estradiol-replaced animals following distal somatic injury and stress that mimics some characteristics reported in patients with temporomandibular disorder and comorbid irritable bowel syndrome. This new animal model is a powerful experimental tool that can be employed to gain further mechanistic insight into overlapping pain conditions.PerspectiveThe majority of patients with temporomandibular disorder report symptoms consistent with irritable bowel syndrome. Stress and female prevalence are common to both conditions. In a new experimental paradigm in ovariectomized rats with estradiol replacement, masseter inflammation followed by stress induces visceral hypersensitivity that persists for months, modeling these comorbid pain conditions.     

GPER-mediated,oestrogen-dependent visceral hypersensitivity in stressed rats is associated with mast cell tryptase and histamine expression

Visceral hypersensitivity (VH) is common in irritable bowel syndrome (IBS), and female patients are more likely to seek healthcare services for IBS-related abdominal pain. Oestrogen has been reported to mediate pain modulation via its receptor, and mast cells are known to participate in the development of visceral hypersensitivity. Our previous studies showed that the G-protein-coupled oestrogen receptor (GPER, also known as GPR30) was expressed by mast cells in human colonic tissues and was associated with IBS type and severity of visceral pain. However, whether GPER is involved in oestrogen-dependent visceral hypersensitivity via mast cell degranulation is still unknown. Rats were subjected to wrap partial restraint stress to induce visceral hypersensitivity and were ovariectomized (OVX) to eliminate the effects of oestrogen on visceral hypersensitivity. OVX rats were treated with oestrogen, an oestrogen receptor α and β antagonist (ICI 182.780), a GPER antagonist (G15) or a GPER agonist (G1), to evaluate the effects of oestrogen via its receptor. The colorectal distention test was performed to assess visceral sensitivity. Immunofluorescence studies were performed to evaluate GPER and mast cell tryptase co-expression. Mast cell number with degranulation was detected by specific staining. Mast cell tryptase expression in rat colon was also investigated by Western blot and immunohistochemistry. Substance P and histamine expression were examined by ELISA. GPER was expressed by the majority of tryptase-positive mast cells in the colonic mucosa. Stressed rats showed increased visceral sensitivity, increased mast cell degranulation, mast cell tryptase expression, and increased colon histamine levels. Ovariectomy reduced stress-induced VH in female rats and decreased mast cell degranulation, mast cell tryptase expression, and histamine levels, whereas oestrogen replacement reversed these effects. In OVX rats, the GPER antagonist G15 counteracted the enhancing effects of oestrogen on stress-induced VH, mast cell degranulation, mast cell tryptase, and histamine expression, whereas VH was preserved after treatment with ICI 182.780. On the other hand, pretreatment with the selective GPER agonist G1 at doses between 1 and 20 μg/kg significantly increased VH, mast cell tryptase, and histamine expression in OVX-stressed rats, mimicking the effects of oestrogen. GPER plays a pivotal role in the regulation of mast cell degranulation, mast cell tryptase expression, and histamine levels and contributes to the development of colonic hypersensitivity in a female rat model of IBS.     

Estrogen alters spinal NMDA receptor activity via a PKA signaling pathway in a visceral pain model in the rat

Pain symptoms in several chronic pain disorders in women, including irritable bowel syndrome, fluctuate with the menstrual cycle suggesting a gonadal hormone component. In female rats, estrogens modulate visceral sensitivity although the underlying mechanism(s) are unknown. In the present study the effects of 17-β estradiol on N-methyl-d-aspartate (NMDA) receptor signaling of colorectal nociceptive processing in the spinal cord were examined. Estrogen receptor alpha and the NR1 subunit of the NMDA receptor are co-expressed in dorsal horn neurons, supporting a direct action of estradiol on NMDA receptors. Intrathecal administration of the NMDA receptor antagonist d(−)-2-amino-5-phosphonopentanoic acid (APV) dose-dependently attenuated the visceromotor response with greater potency in ovariectomized (OVx) rats compared to OVx with estradiol replacement (E2) rats. Estradiol significantly increased protein expression of NR1 in the lumbosacral spinal cord compared to OVx rats. Colorectal distention significantly increased phosphorylation of NR1ser-897, a PKA phosphorylation site on the NR1 subunit in E2, but not OVx rats. Intrathecal administration of a PKA inhibitor significantly attenuated the visceromotor response, decreased NR1 phosphorylation and increased the potency of APV to attenuate the visceromotor response compared to vehicle-treated E2 rats. These data suggest that estradiol increases spinal processing of visceral nociception by increasing NMDA receptor NR1 subunit expression and increasing site-specific receptor phosphorylation on the NR1 subunit contributing to an increase in NMDA receptor activity.     

Stress-induced hyperalgesia is associated with a reduced and delayed GABA inhibitory control that enhances post-synaptic NMDA receptor activation in the spinal cord

GABA and glutamate are both affected by stress and are involved in nociception. Thus, we determined whether stress-induced enhancement of inflammatory hyperalgesia is mediated by an imbalance between glutamate and GABA neurotransmission. Male rats were subjected daily to 10 to 20 minutes per day of either forced swimming (FS) or sham swimming for 3 consecutive days; nonconditioned rats served as controls. Some rats were treated i.p. with ketamine (5 mg/kg), diazepam (2 mg/kg), flumazenil (0.1 mg/kg), or vehicle (0.9% NaCl), 30 to 60 minutes before each conditioning session or nociception assessment. Pain behavior, spinal nociceptive neuronal activation and GABA and glutamate release were respectively evaluated by the formalin test, the expression of c-Fos and in vivo microdialysis of superficial laminae of the lumbar spinal cord, 48 hours after the last conditioning session. Nitric oxide metabolites (NO_x) were determined as markers of post-synaptic NMDA receptor activation. FS stress enhanced formalin-induced hyperalgesia, increased pain-elicited c-Fos expression, decreased basal and delayed pain-induced GABA release, and increased basal and induced glutamate release. Hyperalgesia and c-Fos overexpression were blocked only by prestress treatment with diazepam and post-stress treatment with ketamine, whereas changes in GABA and glutamate release were reversed by prestress treatment with diazepam. Diazepam effects were blocked by flumazenil. NO_x increased in lumbar spinal cord of FS rats by a mechanism antagonized by ketamine. Thus, stress-induced hyperalgesia is initiated by a decreased and delayed GABA release and GABA-A receptor activation, whereas it is maintained by increased glutamate release and NMDA glutamate receptor activation at the spinal level.     

Stress-induced visceral hypersensitivity in female rats is estrogen-dependent and involves tachykinin NK1 receptors

Hormonal cycling may be related to a higher incidence of pain syndrome in female. As tachykinins are pivotal in stress-induced colonic dysfunction, we investigated whether ovarian steroids influence stress-induced visceral hypersensitivity to rectal distension (RD) in female rats and further, whether this influence involves NK1 receptors. Female Wistar rats, either intact or ovariectomized (OVX), were equipped for abdominal muscle electromyography and submitted to 2-h partial restraint stress (PRS) or sham-PRS. First, the effect of PRS was evaluated in intact rats. Second, abdominal response to RD was recorded in OVX rats treated with either, progesterone, 17beta-estradiol, 17beta-estradiol-plus-progesterone, or vehicle, in both basal and PRS conditions. Third, the NK1 receptor-antagonist, SR140333, was tested in PRS-intact and PRS-OVX rats under 17beta-estradiol or 17beta-estradiol-plus-progesterone treatment. PRS induced visceral hypersensitivity to RD and this effect was prevented by ovariectomy. OVX rats treated with 17beta-estradiol or 17beta-estradiol-plus-progesterone, but not progesterone alone, exhibited visceral hypersensitivity after PRS similar to that of intact rats. Both stress-induced visceral hypersensitivity in intact rats and the hormonally-restored visceral hyper-responsiveness of OVX rats were antagonized by SR140333. It is concluded, therefore, that stress-induced visceral hypersensitivity in female rats is estrogens-dependent and mediated through NK1 receptor activation.     

Differential Activation of Colonic Afferents and Dorsal Horn Neurons Underlie Stress-Induced and Comorbid Visceral Hypersensitivity in Female Rats

Chronic Overlapping Pain Conditions, including irritable bowel syndrome (IBS) and temporomandibular disorder (TMD), represent a group of idiopathic pain conditions that likely have peripheral and central mechanisms contributing to their pathology, but are poorly understood. These conditions are exacerbated by stress and have a female predominance. The presence of one condition predicts the presence or development of additional conditions, making this a significant pain management problem. The current study was designed to determine if the duration and magnitude of peripheral sensitization and spinal central sensitization differs between restraint stress-induced visceral hypersensitivity (SIH) and chronic comorbid pain hypersensitivity (CPH; stress during pre-existing orofacial pain). SIH in female rats, as determined by the visceromotor response, persisted at least four but resolved by seven weeks. In contrast, CPH persisted at least seven weeks. Surprisingly, colonic afferents in both SIH and CPH rats were sensitized at seven weeks. CPH rats also had referred pain through seven weeks, but locally anesthetizing the colon only attenuated the referred pain through four weeks, suggesting a transition to colonic afferent independent central sensitization. Different phenotypes of dorsal horn neurons were sensitized in the CPH rats seven weeks post stress compared to four weeks or SIH rats. The current study suggests differential processing of colonic afferent input to the lumbosacral spinal cord contributes to visceral hypersensitivity during comorbid chronic pain conditions.PerspectiveChronic Overlapping Pain Conditions represent a unique challenge in pain management. The diverse nature of peripheral organs hinders a clear understanding of underlying mechanisms accounting for the comorbidity. This study highlights a mismatch between the condition-dependent behavior and peripheral and spinal mechanisms that contribute to visceral pain hypersensitivity.     

Estradiol and testosterone differently affect visceral pain‐related behavioural responses in male and female rats

In the study of pain, the presence of sex differences is well known, with female subjects being more affected in a number of chronic painful conditions; however, the underlying mechanisms and the involvement of gonadal hormones, are still controversial. This study evaluated visceral pain in a validated rat model of artificial calculosis and the effects of estradiol and testosterone administration. Adult male and female rats were divided into groups and treated with one of the substances or Oil (vehicle) for 5 days, starting 2 days before surgery, with half receiving an artificial calculosis (Stone) and half only a sham (Sham) procedure. The animals’ behaviour (ureteral crises, frequency and duration) were recorded for 72 h; estradiol and testosterone plasma levels were determined in all groups at the end of the observation period. After surgery, only Stone rats showed ureteral pain crises, with a significant sex difference in the Oil‐treated groups in which the number and duration of crises were higher in females than in males. This difference was not present in the estradiol‐treated groups in which ureteral crises were decreased only in females while testosterone treatment had no effect in either sex. Estradiol and testosterone plasma levels were affected by treatments in both sexes. These results confirm that, also in this model of visceral pain, females experience more pain than males; moreover, they show that supraphysiological levels of estradiol, but not of testosterone, are analgesic only in females. A dose and sex‐dependent efficacy of gonadal hormones is suggested and discussed.     

