A novel animal model for understanding secondary traumatic stress and visceral pain in male rats.

Empathetic relationships and the social transference of behaviours have been shown to occur in humans, and more recently through the development of rodent models, where both fear and pain phenotypes develop in observer animals. Clinically, observing traumatic events can induce 'trauma and stressor-related disorders' as defined in the DSM 5. These disorders are often comorbid with pain and gastrointestinal disturbances; however, our understanding of how gastrointestinal - or visceral - pain can be vicariously transmitted is lacking. Visceral pain originates from the internal organs, and despite its widespread prevalence, remains poorly understood. We established an observation paradigm to assess the impact of witnessing visceral pain. We utilised colorectal distension (CRD) to induce visceral pain behaviours in a stimulus rodent while the observer rodent observed. Twenty four hours post-observation, the observer rodent's visceral sensitivity was assessed using CRD. The observer rodents were found to have significant hyperalgesia as determined by lower visceral pain threshold and higher number of total pain behaviours compared with controls. The behaviours of the observer animals during the observation were found to be correlated with the behaviours of the stimulus animal employed. We found that observer animals had hypoactivity of the hypothalamic-pituitary-adrenal (HPA) axis, highlighted by reduced corticosterone at 90 minutes post-CRD. Using c-Fos immunohistochemistry we showed that observer animals also had increased activation of the anterior cingulate cortex, and decreased activation of the paraventricular nucleus, compared with controls. These results suggest that witnessing another animal in pain produces a behavioural phenotype and impacts the brain-gut axis.

A novel visceral pain model of uterine cervix inflammation in rat.

Treatment of visceral pain originating from the uterine cervix is a substantial clinical problem. The underlying mechanisms of such visceral pain remain unclear mainly due to a lack of reliable model. This study aimed to develop and evaluate the performance of a rat model of pain induced by uterine cervix inflammation. Rats were randomized to six groups according to the solution injected into the uterine cervix: normal saline, vehicle, capsaicin (0.3 mg, 0.6 mg, 0.9 mg), capsaicin 0.9 mg + morphine (n = 15 in each group). Spontaneous behaviors after cervical injection were recorded by a computerized video system and analyzed offline. An equation for calculating a novel pain score was derived from particular behaviors, based on Pearson's correlation analysis and regression analysis. c-Fos expression in the spinal cord was detected. The pain score and c-fos expression in the spinal cord were highest in the 0.9 mg capsaicin group and lowest in the normal saline and vehicle groups (P < 0.05). Intrathecal morphine significantly decreased the pain score (P < 0.05) and c-fos expression in the spinal cord (P < 0.05). Injection of capsaicin into the uterine cervix in rats could be a practical model of inflammatory cervical pain, which can be evaluated using our novel pain score. This model will provide further insight into the mechanism underlying visceral pain originating from the uterine cervix.

Histone H3K9 methylation regulates chronic stress and IL-6-induced colon epithelial permeability and visceral pain.

BACKGROUND: Chronic stress is associated with activation of the HPA axis, elevation in pro-inflammatory cytokines, decrease in intestinal epithelial cell tight junction (TJ) proteins, and enhanced visceral pain. It is unknown whether epigenetic regulatory pathways play a role in chronic stress-induced intestinal barrier dysfunction and visceral hyperalgesia. METHODS: Young adult male rats were subjected to water avoidance stress ± H3K9 methylation inhibitors or siRNAs. Visceral pain response was assessed. Differentiated Caco-2/BBE cells and human colonoids were treated with cortisol or IL-6 ± antagonists. Expression of TJ, IL-6, and H3K9 methylation status at gene promoters was measured. Transepithelial electrical resistance and FITC-dextran permeability were evaluated. KEY RESULTS: Chronic stress induced IL-6 up-regulation prior to a decrease in TJ proteins in the rat colon. The IL-6 level inversely correlated with occludin expression. Treatment with IL-6 decreased occludin and induced visceral hyperalgesia. Chronic stress and IL-6 increased H3K9 methylation and decreased transcriptional GR binding to the occludin gene promoter, leading to down-regulation of protein expression and increase in paracellular permeability. Intrarectal administration of a H3K9 methylation antagonist prevented chronic stress-induced visceral hyperalgesia in the rat. In a human colonoid model, cortisol decreased occludin expression, which was prevented by the GR antagonist RU486, and IL-6 increased H3K9 methylation and decreased TJ protein levels, which were prevented by inhibitors of H3K9 methylation. CONCLUSIONS & INFERENCES: Our findings support a novel role for methylation of the repressive histone H3K9 to regulate chronic stress, pro-inflammatory cytokine-mediated reduction in colon TJ protein levels, and increase in paracellular permeability and visceral hyperalgesia.

Identification of experimental bladder sensitivity among dysmenorrhea sufferers.

BACKGROUND: Dysmenorrhea is a common risk factor for chronic pain conditions including bladder pain syndrome. Few studies have formally evaluated asymptomatic bladder pain sensitivity in dysmenorrhea, and whether this largely reflects excess pelvic symptom reporting due to comorbid psychological dysfunction. OBJECTIVE: We sought to determine whether bladder hypersensitivity is more common among women reporting moderate or greater dysmenorrhea, without chronic pain elsewhere, after accounting for anxiety and depression. Demonstrating this would suggest that dysmenorrhea might be an early clue for visceral or widespread pain hypersensitivity and improve understanding of potential precursors to bladder pain syndrome. STUDY DESIGN: We compared cohorts of regularly menstruating women, without symptoms of chronic pain elsewhere, reporting (1) moderate-to-severe dysmenorrhea (n = 98) and (2) low levels or no menstrual pain (n = 35). Participants underwent rapid bladder filling following a standard water ingestion protocol, serially rating bladder pain and relative urgency during subsequent distension. Potential differences in bladder volumes were controlled for by sonographic measurement at standard cystometric thresholds. Bladder sensitivity was also measured with complementary measures at other times separately including a simplified rapid filling test, palpation of the bladder wall, and through ambulatory self-report. Anxiety and depression were evaluated with the National Institutes of Health Patient-Reported Outcomes Measurement Information System measures. RESULTS: Women with moderate-to-severe dysmenorrhea reported more urinary symptoms than controls and had a lower maximum capacity (498 ± 18 mL vs 619 ± 34 mL, P < .001) and more evoked bladder filling pain (0-100 visual analog scale: 25 ± 3 vs 12 ± 3, P < .001). The dysmenorrhea-bladder capacity relationship remained significant irrespective of menstrual pain severity, anxiety, depression, or bladder pain (R2 = 0.13, P = .006). Severity of menstrual pain predicted evoked bladder pain (R2 = 0.10, P = .008) independent of anxiety (P = .21) and depression (P = .21). Women with moderate-to-severe dysmenorrhea exhibiting provoked bladder pain (24/98, 24%) also reported higher pain during the screening rapid bladder test (P < .001), in response to transvaginal bladder palpation (P < .015), and on prospective daily diaries (P < .001) than women with dysmenorrhea without provoked bladder pain. CONCLUSION: Women experiencing moderate-to-severe dysmenorrhea also harbor a higher pain response to naturally evoked bladder distension. Noninvasive bladder provocation needs to be tested further longitudinally in those with dysmenorrhea to characterize the course of visceral sensitivity and determine if it may help predict individuals at risk for developing subsequent pain in the bladder or elsewhere.

P2X3 receptor-mediated visceral hyperalgesia and neuronal sensitization following exposure to PTSD-like stress in the dorsal root ganglia of rats.

BACKGROUND: Patients with posttraumatic stress disorder (PTSD) often share co-morbidity with chronic pain conditions. Recent studies suggest a role of P2X3 receptors and ATP signaling in pain conditions. However, the underlying mechanisms of visceral hyperalgesia following exposure to PTSD-like stress conditions remain unclarified. METHODS: The behavior and hormones relevant for PTSD were studied. Visceromotor responses (VMR) and the abdominal withdrawal reflexes (AWR) to colorectal distention (CRD) were recorded to determine P2X3-receptor-mediated alteration of hyperalgesia following single-prolonged stress (SPS) exposure. Immunofluorescence, Western blotting, and patch-clamp were used. KEY RESULTS: The escape latency, adrenocorticotropic hormone and cortisol were increased on days 7-14. Visceromotor responses and AWR was reduced at day 1 in SPS rats but increased to higher levels than in controls after exposure to day 7. Intrathecal administration of the P2X3-receptor antagonist TNP-ATP abolished the CRD response. Based on immunofluorescence and Western blotting analysis, SPS-treated rats exhibited reduced P2X3 expression in dorsal root ganglia (DRG) after day 1 compared with controls. P2X3 expression in DRG was enhanced on day 7 after SPS and the increase of the P2X3 expression was maintained on day 14 and 21 compared with controls. The P2X3-receptor agonist α,ß-me ATP (10 µM) induced a fast desensitizing inward current in DRG neurons of both control and SPS-treated rats. The average peak current densities in SPS-treated group were increased 3.6-fold. TNP-ATP (100 nM) markedly blocked all fast α,ß-me ATP-induced inward currents in the DRG neurons both in control and SPS-treated rats. CONCLUSIONS & INFERENCES: The data indicate an important role of P2X3 signaling in visceral hyperalgesia following PTSD-like stress.

Effects of intraperitoneally administered L-histidine on food intake, taste, and visceral sensation in rats.

To evaluate relative factors for anorectic effects of L-histidine, we performed behavioral experiments for measuring food and fluid intake, conditioned taste aversion (CTA), taste disturbance, and c-Fos immunoreactive (Fos-ir) cells before and after i.p. injection with L-histidine in rats. Animals were injected with saline (9 ml/kg, i.p.) for a control group, and saline (9 ml/kg, i.p.) containing L-histidine (0.75, 1.5, 2.0 g/kg) for a L-histidine group. Injection of L-histidine decreased the average value of food intake, and statistically significant anorectic effects were found in animals injected with 1.5 or 2.0 g/kg L-histidine but not with 0.75 g/kg L-histidine. Taste abnormalities were not detected in any of the groups. Animals injected with 2.0 g/kg L-histidine were revealed to present with nausea by the measurement of CTA. In this group, a significant increase in the number of Fos-ir cells was detected both in the area postrema and the nucleus tractus solitarius (NTS). In the 0.75 g/kg L-histidine group, a significant increase in the number of Fos-ir cells was detected only in the NTS. When the ventral gastric branch vagotomy was performed, recovery from anorexia became faster than the sham-operated group, however, vagotomized rats injected with 2.0 g/kg L-histidine still acquired CTA. These data indicate that acute anorectic effects induced by highly concentrated L-histidine are partly caused by induction of nausea and/or visceral discomfort accompanied by neuronal activities in the NTS and the area postrema. We suggest that acute and potent effects of L-histidine on food intake require substantial amount of L-histidine in the diet.

Differential expression of ATP-gated P2X receptors in DRG between chronic neuropathic pain and visceralgia rat models.

There are divergences between neuropathic pain and visceralgia in terms of the duration, location, and character of hyperalgesia. It is generally recognized that nociceptive receptors, including P2X receptors, may play different roles in nociceptive mechanisms. The different roles of P2X1-7 receptors have not been fully understood both in neuropathic pain and visceral hyperalgesia. In order to explore the different expressions of P2X1-7 receptors in these two hyperalgesia models, the lumbosacral dorsal root ganglion (DRG) neurons from rat sciatic nerve chronic constriction injury (CCI) model and neonatal colorectal distention (NCRD) model were studied (both the primary nociceptive neuron afferents of those two models projected to the same segment of spinal cord). Both immunohistochemistry (IHC) technique and real-time fluorescence quantitative polymerase chain reaction (RT-PCR) technology were applied to analyze the protein expression levels and nucleic acid of P2X1-7 receptors. We found that except P2X2 and P2X3, the expression levels of P2X1 and P2X5 receptors increased in neuropathic pain while those expression levels of P2X4, P2X6, and P2X7 receptors increased in visceral pain. Our results also suggested that in addition to P2X2/3 heteromeric, other P2X subunits may also involved in generation heteromeric such as P2X1/5 and/or P2X2/5 in neuropathic pain and P2X4/6 and/or P2X4/7 in visceral pain.

Roles of prefrontal cortex and paraventricular thalamus in affective and mechanical components of visceral nociception.

