Changes Of Colonic Transit,rectal Sensitivity And The Expression Of Signal Molecules In The Distal Colon Of Mice After Pelvic Nerve Denervation

Background:Dysfunction of colorectal motility caused by injury of pelvic nerve is very common in colorectal surgery.It often can cause symptoms like abdominal distension and pain,intractable constipation,poor stool output and straining feeling in anus and so on,which has a strong impact on patients’ quality of life and is very hard to be dealt with[1].There are many key points which can lead in injury of autonomic nerve in laparoscopic colorectal surgery.A study shows that the occurrence rate of dysfunction of sphincter ani by dissociating rectum in rectal resection for lower rectal cancer reaches up to 71%[2].It has been observed clinically that some patients suffered from colorectal dysfunction could recover as time went on,which suggests that there may be an adaptive restoration mechanism in the organism.A study found that the colonic transit of PND(pelvic nerve denervation)rats can recover in about a week[3].We found that the expression of TRPA1 protein in the distal colonic mucosa of PND rats obviously decreased after operation,but as time went on it showed a sign of recovery[4].And the mechanism of the recovery of colonic transit has a close relative with the expression level of TRPA1 protein in the distal colonic mucosa in rats.This suggests that TRPA1 may play an important role in the adaptive restoration mechanism of colorectal motility in PND rats.TRPA1 is a molecular sensor expressed in EC(enterochromaffin)cells and can perceive physical or chemical stimuli and mediate EC cells to release 5-HT to regulate and control intestinal motility[5].While among the numerous 5-HT receptors,5-HT3 and 5-HT4 receptors are mainly concerned in the sensory neurons of colonic mucosa[6].This study aims to establish the PND model in mice,to deeply research the role of TRPA1 in the adaptive restoration mechanism of colorectal motility in PND mice by TRPA1 knockout mice,and to observe the alterations of 5-HT3 and 5-HT4 receptor.Objective:1.To establishment pelvic nerve denervation model in mice.2.To observe the alterations of TRPA1 expression in distal colon in PND mice.3.To research the role of TRPA1 in the adaptive restoration mechanism of colorectal motility in PND mice by TRPA1 knockout mice.Methods:1.Experimental subjects and groups(1)108 adult male C57 mice were randomly divided into 3 groups.36 mice for colonic transit test,18 mice in the sham operation group and 18 mice in the PND group.36 mice for visceral sensitivity test,18 mice in the sham operation group and 18 mice in the PND group.36 mice for the detection of TRPA1 in distal colon,18 mice in the sham operation group and 18 mice in the PND group.(2)Establish PND models with 18 WT mice and 18 TRPA1 knockout mice for the detection of 5-HT3 and 5-HT4 receptor in distal colon.2.Model preparation(1)PND group: transect the pelvic nerves after a laparotomy.(2)Sham operation group: do not transect the pelvic nerves after a laparotomy.(3)Colonic transit test: embed a silicone tube in cecum near colon.Visceral sensitivity test: embed two platinum electrodes in obliquus extern abdominis.3.Detection index(1)Colonic transit test: assess colonic transit respectively at POD 1,3,and 7 by injecting methylene blue into colon and calculating the percentage of colored colon by methylene blue in the whole colon 20 minutes after injection.(2)Visceral sensitivity test: the alteration of visceral sensitivity was evaluated by measuring the viscero-motor response(VMR)to graded colorectal distension(CRD)at POD 1,3,and 7.(3)Western Blot: detect the expression level of the signal molecule in the distal colonic mucosa at POD 1,3,and 7.Results:(1)Uroschesis occered to all the mice of the PND group and we had to massage the bladder to assist the urination of them.Colonic transit test showed that the colonic transit of the PND group reduced significantly at POD 1 and 3 compared with the sham operation group(P<0.001,P=0.017),while the colonic transit of the PND group recovered as time went on.At POD 7,there was little difference between the colonic transit of the PND group and the sham operation group(P>0.05).Visceral sensitivity test showed that compared with the sham operation group,the rectal sensitivity of the PND group reduced significantly at POD 1,3,7(P=0.011,0.029,0.007),but the rectal sensitivity of the PND group also recovered as time went on.(2)The expression of TRPA1 protein in the distal colon of the PND group reduced significantly at POD 1 and 3 compared with the sham operation group(P<0.001,P=0.001),while the expression of TRPA1 protein of the PND group recovered as time went on.At POD 7,there was little difference between the expression of TRPA1 protein of the PND group and the sham operation group(P>0.05).(3)After pelvic nerve denervation,both in TRPA1 knockout mice and WT mice,the expression of 5-HT3 and 5-HT4 receptor protein in the distal colon recovered as time went on.The expression of 5-HT3 receptor protein in the distal colon of TRPA1 knockout mice reduced significantly at POD 1 and 3 compared with WT mice(P=0.02,0.048).The expression of 5-HT4 receptor protein in the distal colon of TRPA1 knockout mice reduced significantly at POD 1,3and 7 compared with WT mice(P<0.001,P=0.036,0.011).Conclusions:(1)The colonic transit and the rectal sensitivity will reduce significantly after PND,but these changes can recover as time goes on.(2)Denervation of pelvic nerves can obviously reduce the expression of TRPA1 protein in the distal colon of mice,but this change can recover over time.(3)After PND,the expression of 5-HT3 and 5-HT4 receptor of TRPA1 knockout mice can recover over time,but not so fast as that of WT mice.