Neonatal Handling Produces Sex Hormone-Dependent Resilience to Stress-Induced Muscle Hyperalgesia in Rats

Neonatal handling (NH) of male rat pups strongly attenuates stress response and stress-induced persistent muscle hyperalgesia in adults. Because female sex is a well established risk factor for stress-induced chronic muscle pain, we explored whether NH provides resilience to stress-induced hyperalgesia in adult female rats. Rat pups underwent NH, or standard (control) care. Muscle mechanical nociceptive threshold was assessed before and after water avoidance (WA) stress, when they were adults. In contrast to male rats, NH produced only a modest protection against WA stress-induced muscle hyperalgesia in female rats. Gonadectomy completely abolished NH-induced resilience in male rats but produced only a small increase in this protective effect in female rats. The administration of the antiestrogen drug fulvestrant, in addition to gonadectomy, did not enhance the protective effect of NH in female rats. Finally, knockdown of the androgen receptor by intrathecal antisense treatment attenuated the protective effect of NH in intact male rats. Together, these data indicate that androgens play a key role in NH-induced resilience to WA stress-induced muscle hyperalgesia.

Sexually Dimorphic Effects of Unpredictable Early Life Adversity on Visceral Pain Behavior in a Rodent Model

Visceral pain is the hallmark feature of irritable bowel syndrome (IBS), a gastrointestinal disorder, which is more commonly diagnosed in women. Female IBS patients frequently report a history of early life adversity (ELA); however, sex differences in ELA-induced visceral pain and the role of ovarian hormones have yet to be investigated. Therefore, we tested the hypothesis that ELA induces visceral hypersensitivity through a sexually dimorphic mechanism mediated via estradiol. As a model of ELA, neonatal rats were exposed to different pairings of an odor and shock to control for trauma predictability. In adulthood, visceral sensitivity was assessed via a visceromotor response to colorectal distension. Following ovariectomy and estradiol replacement in a separate group of rats, the visceral sensitivity was quantified. We found that females that received unpredictable odor-shock developed visceral hypersensitivity in adulthood. In contrast, visceral sensitivity was not significantly different following ELA in adult males. Ovariectomy reversed visceral hypersensitivity following unpredictable ELA, whereas estradiol replacement reestablished visceral hypersensitivity in the unpredictable group. This study is the first to show sex-related differences in visceral sensitivity following unpredictable ELA. Our data highlight the activational effect of estradiol as a pivotal mechanism in maintaining visceral hypersensitivity.PerspectiveThis article directly implicates a critical role for ovarian hormones in maintaining visceral hypersensitivity following ELA, specifically identifying the activational effect of estradiol as a key modulator of visceral sensitivity. These data suggest that ELA induces persistent functional abdominal pain in female IBS patients through an estrogen-dependent mechanism.     

Effects of gonadal hormones on the peripheral cannabinoid receptor 1 (CB1R) system under a myositis condition in rats

In this study, we assessed the effects of peripherally administered cannabinoids in an orofacial myositis model, and the role of sex hormones in cannabinoid receptor (CBR) expression in trigeminal ganglia (TG). Peripherally administered arachidonylcyclopropylamide (ACPA), a specific CB1R agonist, significantly attenuated complete Freund’s adjuvant (CFA)-induced mechanical hypersensitivity in the masseter muscle in male rats. The ACPA effect was blocked by a local administration of AM251, a specific CB1R antagonist, but not by AM630, a specific CB2R antagonist. In female rats, a 30-fold higher dose of ACPA was required to produce a moderate reduction in mechanical hypersensitivity. CFA injected in masseter muscle significantly upregulated CB1R mRNA expression in TG in male, but not in female, rats. There was a close correlation between the CB1R mRNA levels in TG and the antihyperalgesic effect of ACPA. Interleukin (IL)-1β and IL-6, which are elevated in the muscle tissue following CFA treatment, induced a significant upregulation of CB1R mRNA expression in TG from male rats. The upregulation of CB1R was prevented in TG cultures from orchidectomized male rats, which was restored by the application of testosterone. The cytokines did not alter the CB1R mRNA level in TG from intact as well as ovariectomized female rats. Neither estradiol supplement nor estrogen receptor blockade had any effects on CB1R expression. These data indicate that testosterone, but not estradiol, is required for the regulation of CB1Rs in TG under inflammatory conditions, which provide explanations for the sex differences in the antihyperalgesic effects of peripherally administered cannabinoids.     