Visceral pain represents a major clinical challenge in the management of many gastrointestinal disorders, eg, pancreatitis. However, cerebral neurobiological mechanisms underlying visceral nociception are poorly understood. As a representative model of visceral nociception, we applied cerulein hyperstimulation in C57BL6 mice to induce acute pancreatitis and performed a behavioral test battery and c-Fos staining of brains. We observed a specific pain phenotype and a significant increase in c-Fos immunoreactivity in the paraventricular nucleus of the thalamus (PVT), the periaqueductal gray, and the medial prefrontal cortex (mPFC). Using neuronal tracing, we observed projections of the PVT to cortical layers of the mPFC with contacts to inhibitory GABAergic neurons. These inhibitory neurons showed more activation after cerulein treatment suggesting thalamocortical "feedforward inhibition" in visceral nociception. The activity of neurons in pancreatitis-related pain centers was pharmacogenetically modulated by designer receptors exclusively activated by designer drugs, selectively and cell type specifically expressed in target neurons using adeno-associated virus-mediated gene transfer. Pharmacogenetic inhibition of PVT but not periaqueductal gray neurons attenuated visceral pain and induced an activation of the descending inhibitory pain pathway. Activation of glutamatergic principle neurons in the mPFC, but not inhibitory neurons, also reversed visceral nociception. These data reveal novel insights into central pain processing that underlies visceral nociception and may trigger the development of novel, potent centrally acting analgesic drugs.

Pain sensation in pancreatic diseases is not uniform: the different facets of pancreatic pain.

AIM: To systematically characterize specific pain patterns in the most frequent pancreatic diseases. METHODS: Pain in patients with chronic pancreatitis (n = 314), pancreatic cancer (n = 469), and other pancreatic tumors (n = 249) including mucinous (n = 20) and serous cystadenoma (n = 31), invasive (n = 37) and non-invasive intraductal papillary mucinous neoplasia (IPMN; n = 48), low stage (n = 18) and high stage neuroendocrine neoplasia (n = 44), and ampullary cancer (n = 51) was registered and correlated with clinicopathological data. Survival times were estimated by the Kaplan-Meier method. Patients alive at the follow-up time were censored. Survival curves were compared statistically using the log-rank test. RESULTS: Forty-nine point one percent of pancreatic cancer patients revealed no pain, whereas in chronic pancreatitis only 18.3% were pain free. In contrary, moderate/severe pain was registered in 15.1% in pancreatic cancer patients that was increased in chronic pancreatitis with up to 34.2%. Serous cystadenoma was asymptomatic in most cases (58.1%), whereas 78.9% of all mucinous cystadenoma patients suffered pain. In neuroendocrine neoplasia pain was not a key clinical symptom since 64% of low stage neuroendocrine neoplasia and 59% of high stage neuroendocrine neoplasia patients were pain free. Cancer localization in the pancreatic body and patients with malignant pancreatic neoplasms were associated with more severe pain. Tumor grading and stage did not show any impact on pain. Only in pancreatic cancer, pain was directly associated with impaired survival. CONCLUSION: Pancreatic pain depicts different patterns of abdominal pain sensation according to the respective pancreatic disorder and does not allow a unification of the term pancreatic pain.

Biological and psychological predictors of visceral pain sensitivity in healthy premenopausal women.

BACKGROUND: Factors that are associated with pain perception remain incompletely understood, especially in the visceral pain field. Therefore, the current study aimed to investigate possible psychological and biological predictors of visceral pain sensitivity in healthy subjects. METHODS: In a sample of 59 healthy premenopausal female subjects on hormonal contraceptives, measures of gastrointestinal (GI) symptoms in daily life, trait and state anxiety, depression, serum cortisol concentrations and serum levels of interleukin-6 (IL-6) were obtained, followed by assessment of rectal distension pain sensitivity measures (i.e., rectal distension sensory threshold, pain threshold and pain ratings for discrete rectal distension stimuli). RESULTS: Regression analyses showed that more GI symptoms in daily life predicted a lower pain threshold. Higher levels of state anxiety predicted a lower pain threshold. Higher cortisol concentrations predicted lower pain ratings. IL-6 was positively related to GI symptoms but was a non-significant predictor of pain threshold in the multiple regression analysis. CONCLUSIONS: Similar to findings in patients with functional GI symptoms, we showed that subclinical GI symptoms predict visceral pain sensitivity. In line with somatic pain findings, state but not trait anxiety was found to predict visceral pain sensitivity. Our finding on serum cortisol as positive predictor of pain sensitivity might be interpreted in light of immunosuppressive effects of cortisol. Our finding on the role of IL-6 in GI symptoms is promising for understanding GI complaints in patients and needs further investigation.