Bladder Urothelium Injury Model Induced In Rats By Exposure To Protamine Sulfate And Research Its Related Mechanism

Obiect:1. An acute and chronic animal model for injured bladder urothelium in rats was developed using intravesical infusion of protamine sulfate (PS).2. Research the changes of bladder urothelium,GAGs layer, mucosa permeability and mast cells after infusion of PS and the relationship of these changes, establish the investigative basis for bladder inflammatory disease, especially for Interstitial Cystitis.Materials and Methods:1. ObjectFemale Sprague-Dawley rats weighing 228-267g,,age 6-8 week,were used in this study.2. Main reagents and soucreProtamine sulfate Grade X:Sigma Chemical, St. Louis, Mo;goat anti-rat Sulfated Glycosaminoglycan ELISA kit, goat anti-rat Hyaluronic Acid ELISA kit:ADL,Inc. Periodic acid-Schiff stain kit:Guangdong shi li ke mao Limited Company.2×106 cmp/ml [14C]urea:Tainjin radiologic Institutes.3. Methods50mg PS diluted into 1 ml NS (0.4 ml) or NS alone(0.4 ml) was instilled transurethrally into the bladder for 30 min to establish an acute bladder urothelium injury model.50mg PS diluted into 1 ml NS (0.4 ml) or NS alone(0.4 ml) was instilled transurethrally into the bladder for 30 min,after 60min, intravesical infusion of 50mg/ml PS again. After two days repeat these handling, in all 20 circulates to establish an chronic bladder urothelium injury model.After injury model established, HE stain,toluidin blue stain, transmission electron microscope, Periodic acid-Schiff stain and mensuration of mucosa permeability were done.4.Statistical analysisStatistical analysis was carried out using SPSS16.0.A comparison between the groups was done using Student's t test for parametric data. The dates of different time in one group were compared with an analysis of variance (ANOVA). Values of p<0.05 were regarded as significant.Resulst:1. An acute animal model for injured bladder urothelium in rats was developed using intravesical infusion of 50mg/ml PS.In the model, the surface bladder urothelium slough, the construction of umbrella cell and tight junctions appare damaged, microvillus formation, abundant PMN and slight mast cell (P>0.05) infiltrated in the submucous layer and muscular layer, edema and vasocongestion in the bladder wall.By day 7 after exposure, the entire bladder wall appeared normal.2. An chronic animal model for injured bladder urothelium in rats was developed using repeatly intravesical infusion of 50mg/ml PS for 60 days.On the basic of acute animal model,the chronic animal model appares bladder wall thicken, mast cell infiltrate significant(P<0.05). Transmission electron microscope shows surface bladder urothelium become flatten, tight junctions appare widen and an increased number of both granulated and partial granulated mast cells.These changes are persistent.Bladder wall shows injury, repair and proliferation.3.PAS stain shows there is a continuous and homogeneous PAS+layer on the surface of umbrella cell. By day 1 after once exposure, PAS+layer to disappear,after 3 day,the PAS+layer discontinuous appare. By day 7 after exposure, the continuous and homogeneous PAS+layer appeared normal. The urinary HA/Cr levels were greater on all days after PS injection compared with the corresponding controls. The level of urinary S-GAG/Cr on days 1 and 7 in the PS group were greater than in the control group, (P<0.05)the HA/Cr level on day 1 was increased(P<0.05).After the injury of bladder urothelium,the intermediate cells maturate following by the increase of synthesis of GAGs.4. PS leads to a striking increase of urea permeability of bladder mucous layer at days 1(P<0.05),with recovery by day 7 (P>0.05). By day 7 after once exposure,the bladder urothelium recovery the impermeability of micromolecular solutes.Conclusions1. Urinary solutes are deleteriousness. The integrality of specialized structure of umbrella cell and tight junctions maintenance the impermeability of urine.The injury of bladder urothelium is principal step of whole process, inflammation of inflammation and activation of mast cells are both secondary reaction of damage of urothelium.2.S-GAG and its water molecular layer on the surface of bladder urothelium is imporant protection of umbrella cell and tight junctions,which can exclusion micromolecular toxic solutes form the surface of bladder.When S-GAG were neutralized by PS,the water molecular layer were injury,which cause urothelium damage. Dissolution of S-GAG and degradation of HA may take part in inflammatory reaction and repair of injury.3.In the acute and chronic animal model for injured bladder urothelium in rats was developed using intravesical infusion of PS,bladder wall structure, component of GAGs and permeability appare transient or persistent changes.These changes are similar to feature of IC, support the conclusion of dysfunction bladder urothelium may play an principal role in pathogenesis of IC.Meanwhile establish the investigative basis for bladder inflammatory disease, especially for Interstitial Cystitis.