Sex differences in spinal processing of transient and inflammatory colorectal stimuli in the rat

Sex differences in the spinal processing of somatic and visceral stimuli contribute to greater female sensitivity in many pain disorders. The present study examined spinal mechanisms that contribute to sex differences in visceral sensitivity. The visceromotor response to colorectal distention (CRD) was more robust in normal female rats and after intracolonic mustard oil compared with that in male rats. No sex difference was observed in the CRD-evoked response of lumbosacral (LS) and thoracolumbar (TL) colonic afferents in normal and mustard oil-treated rats, but there was a sex difference in spontaneous activity that was exacerbated by intracolonic mustard oil. The response of visceroceptive dorsal horn neurons to CRD was greater in normal female rats in the LS and TL spinal segments. The effect of intracolonic mustard oil on the CRD-evoked response of different phenotypes of visceroceptive dorsal horn neurons was dependent on sex and segment. The NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (APV) dose-dependently attenuated the visceromotor response in normal rats with greater effect in male rats. Correspondingly, there was greater cell membrane expression of the GluN1 subunit in dorsal horn extracts in female rats. After intracolonic mustard oil, there was no longer a sex difference in the effect of APV nor GluN1 expression in LS segments, but greater female expression in TL segments. These data document a sex difference in spinal processing of nociceptive visceral stimuli from the normal and inflamed colon. Differences in dorsal horn neuronal activity and NMDA receptor expression contribute to the sex differences in the visceral sensitivity observed in awake rats.     

The organizational and activational effects of sex hormones on tactile and thermal hypersensitivity following lumbar nerve root injury in male and female rats

Considerable evidence exists for sex differences in human pain sensitivity. Women typically report a higher incidence of various painful conditions and report that the conditions are more painful when compared to men. In the present study, we sought to determine whether sex differences in pain sensitivity are observed using a lumbar radiculopathy model of low back pain in the rat and whether removal or alteration of gonadal hormones at specific timepoints can modulate these sex differences. Pubertal and adult male and female Sprague-Dawley rats were castrated 2 or 6 weeks prior to L5 nerve root injury to determine the activational hormonal effects. In a separate study, neonatal male and female Sprague-Dawley rats were either castrated or injected with testosterone, respectively, on postnatal day one to determine the organizational effects of gonadal hormones on L5 nerve root injury-induced behavioral hypersensitivity. Our results demonstrate that there was a statistically significant sex difference in the magnitude of mechanical allodynia and thermal hyperalgesia following experimentally induced radiculopathy in the rat: females demonstrated decreased thresholds to tactile and thermal stimuli as compared to males. Furthermore, the enhanced female hypersensitivity was reversed in pubertal and adult animals ovariectomized 6 weeks, but not 2 weeks prior to L5 nerve root injury. Our results demonstrate that the activational effects of gonadal hormones mediate the enhanced female tactile and thermal hypersensitivity following L5 nerve root injury. These results suggest that manipulation of gonadal hormones may be a potential source for novel therapies for chronic pain in women.     

Astrocytic ERK/STAT1 Signaling Contributes to Maintenance of Stress-Related Visceral Hypersensitivity in Rats

