Figure 1-6940.

A:Ex vivo model of bladder distension. Bladders of female Sprague-Dawley rats (100–120 g) were surgically exposed, and ureters were ligated. Bladders were catheterized per urethra and removed with sutures that firmly held the catheters in the urethra. For blocking, 1-hour pre-incubations were performed in medium containing the relevant inhibitors. After blocking, bladders were distended by filling the bladder with culture medium to 40 cm of hydrostatic pressure using water manometry or were not distended. Sham controls included ligated, catheterized, but uninflated bladders. White arrows indicate the ligated ureters. Black arrows point to the catheterized urethra, which has been sutured to hold the catheter in place during distension. B: Bladder hyperdistension stimulates in situ gelatinase activity. Ex vivo bladders were examined by in situ zymography. FITC-gelatin layered onto cryosections of ex vivo bladders is digested by MMP activity. Nonfluorescent dark zones (arrowheads) represent digestion of the FITC-gelatin, localizing regions of gelatinase A/B activity. Bottom panel: Hoechst stain of serial section to show nuclei. Ur, urothelium; LP, lamina propria; D, detrusor; S, serosa. Original magnification, ×125. C: Net gelatinase activity of bladders is increased during distension ex vivo. Gelatinases in the conditioned medium released the quenched activity of FITC in the DQ-gelatin. Doxycycline inhibited the increase in gelatinase A/B activity from distended bladders, P = 0.0126; n = 3. D: Secretion of active MMP-2 is increased in distended bladders and inhibited by doxycycline. Pro- and active-MMP-2 are analyzed by Western blotting in the conditioned medium of distended (D) and nondistended (ND) bladders +/− Doxycycline (Dox) pretreatment for 1 hour. E: Bladder distension stimulates in situ BSMC proliferation. BSMC proliferation was assessed by BrdU incorporation and detected using anti-BrdU and anti-mouse-Alexa 488 antibody on cryosections. Bladders were distended by hydrodistension in serum-free medium with BrdU or were not distended. Distended bladders showed increased BrdU incorporation compared with undistended. Ur, uroepithelium; D, detrusor smooth muscle layer; L, lumen. n = 3. Original magnification, ×125.