The Role Of Estrogen On Chronic Visceral Hypersensitivity In The IBS Model Rats And The Mechanism Of It

Irritable bowel syndrome(IBS) is a common and frequently encountered disease,lt has greatly affected the life quality of the patients,and also has great influence on Chinese and global social economy.The etiology and pathology of IBS are still not clear,so we still have no effective treatment for this disease.IBS is 2 - 3 times more prevalent in women than in men.The severity of pain symptoms in women with IBS fluctuates with the menstrual cycle.Therefore many researchers paid close attention to the relation between gender and pain.Women are easier to suffer from different kinds of pain disease.It was assumed that women are easier to be nervous and to go to see a doctor was the main reason.But the recent reports suggested,there were no difference in many psychiatry and psychology scores between men and women. The body surface pain threshold of women is significantly lower than that of men.But there were few researches on whether there was difference in visceral pain threshold between man and woman,and the results of these researches were contradictory.In the researches which considered there were difference in visceral pain threshold between man and woman,most of them thought gonadal hormone was the reason of the difference.Many researchers only used the estrous cycle of animal or the menstrual cycle of human being to deduce the gonadal hormone level,but didn't measure the gonadal hormone level directly.Although there is some corresponding relation between these physiological cycle and the gonadal hormone level, they can' t reflect the gonadal hormone level completely.As it was reported,though menstrual cycle didn't influence thermo-pain refleetion,but high estrogen level inplasma didhaverelationwithbody surface thermo-pain.So it is more convinced to measure estrogen level than to deduce the gonadal hormone level by physiological cycle.In this study,the estrogen level as well as the estrus cycle of rats were measured.There was pheriphery and central nervous system interaction in pain transmit.Gonadal hormone can interact with this system on different levels.Many studies indicated that the activation of NMDA receptors played important roles in the producing and maintaining of hyperalgia and allodynia.Bin Tang reported in acute visceral pain animal model,estrogen receptor alpha and the NMDAR1 subunit of the NMDA receptor were 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(AP-5) 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 NMDAR1 in the lumbosacral spinal cord compared to OVx rats. These datas suggest that estradiol increases spinal processing of visceral nociception by increasing NMDA receptor NMDAR1 subunit expression in acute visceral pain animal model.As we all know,chronic visceral pain has chronic visceral hypersensitivity,the pathogenesy of which is by far more complex than those of acute visceral pain.Acute visceral pain model can hardly simulate the chronic visceral hypersensitivity state of IBS patients.In 2000 Al-Chaer first reported a new model.It was constructed by neonatal mechanical colorectal irritants,the adult rats had visceral hypersensitivity for a long time. Al-Chaer' s model can simulate the characteristics of IBS patients.It provided a good animal model for the research on IBS pathogenesy.The nervous system transmitters and receptors functional status of this model is greatly different from those of the acute model.NMDA receptor expressions in the dorsal root gangalia(DRG) and spinal cord visceral correlated neurons of this chronic visceral pain animal model were upregulated.Under this condition,can estradiol influence chronic visceral pain by regulate the expression and phosphorylation of NMDA receptors?In this study,the chronic visceral pain and serum estrogen level of female IBS model rats were detected,the relation between them were analyzed.The chronic visceral pain degree of high estrogen and low estrogen female model rats were compared,ln order to avoid the interference of progestogen and other gonadal hormones,only ovariectomized rats and ovariectomized with estrogen substitute rats were used to determine the effect of estrogen on chronic visceral pain and the potential mechanism of it.The inhibit effects of AP-5(NMDA receptor antagonist) on chronic visceral pain and the NMDA receptor expression in the lumbosacral spinal cord and DRG between OVx rats and E2 rats were compared.This study will provide theory basis to the pathogenetic research and the clinical diagnosis and therapy of female IBS patients.1 The characteristics and evaluation of the chronic visceral pain model1.1 Pain thresholdMale and female model rats were made by improved Al-Chare' s method. The pain thresholds of model female rats were significantly lower than those of control female rats.The pain thresholds of model male rats were significantly lower than those of control male rats.No significant difference was found between the male and female model rats' pain thresholds. 