The rostral anterior cingulate cortex (rACC) has been found to be an important brain region in mediating visceral hypersensitivity. However, the underlying mechanisms remain unclear. This study aimed to explore the role of astrocytes in the maintenance of visceral hypersensitivity induced by chronic water avoidance stress (WAS) as well as the potential signaling pathway that activates astrocytes in the rACC. We found that ACC-reactive astrogliosis resulted in the overexpression of c-fos, TSP-1, and BDNF in stress-related visceral hypersensitivity rats. Visceral hypersensitivity was reversed by pharmacological inhibition of astrocytic activation after WAS, as were the overexpression of c-fos, TSP-1 and BDNF. Activation of the astrocytic Gi-pathway increased the visceral sensitivity and expression of c-fos, TSP-1, and BDNF. Visceral hypersensitivity was also ameliorated by the pharmacological inhibition of ERK and STAT1 phosphorylation after WAS. Furthermore, inhibition of the ERK-STAT1 cascade reduced astrocytic activation. These findings suggest that astrocytic ERK/STAT1 signaling in the rACC contributes to the maintenance of stress-related visceral hypersensitivity.PerspectiveVisceral hypersensitivity is a key factor in the pathophysiology of irritable bowel syndrome. This study highlights the important role of astrocytic ERK/STAT1 signaling in activating astrocytes in the rostral anterior cingulate cortex, which contributes to visceral hypersensitivity.     

Vagal Afferents Mediate Antinociception of Estrogen in a Rat Model of Visceral Pain: The Involvement of Intestinal Mucosal Mast Cells and 5-Hydroxytryptamine 3 Signaling

Estrogen reportedly facilitates visceral nociception at the spinal or supraspinal level. The present study was aimed to investigate whether estrogen modulates visceral pain through the vagal pathway. Ovariectomized rats received estradiol, which was administered subcutaneously (to act through both the vagal and spinal pathways) or intraduodenally (to preferentially act through the vagal pathway). Luminally applied estradiol induced a rapid and significant decrease in the visceromotor response to colorectal distension, with increased c-Fos expression in nodose ganglion neurons. Systemically injected estradiol increased visceromotor response and c-Fos expression in both nodose and dorsal root ganglion (T6-12) neurons. The antinociceptive effect of estrogen was abolished by surgical vagotomy or chemical denervation of vagal afferents. Both luminally and systemically administered estradiol elicited selective 5-hydroxytryptamine secretion from the duodenum. Granisetron, a 5-hydroxytryptamine 3 receptor antagonist, reversed the antinociceptive effect of estrogen. Intestinal mucosal mast cell stabilizers prevented estradiol-induced antinociception and 5-hydroxytryptamine secretion. Ultrastructural analysis revealed that estradiol caused piecemeal degranulation of intestinal mucosal mast cells. The actions of estradiol were inhibited by an estrogen receptor β antagonist and mimicked by an estrogen receptor β agonist. These results suggest that estrogen can trigger vagus-mediated antinociception, which is masked by its spinally mediated pronociception.PerspectiveThis study is the first to show a vagus-mediated estrogenic antinociception, in which the nongenomic estrogen receptor β–mediated, intestinal mucosal mast cell–derived 5-hydroxytryptamine/5-hydroxytryptamine 3 receptor pathway is involved. This work may provide new insights into the sex hormone modulation of visceral sensitivity related to irritable bowel syndrome and indicate potential therapeutic targets to manage this disease.     

Sex differences in thermal nociception and prostaglandin-induced thermal hypersensitivity in rhesus monkeys.

The present study examined thermal nociception in 4 male and 4 female rhesus monkeys. In the first experiment, monkeys were tested 5 days/week for 4 consecutive weeks in a warm-water, tail-withdrawal assay of thermal nociception. Thermal nociception did not vary by sex or menstrual cycle phase. However, male monkeys tended to be slightly more sensitive to thermal stimuli than female monkeys in the follicular phase of the menstrual cycle. Thermal nociception did not correlate with estradiol or progesterone levels in female monkeys. In the second experiment, thermal hypersensitivity was induced by administering prostaglandin E(2) (0.0032 to 0.1 mg subcutaneously) into the tail. Prostaglandin E(2) produced slightly greater thermal hypersensitivity in male than in follicular phase female monkeys, but male and luteal phase female monkeys did not differ, and there was not a significant difference between follicular and luteal phase female monkeys. Exposure to the behavioral procedures produced similar increases in blood levels of the stress-related hormones adrenocorticotropic hormone and cortisol in male and female monkeys, which suggests that measures of thermal nociception or thermal hypersensitivity were not confounded by sex differences in stress responses. These results suggest that sex and gonadal hormone levels have only a minor influence on thermal nociception or thermal hypersensitivity in rhesus monkeys. PERSPECTIVE: These modest effects of sex and gonadal hormone levels on measures of pain in non-human primates could be interpreted to support the hypothesis that sex differences in pain perception in humans are due more to sociocultural factors than to a biological imperative.     

