| ObjectivePelvic nerve injury caused by birth, surgery or trauma is the common reasons of clinical colorectal dysfunction, which seriously influence the patient’s quality of life. It is hard to treat in the clinical practice. According to the animal experiments and clinical observation, the abnormal colorectal motility following pelvic nerves injury have a trend of adaptive recovery. However, the detailed regulatory mechanism is not clear. If we can clarify the regulatory mechanism of adaptative recovery, it is possible to find a intervention measures to accelerate or strengthen the adaptative recobery process. This study aims to examine the colonic transit and colonic mucosa TRPA1 protein expression after the pelvic nerves denervation, and to explore the relationship between the TRPA1 protein expression and the colonic transit recovery.Methods1.Subjects and groups96 adult male Sprague-Dawley rats were used in this experiment. the rats were only 6 weeks of age, body weight(200±10)g, and were randomly divided into 2 groups. Among them, 42 rats were used for the colonic transit study, which were divided into sham-operated and operated groups, each group contained 21 rats. Other 54 rats, which were divided into control group, sham-operated group and operated group, each contained 18 rats, were used to detect the colon mucosa TRPA1 protein expression,2.Model preparation2.1 Operated group:In this group, a ventral midline incision, about 3cm, was performed with the small intestinal isolated by a piece of wet gauze. The bladder, rectum, seminal vesicle and prostate were identified under anatomical lens. Behind the protate, the pelvic nerve separated gently with the swab was cut off. Close the incision with a 3-0 silk suture.2.2 Sham-operated group:Rat in this group take a laparotomy. Pelvic nerves were similarly exposed but not cut.Then close the incision with a 3-0 silk suture.Be sure to control models with the same surgical incision length.2.3 Control groupThis group without any treatment.2.4 Rats that used to detect the colonic transit need to be put a catheter into the cecum before closing abdomen. First,make a stoma in the cecum,then put a silicone catheter to the proximal colon through the stoma,next pouch suture the stoma with a 3-0 silk suture and fix the tube on the cecum. Finally,the proximal portion of the tube was brought through the left abdominal wall and subcutaneous tunnel to the posterior neck,fixed to the skin.3.Research indexs3.1 Using the method of radioisotope to evaluate the colonic transit.On the 1, 3 and 7 day post-operation, 0.2mL sodium chromate(51Cr) was injected into the colon throught the catheter. After 3h, the rats were killed and the whole colon were taken out and split into 10 parts. At the same time, feces during the 3 hours were collected and marked as part 11. Each part were measured by gamma counter measure. Finally, the geometric center(GC) of the distribution of 51 Cr were calculated by.3.2 Western Blotting: to detectthe TRPA1 protein expression of the proximal and distal colonic mucosa.Results1.Changes of colonic transit after pelvic nerves denervation.1.1 In operated group, colonic transit on postoperative day(POD) 3 showed significant recovery compared with POD1(p < 0.05). That of POD7 showed further recovery compared with POD3(p < 0.05).1.2 In sham-operated group, colonic transit on postoperative day(POD) 3 showed significant recovery compared with POD1(p < 0.05). While there was no significant difference between colonic transit on POD7 and POD3(p >0.05).1.3 At POD1, operated group have significantly delayed colonic transit compared with sham-operated group(p < 0.05). At POD3, differences between the two groups just in the same situation(p < 0.05). At POD7, there was no significant difference between the two groups(p >0.05).2. Changes of TRPA1 protein expression of the proximal colonic mucosa2.1 Comparing with TRPA1 protein expression of control group, sham-operated group and operated group, differences on POD1 were statistically significant(F=73.497,p<0.05). Use LSD analysis to compare each two groups. Comparing with that of control group, TRPA1 protein expression of sham-operated group and operated group both showed significant down regulation(p<0.05).Operated group expression showed more significant down regulation compared with sham-operated group(p<0.05). On POD3 and POD7, there was no significant difference between the three groups(F=2.087,0.656,p >0.05).2.2 Comparing with the expression on POD1, POD3 and POD7, differences in sham-operated group and differences in operated group both were statistically significant(F=5.651,41.005,p<0.05).These two groups showed the same situation. TRPA1 protein expression on POD3 were obviously higher than that on POD1(p<0.05). There were no statistically significant difference between POD3 and POD7(p>0.05).3. Changes of TRPA1 protein expression of the distal colonic mucosa3.1 Comparing with TRPA1 protein expression of control group, sham-operated group and operated group, differences on POD1 were statistically significant(F=58.773,p<0.05). Use LSD analysis to compare each two groups. Comparing with that of control group, TRPA1 protein expression of sham-operated group and operated group both showed significant down regulation(p<0.05).Operated group expression showed more significant down regulation compared with sham-operated group(p<0.05). On POD3, differences between three groups were statistically significant(F=8.680,p<0.05). There were no statistically significant difference between sham-operated group and control group(p>0.05).Comparing with sham-operated group and control group, TRPA1 protein expression of operated group showed statistically significant down regulation(p<0.05).On POD7, there was no significant difference between the three groups(F=1.319,p >0.05).3.2 Comparing with the expression on POD1, POD3 and POD7, differences in sham-operated group were statistically significant(F=16.975,p<0.05).Expression on POD3 were obviously higher than that on POD1(p<0.05). There were no statistically significant difference between POD3 and POD7(p>0.05).3.3 Comparing with the expression on POD1, POD3 and POD7, differences in operated group were statistically significant(F=50.417,p<0.05).Expression on POD3 were obviously higher than that on POD1(p<0.05). That of POD7 showed further up regulation compared with POD3(p < 0.05).Conclusion1. Transection of pelvic nerves delayed colonic transit significantly,and a clear trend to recovery was observed after surgery over time.2. The TRPA1 protein expression of the distal colonic mucosa was decreased significantly after pelvic nerves transection,and it demonstrated gradually creasing after surgery over time.3. The colonic transit and the TRPA1 protein expression of the distal colonic mucosa after pelvic nerves transection showed highly consistency in recovery trend.In conclusion, we found that the TRPA1 protein expression of the distal colonic mucosa was decreased significantly after pelvic nerves transection,and it showed highly consistency in recovery trend with the colonic transit. Accordingly, we think the colonic transit’s adaptability of recovery after pelvic nerves injury associated with changes of the expression of TRPA1 protein in distal colonic mucosa. |
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