The effect of programmed cryopreservation on immunogenicity of bladder mucosa in New Zealand rabbits.

A significant reduction in immunogenicity has been observed in some frozen–thawed tissues after cryopreservation. The objective of this study was to evaluate the effects of programmed cryopreservation on immunogenicity of rabbit bladder mucosa and on the extent of immunological rejection caused by the allograft. This study would provide theoretical support for the application of allogenic frozen–thawed bladder mucosa in the treatment of urethral stricture. Forty-two adult male New Zealand rabbits were used in this study. The immunogenicity was detected by mixed lymphocyte reaction using the allograft of bladder mucosa (fresh and frozen–thawed) and spleen lymphocytes. Twelve urethral stricture models were established in New Zealand rabbits for substitution urethroplasty using the allograft of bladder mucosa, which were divided into fresh and frozen–thawed group. Two weeks after operation, lymphocyte proliferation was detected in both blood and spleen of recipient rabbits. At the same time, immunohistochemical staining of urethral allograft was performed and the expression of CD3, CD4 and CD8 were observed. The mRNA of bladder mucosa (fresh and frozen–thawed) was extracted and the expressions of RLA-I, RLA-II and RLA-III gene were detected by real-time PCR. By mixed lymphocyte reaction, we found that allogenic lymphocyte proliferation stimulated by frozen–thawed bladder epithelial cells was significantly weaker than that of the fresh cells. The blood and spleen lymphocytes from fresh bladder mucosa group showed significantly higher proliferation rate than frozen–thawed group. Compared with the fresh group, the expression of CD3+ and CD8+ T cells infiltrated in the operation locus of bladder mucosa urethroplasty was significantly decreased in the frozen–thawed group. However, the expressions of RLA genes did not change significantly after the freeze–thaw procedure. This study demonstrates for the first time that a programmed freeze–thaw procedure of rabbit bladder mucosa could reduce its immunogenicity in allogenic bladder mucosa urethroplasty and thus restrict the extent of immunological rejection, therefore, provides theoretical support for the application of frozen–thawed bladder mucosa in the treatment of urethral stricture.