Dual role of 5-HT3 receptors in a rat model of delayed stress-induced visceral hyperalgesia

Despite its beneficial effect in IBS patients, the mechanism of action of the 5-HT3 receptor (5-HT3R) antagonist alosetron is still incompletely understood. We aimed to characterize the effect and site(s) of action in a model of stress-induced sensitization of visceral nociception in rats. Adult male Wistar rats were equipped for recording of visceromotor response (VMR) to phasic colorectal distension (CRD; 10-60 mmHg). VMR to CRD was recorded 24 h after an acute session of water avoidance (WA) stress (post-WA). Baseline and post-WA responses were measured in rats exposed to WA or sham-WA, treated with alosetron at 0.3 mg/kg subcutaneously (s.c.) 25 nmol intrathecally (i.t.) or vehicle before post-WA CRD. Some rats were treated with capsaicin/vehicle on the cervical vagus nerve and received alosetron (0.3 mg/kg, s.c.) 15 min before post-WA CRD. WA stress led to visceral hyperalgesia 24 h later. Alosetron (0.3 mg/kg, s.c.), failed to inhibit WA-induced exacerbation of VMR to CRD. Stress-induced visceral hyperalgesia was abolished when alosetron was injected intrathecally (P<0.05) in intact rats or subcutaneously (0.3 mg/kg) in capsaicin-pretreated animals (P<0.05). Capsaicin-pretreatment did not affect the exacerbating effect of stress on visceral sensitivity. Alosetron had no inhibitory effect on normal visceral pain responses when administered subcutaneously or intrathecally. We demonstrated that 5-HT3Rs on central terminals of spinal afferents are engaged in the facilitatory effect of stress on visceral sensory information processing. In addition, we showed that stress-induced sensitization of visceral nociception is independent of 5-HT3R activation on vagal afferents.     

Sex differences in formalin-induced pain in prenatally stressed infant rats

The aim of this work was to study the effects of prenatal stress on nociceptive responses in the formalin test in female and male infant (7-day-old) Long-Evans hooded rats. Prenatally stressed infant rats displayed biphasic flinching+ shaking behavior whereas non-stressed animals showed only a weak second phase. Pain sensitivity in prenatally stressed males was significantly greater than that of prenatally non-stressed males during the second phase only; there were no differences in pain sensitivity between prenatally stressed and non-stressed females. Moreover prenatally stressed male rats pups demonstrated that the second phase of the response to formalin was enhanced relative to the second phase in stressed females. The current and previous data [Butkevich IP, Barr GA, Mikhailenko VA, Otellin VA. Increased formalin-induced pain and expression of fos neurons in the lumbar spinal cord of prenatally stressed infants rats. Neurosci Lett 2006a;403:222-226] show increased tonic pain in prenatally stressed infant rats and a large increase in the number of formalin-induced fos-like immunoreactivity in the spinal cord dorsal horn. There is a concomitant decrease in serotonin-like immunoreactivity in the lumbar spinal cord dorsal horn [Butkevich IP, Barr GA, Otellin VA. Effect of prenatal stress on behavioral and neural indices of formalin-induced pain in infant rats. Abstracts, 35th Annual Meeting of Soc. For Neurosci. 2005a. Program No. 512.4 Washington, DC: Society for Neuroscience]. Given the decreased level of perinatal testosterone in prenatally stressed rats to which infant males are more sensitive than females, we suggest that these hormonal, behavioral and neuronal indices are strongly interrelated in prenatally stressed 7-day-old rat pups and that the decreased surge of testosterone may contribute to the increased behavioral response in the second phase in male rat pups. Mechanisms underlying the behavioral pain response induced by inflammation in prenatally stressed rat pups are characterized by sexual dimorphism even prior to the activational effects of sex hormones.     

