Estrogen Mediates Visceral Hypersensitivity Induced By Psychological Stress In Female Rats And Involves NR2B

BackgroundIrritable bowel syndrome (IBS) is a disorder characterized most commonly by cramping, abdominal pain, bloating, constipation, and diarrhea. As many as 20 percent of the adult population, or one in five Americans, has symptoms of IBS, making it one of the most common disorders diagnosed by doctors. It occurs more often in women than in men, and it begins before the age of 35 in about 50 percent of people. There is no specific test for IBS, although diagnostic tests may be performed to rule out other diseases.Visceral hypersensitivity (VH) is widely regarded as the reason for the development of functional gastrointestinal diseases, including functional dyspepsia and irritable bowel syndrome. N-methyl-D-aspartate (NMDA) receptor is involed in VH. The NMDA receptor subumit 2B (NR2B) takes the most important role in pain regulation and neuron plastic.A number of chronic pain syndromes, including IBS, fibromyalgia, and temporomandibular disorders, are more prevalent in women compared with men. In many cases, the severity of symptoms fluctuate with the menstrual cycle, suggesting that gonadal steroid levels impact pain severity. For example, IBS is two to three times more prevalent in women than men. Acute experimental pain studies in humans also report that threshold and tolerance to experimental pain is modulated across the menstrual cycle or by hormone replacement therapy. Whether the increasing severity of symptoms is attributable to elevated or depressed levels of a particular hormone or the cyclical variation of hormone levels across the menstrual cycle is not clear. Water avoidance stress (WAS), as a psychological stress, developed persistent VH in male rats. Ovarian steroids may, indeed, affect the hypothalamicpituitary- adrenal (HPA) axis response to stress in female rats by regulating the synthesis of stress mediators such as CRF or ACTH. Sex steroids also differentially regulate the expression of NMDA receptors, and the implication of NR2B has been demonstrated in VH.To explore the mechanism of estrogen on the altered VMR to colorectal distension in water avoidance stressed female rat, we further evaluated whether NR2B are involved in the estrogen contribution to stress-induced VH.Methods and results1 . Repeated exposure to water avoidance stress in female rats: a model for visceral hyperalgesiaTo better understand the mechanisms underlying this relationship between estrogen and VH we aimed to characterize changes in visceral nociception in rats exposed to 10 days of chronic psychological stress. Separate groups of Wistar female rats were surgically equipped with electrodes in the abdominal muscles for EMG recording 5 days before the beginning of the experiment. Female Wistar rats were submitted daily to either 1 h WAS or sham WAS for 10 consecutive days. The visceromotor response (VMR) to colorectal distension (CRD) at baseline and 4 days following chronic WAS finished. Colonic samples and plasma were collected to evaluated for structural and plasma CRF/ACTH changes under WAS. The brain sections were taken for neuronal activity in amygdala and paraventricular nucleus as assessed by c-Fos-like immunoreactivity (c-FLI).Our results shew that rats exposed to chronic WAS (but not sham stress) developed persistent visceral hyperalgesia. WAS rats plasma (ACTH 77.19 + 22.71) pg/ml, was lower than shamWAS (148.71±50.37) pg/ml (P<0.01) . The level of plasma CRF between WAS and sham WAS rats was not diffenent significantly. C-FLI of the WAS rats 25.64 + 5.39 was more than that of shamWAS rats 19.62 + 3.62 in amygdala (p<0.05), but not in paraventricular nucleus(26.12±5.74 VS 22.43 + 3.87).2. Estrogen mediates visceral hypersensitivity in female ratsBilateral ovariectomy (OVX) was performed 10 days before WAS. In order to investigate the effect of estrogen on visceral sensitivity induced by WAS, OVX rats received intracerebroventricular (ICV) infusion of estradiol (E2, water solutable estrogen, 1.0ug/5ul, per rat) or normal saline (NS, 5ul, per rat) 30 min before WAS or sham WAS. All groups underwent colorectal distension 4 days after WAS or sham-WAS. CRF/ACTH of those rats' plasma was measured The brains were taken for assess neuronal activity in amygdala and paraventricular nucleus as c-FLI. The dorsal root ganglia (DRG) LI /SI sections were taken for asscess P2X₃ immunoreactivity stained by fluorescein isothiocyanate-lectin.Our results shew that VH induced WAS was prevented by OVX. Treated with ICV E2, exhibited visceral hypersensitivity after WAS similar to that of intact rats. E2 did not change the level of plasma CRF/ACTH in the WAS rats. C-FLI in amygdala was much more than that of WAS+NS (p<0.05), but not in paraventricular nucleus. The expression of P2X₃ in DRG LI/SI of WAS+E2 was much more than that of WAS+NS (p<0.05)3. Estrogen affects P2X₃,NR2B mRNA expression in rats exposed to WASIn the third series experiment, groups of OVX rats were expose to WAS following ICV infusion E2 or NS. Then, real time PCR was taken for comparison the expression P2X₃ mRNA in DRG and NR2B mRNA in DRG or Anterior cingulate cortex (ACC) between two groups of WAS+E2 and WAS+NS.Our results shew that the expression P2X₃ mRNA of DRG in WAS rats treated with E2, was higher than that of treated with NS rats (p<0.05). The NR2B mRNA expression in ACC of WAS+E2 rats, was higher than that of WAS+E2 rats (p<0.05), but not NR2B mRNA expression in DRG..4. The role of estrogen and NR2B involed in estroen effect ion on VH induced by WASIn this series experiment, groups of OVX rats were expose WAS following ICV infusion E2 and estrogen receptor antagonist ICI 182,780 (WAS+E2+ICI), ICI 182,780 only (WAS+ICI), E2 and NR2B antagonist Ro25-6981 (WAS+E2+Ro), E2 and NMDA receptor antagonist AP5, the VMR was taken to evaluate the visceral hypersensitivity compared with the WAS+E2 and WAS+NS rats in the secod series experiment.The results shew that WAS+E2+ICI rats' VMR is less than WAS+E2 on 80mmHg CRD (p<0.05) . The VMR of WAS+E2+ICI rats, was still more on 60 mmHg,80mmHg CRD than WAS+NS rats (p<0.05) .WAS+E2+AP5 rats reponced less on 60 mmHg,80mmHg CRD than WAS+E2 rats (p<0.05) . Compared with WAS+E2 rats , WAS+E2+Ro rats reponced less on 80mmHg CRD than WAS+E2 rats (p<0.01) .5. Estrogen modulates the visceromotor reflex to acute colorectal stimulation in the female ratsOVX rats were ICV infused with E2 10 ug,E2 1.0 ug,E2 0. lug or NS, and responses to innocuous and noxious intensities of CRD were measured at 30 min and 4 h after injection.Our data shew that estrogen enhanced VH dose dependently. ICI 182,780 partialy blocked the effects of E2 on VH.Another series experiment was taken to assesse the work of estrogen receptors and NR2B antagonist on the VH. OVX rats received ICV infusion of either E2, E2+ICI 182,780, E2+Ro 25-6981, or E2+AP5; Responses to innocuous andnoxious intensities of CRD were measured at 30 min and 4 h after injection.Our data shew that E2+ ICI 182,780, E2+ Ro 25-6981, and E2+AP5 group VMR were less compared with that treated with E2 only. Ro 25-6981 and AP5 blocked the effects of E2.Conclusion1) Chronic water avoidance stress induces visceral hypersensitivity in female rats.2) Estrogen could enhance VH induced by water avoidance stress in female rats.3) Estrogen increases c-FLI in amygdala, P2X₃ immunoreactivity stained neuro and P2X₃ mRNA in DRG, NR2B mRNA in ACC in water avoidance stressed female rats.4) Estrogen mediates visceral hypersensitivity induced by water avoidance stress in female rats and involves NR2B.5) Estrogen modulates the VMR to acute colorectal stimulation in the female rats. NR2B may contribute to alterations in sensory processing in the female rats.