1.2 Abdominal withdrawal reflex scoresThe AWR scores of model male rats were significantly higher than those of control male rats under 20-60mmHg CRD stimulation.The difference of AWR scores disappeared under 80 mmHg CRD stimulation.The AWR scores of model female rats were significantly higher than those of control female rats under 20 mmHg CRD stimulation.The difference of AWR scores disappeared under 40-80 mmHg CRD stimulation.1.3 Tail-flick latencyThe tail-flick latency of both model and control rats were compared.The tail-flick latency of model rats were significantly shorter than those of the control rats.1.4 The colorectal pathological changeThe distal colon descendens and rectal topographic histology of control rats and model rats were compared.Neither control nor model rats showed apparent pathological change under macroscopic observation and microscopic examination.1.5 The relation between serum estrogen level and estrus cycle of the female model ratsVaginal smear of 19 female model rats was observed.Serum estrogen level was detected in preoestrus,estrus,metoestrus and dioestrus.The serum estrogen level in preoestrus was significantly higher than those in estrus,metoestrus and dioestrus.No significant difference was found among the serum estrogen levels in estrus,metoestrus and dioestrus.1.6 Grouping of the animalsBoth model and control rats were divided into high estrogen(E2) group and low estrogen(OVx ) group.Both E2 and OVx rats were ovariectomized.Only E2 rats accepted estrogen(100μg,i.p.)two days before visceral pain measurement.2 The correlation between visceral pain and serum estrogen level 2.1 The electric discharge of male and female model ratsChange rate of electric discharge increased with CRD pressure.The difference of electric discharge change rates among each pressure was significant.Under 15 mmHg,30 mmHg,45 mmHg CRD pressure,electric discharge of EOMA of the model female rats was higher than those of the model male rats.2.2 The relation between electric discharge of EOMA of model female rats and their serum estrogen levelThe mean serum estrogen level of model female rat was 115.70±26.34 pmol/L,the minimun value was 84.50 pmol/L,the maximum was 154.20 pmol/L. A positive relation was detected between the electric discharge of EOMA of model female rats and their serum estrogen level under 20~80mmHg CRD pressure.2.3 The comparism of visceral pain sensitivity between high estrogen rats and OVx rats2.3.1 Vaginal smear results of ratsUnder microscopic examination,vaginal smear of E2 rats(high estrogen) showed many big polygonal keratinocytes,which is a typical estrous manifestation.This indicated high estrogen level in the rat,which can promote the maturation and cornification of the vagina epithelium cells. Vaginal smear of OVx rats(low estrogen) showed many small round epithelium cells,which is a typical dioestrus manifestation.This indicated low estrogen level in the rat.2.3.2 Comparism of electric discharge of EOMAUnder 20mmHg CRD pressure,the electric discharge of EOMAof E2 model rats was significantly higher than those of the OVx model rats.This demonstrated the visceral pain of E2 model rat was more sensitive than those of the OVx model rats.High estrogen level can lead to visceral pain sensitization.On the other hand,the electric discharge of EOMA of E2 control rats and OVx control rats didn't show significant difference under 20mmHg CRD pressure.This indicated estrogen can add to the high visceral pain sensitivity of model rats.3 The inhibited effect of chronic visceral pain by intrathecal administration of AP-5 and its relation with estrogen level.3.1 Intrathecal administration of AP-5 attenuated the visceromotor response3.1.1 Time-effect relationship of AP-5 inhibitionThe peak effect occurred 5 min after AP-5 administration.The effect decreased as time went on.5min,10min,15min after AP-5 administration, the visceromotor response were all significantly lower than those before AP-5 administration.This demonstrated intrathecal administration of AP-5 significantly attenuated the visceral pain of rats.3.1.2 The inhibited effect of intrathecal AP-5 in OVx and E2 model rats.5min,10min,15min,30min after AP-5 administration,the inhibited percent of OVx and E2 model ratsdidn' t show any significant difference.3.2 The expression of NMDARI in OVx and E2 model rats3.2.1 The expression of NMDARI in OVx and E2 model rats' DRGThe NMDARI expression in OVx and E2 model rats' DRG were compared by immunohistochemistry.The positive cells in the rat' s lumbar and sacral DRG were round or elliptic,their kytoplasm stainedbrown.Image analysis showed,there were no significant difference between the average opticals which stands for the NMDAR1 expression in E2 rats and those of the OVx rats.3.2.2 The lumbar and sacral spinal cord expression of NMDAR1 in E2 and OVx ratsThe NMDAR1 expression in OVx and E2 model rats' spinal cord dorsal horn neurons were compared by immunohistochemistry.NMDARI expression in both E2 and OVx rats' lumbar and sacral spinal cord were found.The kytoplasm stained brown.Image analysis showed,there were no significant difference between the average opticals which standed for the NMDAR1 expression in E2 rats' lumbar and sacral spinal cord and those of the OVx rats.3.2.3 Expression of NMDAR1 in intestinal tractNMDAR1 expression in intestinal tract internal nerve plexus was found.Its kytoplasm stained brown.In conclusion,this study indicates:1.Neonatal colonrectal distension can lead to chronic visceral sensitization in adult(after 8 wk) male and female rats.There was no conspicuous pathological change in colorectal topographic histology.This indicated that this model could be used to do research on IBS like chronic functional visceral pain.2.The visceral pain sensitivity of female model rats was higher than that of the male model rats.The discharging reactions of female rats' EOMA to CRD were positively related to serum estrogen level.Visceral pain sensitivity of E2 model rats was higher than that of OVx model rats.These results all support the suppose that estrogen can increase colorectal sensitivity.3.Intrathecal administration of AP-5(NMDA receptor antagonist) attenuated the visceromotor response.There was no significant difference between the effect of AP-5 in E2 and OVx rats.No significant difference was found between the NMDARI expression in E2 and OVx rats' spinal cord dorsal horn neurons.These datas suggest that estradiol doesn't increase processing of visceral nociception by increasing NMDA receptor NMDAR1 subunit expression in spinal cord.