Footshock-Induced Urinary Bladder Hypersensitivity: Role of Spinal Corticotropin-Releasing Factor Receptors

Stress-induced hyperalgesia (SIH), a common clinical observation associated with multiple painful diseases including functional urinary disorders, presently has no mechanistic explanation. Using a footshock treatment, a classic stressor, to magnify physiological responses in a model of urinary bladder pain, we examined one potential group of mediators of SIH, the corticotropin-releasing factor (CRF)-related neuropeptides. Exposure to a footshock treatment produced bladder hypersensitivity in female Sprague-Dawley rats, manifested as significantly more vigorous visceromotor responses (VMRs) to urinary bladder distension (UBD) compared with rats that were exposed to a non-footshock treatment. This bladder hypersensitivity was significantly attenuated by blocking spinal CRF₂ receptors but not CRF₁ receptors. Furthermore, spinal administration of urocortin 2, a CRF₂ receptor agonist, augmented UBD-evoked VMRs in a way similar to what was observed after exposure to Footshock, an effect significantly attenuated by pretreatment with spinal aSVG30, a CRF₂ receptor antagonist. Surprisingly, neither spinal administration of CRF nor the CRF₁ receptor antagonist antalarmin had an effect on bladder nociceptive responses. The results of the present study not only provide further support for a role of stress in the exacerbation of bladder pain but also implicate spinal urocortins and their endogenous receptor, the CRF₂ receptor, as potential mediators of this effect.PerspectiveThis study presents evidence that spinal urocortins and CRF₂ receptors are involved in stress-induced hypersensitivity related to the urinary bladder. This provides a basis for investigating how urocortins mediate SIH, ultimately leading to more effective treatment options for patients with painful bladder syndromes as well as stress-exacerbated chronic pain.     

Sex-specificity and estrogen-dependence of kappa opioid receptor-mediated antinociception and antihyperalgesia

This investigation determined whether the activation of the kappa opioid receptor (KOR) in the spinal cord produces estrogen-dependent, sex-specific modulation of acute and inflammation-induced persistent nociception. We demonstrate for the first time that KOR antinociception and gene expression are enhanced by exogenous or endogenous estrogen in the female. The lack of KOR antinociception and KOR gene expression are not altered by the hormonal status (testosterone or estrogen) in males. Cannulae were implanted intrathecally in male, gonadectomized male (GDX), intact and ovariectomized female (OVX) Sprague-Dawley rats. Estradiol was injected subcutaneously, 48 h before testing (GDX+E and OVX+E). Intrathecal injection of U50,488H, a selective KOR agonist, dose dependently increased heat-evoked tail flick latencies (TFLs) in proestrous and OVX+E groups, but not in male, GDX, GDX+E, OVX, and diestrous groups. Further, estrogen dose-dependently enhanced the effect of U50,488H in OVX rats. KOR selective antagonist, nor-binaltorphimine (Nor-BNI), blocked the antinociceptive effect of U50,488H. U50,488H reversed the carrageenan-induced thermal hyperalgesia in OVX+E rats, but not in male or OVX rats. However, U50,488H treatment did not alter mechanical thresholds in any group, with or without inflammation. KOR gene expression was enhanced in proestrous and OVX+E groups as compared to any other group. We conclude that selective activation of KOR in the spinal cord produces sex-specific, stimulus- and estrogen-dependent attenuation of acute and inflammatory pain in the rat via estrogen-induced upregulation of the KOR gene expression in the spinal cord. These findings may further implicate estrogen dependence of KOR effects in learning, epilepsy, stress response, addiction etc.