Experimental Study On Urethraplasty Using Allograft Of Programmed Frozen-thawed New Zealand Rabbit's Bladder Mucosa

Experimental study on urethraplasty using allograft of programmedfrozen-thawed New Zealand rabbit's bladder mucosaPARTⅠEstablishment of a stable urethral stricture model by simulating the TURenvironment in New Zealand rabbitsObjective:To get the reason for iatrogenic urethral stricture after TUR operation,and simulate the TUR environment to explore the method of building a stable urethralstricture model in New Zealand white rabbits.Methods:Twenty male New Zealand white rabbits were randomly divided into twogroups,each group had 10 rabbits with the average weight of (2.0±0.1)kg.Afterintravenous anesthesia by sodium pentobarbital,we made models by stripping 1.0 cmlong urethral mucosa in groupⅠ,then suturing the incision and fixing the F 8 catheter.In models of groupⅡ,we repeated coagulating the urethral mucosa wound by bipolarcoagulation.We did microsurgery for all models by 10 times Optical microscope.Weobserved the daily micturation condition after operation for both groups.We did theantegrade urethrography at one month and two months postoperatively.Then weobserved the histological change of surgical urethra.Results:Two rabbits died after operation in groupⅠand one in groupⅡ,theremaining rabbits completed the test.One week later the catheter withdraweditself.We found the rabbits in groupⅡwere more irritable than those in groupⅠfromthe second month.At one month postoperatively,we did not find significance urethralstricture in both groups on antegrade urethrogram.But at two months postoperatively,we found significance urethral stricture with narrow lumen and discontinuous mucosa,for all 9 rabbits in groupⅡand only 2 in groupⅠ.We observed the histological changeof the surgical urethra,there was no evidence of epithelial cell growth but collagenfiber deposition.Conclusion:Repeated coagulation is an important reason for iatrogenic urethral stricture after TUR.we establish a way to build a stable,homogeneous and repetitiveurethral stricture model of new Zealand rabbits by simulating the TUR environment,help to research and treat urethral stricture. PARTⅡThe effect of programmed cryopreservation on immunogenicity ofNew Zealand rabbit bladder mucosaObjective:To evaluate the effect of programmed cryopreservation on immunogenicityof rabbit bladder mucosa and the intensity of immunological rejection caused by theallograft.Methods:Six New Zealand white rabbits were divided into three pairs.Each one ofevery pair was randomly marked as A or B.The immunogenicity was examinedthrough mixed lymphocyte cell response using the bladder mucosa (cryopreservationand non-cryopreservation)and spleen from pair of rabbits.Twelve New Zealandrabbit models of urethral stricture were prepared to accept the transplant of bladdermucosa,which were divided into two groups of cryopreservation andnon-cryopreservation group,another six normal rabbits were made as controlgroup.Two weeks later,The lymphocyte proliferation was detected aftertransplantation from transplanted rabbit blood and spleen.At the same time,theurethra from transplanted rabbit for immunohistochemical staining was performed,and the expressions of CD3,CD4 and CD8 were observed.The mRNA of bladdermucosa (cryopreservation and non-cryopreservation)from another six New Zealandrabbits was extracted and the expressions of RLA-Ⅰ,RLA-Ⅱand RLA-Ⅲgenewere detected by real-time PCR.Results:The ability of cryopreserved bladder epithelial cells stimulating allogeneiclymphocyte proliferation from the cryopreservation group was significantly weakerthan that from the non-cryopreservation group.The blood and spleen lymphocytesfrom transplanted rabbit bladder mucosa without cryopreservation showed higherproliferation rate than those with cryopreservation.Compared with thenon-cryopreservation group,the expression of CD3+ and CD8+ T cells infiltrated inthe transplanted locus of bladder mucosa was decreased in the cryopreservation group.The expressions of RLA genes didn't change significantly after cryopreservation. Conclusion:Programmed cryopreservation of rabbit bladder mucosa could reduce itsimmunogenicity in the allotransplantation.and thus restrict the extent ofimmunological rejection. PARTⅢExperimental study on the treatment of urethral stricture using tubularizedrabbit bladder mucosa after programmed cryopreservationObjective:To confirm that cryopreservation technique is able to protect tissuestructure from the ultra low temperature environment.And to observe the surgicaleffect of substitution urethroplasty using allograft cryopreserved rabbit bladdermucosa,and compared with control group,in order to confirm allograft cryopreservedbladder mucosa is good material for substitution urethroplastyMethods:We harvested bladder mucosa from six New Zealand rabbits,then dividedinto two groups(cryopresevation and non-cryopresevation),(1)we compared thehistological change of H.E.stained bladder mucosa before and aftercryopreservation.(2)we compared the electron microscopic change of bladder mucosabefore and after cryopreservation.12 models were randomly divided into two groups,6 rabbits in each group with the average weight of (2.1±0.2)kg.Then we tookanother 6 rabbits,after intravenous anesthesia by 3% sodium pentobarbital,weharvested two pieces of fresh bladder mucosa (10*8mm),and one piece should beprogrammed cryopreserved.In test group,we performed the tubularized graftsubstitution urethroplasty using cryopreserved bladder mucosa for urethral stricture,and in control group we did the same operation but using non-cryopreserved bladdermucosa from the same rabbit.(3)we compared the urethral sample of surgical part atone week postoperatively.(4)we compared the histological changes of H.E.stainedurethral sample at one week postoperatively.(5)we compared the result of antegradeurethrography at two months postoperatively.(6)we compared the H.E.stainedurethral sample at two months postoperatively.Results:There was no obvious histological change and electron microscopic changeof bladder mucosa before and after cryopreservation,but still a few epithelial cellswere shedding.The inflammation reaction of surgical part in test group was obviouslyharder than those in control group in one week after surgery.According to the resultsof antegrade urethrography,urethral continence and lumen diameter were better than those in control group in two months after surgery.Pathological result suggested thattwo weeks after surgery the urethral epithelial cells were growing well,but in controlgroup lots of epithelial cells were necrosis.In two months later there was a lay ofurethral epithelial in test group,but in control group we could only see scar because ofthe severe inflammation.Conclusion:Programmed cryopreserved technique is able to maintain the mucosastructure.Because of the high success rate of surgery,we conclude that cryopreservedbladder mucosa could be used as an allograft substitution for urethroplasty in thetreatment of urethral stricture.