Increased vaginal oxidative stress, apoptosis, and inducible nitric oxide synthase in a diabetic rat model: implications for vaginal fibrosis  Monica G. Ferrini, Ph.D., Gaby Nolazco, M.Sc., Dolores Vernet, Ph.D., Nestor F. Gonzalez-Cadavid, Ph.D., Jennifer Berman, M.D.  Fertility and Sterility  Volume 86, Issue 4, Pages 1152-1163 (October 2006) DOI: 10.1016/j.fertnstert.2006.01.058 Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 Collagen and smooth muscle contents in the vagina from diabetic and control rats. (A) Fresh tissue from rats injected with streptozotocin (diabetes group, filled bars) or with saline (control group, open bars), and killed 3 months later, was homogenized and used for hydroxyproline determinations in hydrolysates. (B) Formalin-fixed paraffin-embedded tissue sections were immunostained for ASMA, counterstained with hematoxylin, and examined at ×40. Bar = 100 μm. (C) Quantitative image analysis was applied to determine the relative intensity of ASMA expression per area. Open bars, control group; filled bars, diabetes group. *P<.05; **P<.01. Ferrini. Diabetes and vaginal fibrosis in the rat. Fertil Steril 2006. Fertility and Sterility 2006 86, 1152-1163DOI: (10.1016/j.fertnstert.2006.01.058) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Expression of profibrotic factors TGFβ1 and PAI-1 in vaginal tissue sections from diabetic and control rats. (A) Tissue sections adjacent to the ones presented in Figure 1 were immunostained for TGFβ1 and PAI-1, counterstained with hematoxylin, and examined at ×40. EP = epithelia; LP = lamina propria; MU = muscularis. Bar = 100 μm. (B) Quantitative image analysis was applied to determine the relative intensity of expression per area. (C) Tissue homogenates used for hydroxyproline determinations were processed by centrifuge, and the supernatants fractionated by polyacrylamide gel electrophoresis and submitted to Western blot, followed by the staining with Ponceau red and immunoreaction with the antibody used above. Each lane represents a separate homogenate. Densitometry evaluation of band intensities was performed and corrected by the intensities of the 90-kDa major band stained with Ponceau red. Open bars, control group; filled bars, diabetes group. *P<.05; **P<.01; ***P<.001. Ferrini. Diabetes and vaginal fibrosis in the rat. Fertil Steril 2006. Fertility and Sterility 2006 86, 1152-1163DOI: (10.1016/j.fertnstert.2006.01.058) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 Evaluation of oxidative stress in blood and vaginal tissue from diabetic and control rats. (A) Whole blood and tissue homogenates were treated with a special buffer to preserve ROS stability, and levels were estimated indirectly by the reduced–oxidized glutathione (GSH–GSSG) ratio. (B) Aliquots of the tissue homogenates used for Figure 2 were submitted to Western blot with an antibody against xanthine oxidoreductase (XOR), as in Figure 2. Each lane represents a separate homogenate. Densitometric evaluation of band intensities on the gel blot (not shown) was performed. (C) Tissue sections adjacent to the ones presented in the preceding figures were immunostained for Cu/Zn-SOD and were counterstained with hematoxylin and examined at ×400. Bar = 50 μm. (D) Quantitative image analysis was applied to determine the relative intensity of expression per area. OD = optical density. Open bars, control group; filled bars, diabetes group. *P<.05; **P<.01. Ferrini. Diabetes and vaginal fibrosis in the rat. Fertil Steril 2006. Fertility and Sterility 2006 86, 1152-1163DOI: (10.1016/j.fertnstert.2006.01.058) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 4 Programmed cell death in vaginal tissue sections from diabetic and control rats by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL). (A) Tissue sections adjacent to the ones presented in the preceding figures were submitted to TUNEL, counterstained with hematoxylin, and examined at ×400. EP = epithelia; LP = lamina propria. Bar = 50 μm. (B) Quantitative image analysis was applied to determine the relative proportion of apoptotic cells per area (apoptotic index × 100). Open bars, control group; filled bars, diabetes group. *P<.05. Ferrini. Diabetes and vaginal fibrosis in the rat. Fertil Steril 2006. Fertility and Sterility 2006 86, 1152-1163DOI: (10.1016/j.fertnstert.2006.01.058) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 5 Induction of iNOS and peroxynitrate formation in vaginal tissue sections from diabetic and control rats. (A) Tissue sections adjacent to the ones presented in the preceding figures were immunostained for iNOS, counterstained with hematoxylin, and examined at ×400. Bar = 50 μm. (B) Quantitative image analysis was applied to determine the relative intensity of expression per area and the proportion of positive cells per area. (C) Tissue homogenates used for other Western blots (Figs. 2 and 3) were fractionated by polyacrylamide gel electrophoresis and submitted to Western blot with the antibody used above, as described on Figure 2. Each lane represents a separate homogenate (left panels). Densitometry evaluation of band intensities was performed (right panels). (D) Tissue sections adjacent to the ones presented in the preceding figures were immunostained for nitrotyrosine, as above (not shown). Quantitative image analysis was applied to determine the relative intensity of expression per area and the proportion of positive cells per area. Open bars, control group; filled bars, diabetes group. *P<.05; **P<.01; ***P<.001. Ferrini. Diabetes and vaginal fibrosis in the rat. Fertil Steril 2006. Fertility and Sterility 2006 86, 1152-1163DOI: (10.1016/j.fertnstert.2006.01.058) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions