1. Translocation of NF-κB and Expression of Cyclooxygenase-2 Are Enhanced by Ketamine-Induced Ulcerative Cystitis in Rat Bladder 
Yung-Shun Juan, Yi-Lun Lee, Cheng-Yu Long, Jhen-Hong Wong, Mei-Yu Jang, Jian-He Lu, Wen-Jeng Wu, Yen-Shun Huang, Wei- Chiao Chang, Shu-Mien Chuang 
The American Journal of Pathology 
Volume 185, Issue 8, Pages (August 2015)
DOI: /j.ajpath
Copyright © 2015 American Society for Investigative Pathology Terms and Conditions

2. Figure 1
The effects of ketamine and cyclooxygenase (Cox)-2 inhibitor treatment on cystometric parameters and voiding behavior. A: Cystometry recordings illustrate micturition pressure, voiding volume, and frequency, including voiding contraction (arrows) and nonvoiding contraction (asterisks). B: Trace recordings illustrate micturition frequency and voiding volume in each treatment group.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2015 American Society for Investigative Pathology Terms and Conditions

3. Figure 2
The effects of ketamine and cyclooxygenase (Cox)-2 inhibitor on bladder damage and interstitial fibrosis. A–C: Representative microimages of saline (A), ketamine (B), and ketamine + COX-2 inhibitor (C) groups are shown by Masson's trichrome stain. A: In the saline group, there are three to five layers of urothelium and only sparse collagen distribution between the DSM bundles. B: The ketamine group shows denuded urothelial mucosa (star), erythrocyte debris under urothelium (arrowhead), mononuclear cell infiltration (yellow arrows), and increased connective tissue fibrosis (black arrow). C: However, the ketamine + Cox-2 inhibitor group shows decreased fibrosis (arrow) and mononuclear cell infiltration. The star indicates urothelium. D–F: Profibrotic proteins of transforming growth factor (Tgf)-β (D), fibronectin (E), and type I collagen (F) were measured by Western blot analysis in bladder tissue. The protein expression was quantified against glyceraldehyde-3-phosphate dehydrogenase (Gapdh). Results were normalized as the saline group = 100%. Data are expressed as means ± SD. n = 10. ∗P < 0.05, ∗∗P < 0.01 versus saline group; ††P < 0.01 versus ketamine group. DSM, detrusor smooth muscle; SL, suburothelial layer; UL, urothelial layer.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2015 American Society for Investigative Pathology Terms and Conditions

4. Figure 3
A–C: Immunofluorescence analysis of NF-κB p65 and cyclooxygenase (COX)-2 expression after treatment with saline (A), ketamine (B), or ketamine + COX-2 inhibitor (C) in bladder tissue. Double immunostaining of NF-κB p65 (green, left panels) and COX-2 (red, arrowheads, middle panels) is shown in urothelial layer (UL; areas within the dotted lines). Nuclear DNA was labeled with DAPI (blue). The merged image from left and middle panels (yellow, right panels) is shown. Erythrocytes (arrows) in the UL and suburothelial layer (SL) are identified in ketamine group. The arrowhead (B) indicates NF-κB and COX-2 co-stained cell. D: Western blots of Cox-2 expression and quantification of the percentage of COX-2 to glyceraldehyde-3-phosphate dehydrogenase (Gapdh) in bladder tissue. E: Real-time quantitative PCR was also applied to assess the COX-2 mRNA expression in the UL of the bladder. Data are expressed as means ± SD. n = 12. ∗∗P < 0.01 versus saline group; ††P < 0.01 versus ketamine group.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2015 American Society for Investigative Pathology Terms and Conditions

5. Figure 4
AlamarBlue and MTT analyses of bladder urothelial cell viability. A–F: Primary urothelial cells treated with ketamine and urine show significant dose-dependent reductions in cell viability (A and B) and MTT test results (C–F). G and H: Quantitative RT-PCR of rat urothelial cells after ketamine and urine treatment was applied to the transcriptional level of cyclooxygenase (Cox)-2 mRNA expression. Data are expressed as means ± SD. n = 10. ∗P < 0.05, ∗∗P < 0.01 versus saline group.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2015 American Society for Investigative Pathology Terms and Conditions

6. Figure 5
A–F: Immunofluorescence analysis of NF-κB p65 and cyclooxygenase (Cox)-2 expression in rat primary urothelial cells after 4-hour treatment with ketamine (A–C) or urine (D–F) obtained from rats treated with saline (A and D), ketamine (B and E), or ketamine + COX-2 inhibitor (C and F). Double-labeled immunofluorescence analysis of NF-κB p65 (fluorescein isothiocyanate; green, left panels), Cox-2 (rhodamine; red, middle panels), and ectodermal dysplasia (Ed)-1 (rhodamine, red, middle panels) proteins was performed. Nuclear DNA was labeled with DAPI (blue). The merged image from left and middle panels (yellow, right panels) is shown. Inset, B and E: NF-κB p65 with ED-1 co-staining. The arrows refer to NF-κB p65+/COX-2+ co-staining in urothelial cells, and the arrowheads indicate NF-κB p65+/ED-1+ co-stained in macrophages. G: Quantitative analysis of NF-κB p65+ cells colabeled with COX-2 or Ed-1 shows that ketamine and urine treatment stimulates NF-κB p65 and COX-2 expression and activates macrophage in urothelium. Data are expressed as means ± SD. n  = 10. ∗P < 0.05, ∗∗P < 0.01 versus saline group.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2015 American Society for Investigative Pathology Terms and Conditions

7. Figure 6
The effects of ketamine and urine treatment on NF-κB p65 translocation and cyclooxygenase (Cox)-2 expression on primary urothelial cell culture. A–F: Confocal microscopy was performed to examine the nuclear and cytoplasmic morphology of urothelial cells as well as NF-κB p65 and Cox-2 expression after treatment with ketamine (A–C) and urine (D–F). Double-labeled immunofluorescence study on primary urothelial cells with NF-κB p65 (fluorescein isothiocyanate; green, left panels) and Cox-2 (rhodamine; red, arrows) proteins was performed. Nuclear DNA was labeled with DAPI (blue). The merged image from left and middle panels (yellow, right panels) is shown. G–I: Protein levels of NF-κB p65 in nuclear and cytoplasmic extracts were investigated using Western blot analysis after urine treatment. Data are expressed as means ± SEM. n = 10. ∗P < 0.05, ∗∗P < 0.01 versus saline group; ††P < 0.01 versus ketamine group.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2015 American Society for Investigative Pathology Terms and Conditions

8. Figure 7
Urine ketamine and its metabolites induce NF-κB translocation and regulate cyclooxygenase (COX)-2 gene (PTGS2) in human J82 cells. A–C: Double-labeled immunostaining analysis detects the expression of NF-κB p65 (fluorescein isothiocyanate; green, arrows, left panels) and COX-2 (rhodamine; red, arrows, middle panels) in human J82 cells stimulated with urine for 4 hours after treatment with saline (A), ketamine (B), or ketamine + COX-2 inhibitor (C). Nuclear DNA was labeled with DAPI (blue). The merged image from left and middle panels (yellow, right panels) is shown. The arrows (right panels) indicate NF-κB p65/COX-2 co-staining. D: Quantitative analysis of percentage of NF-κB p65+ cells co-stained with COX-2. ∗∗P < 0.01 versus saline group; ††P < 0.01 versus ketamine group. Scale bar = 100 μm.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2015 American Society for Investigative Pathology Terms and Conditions

9. Figure 8
NF-κB binding site is an essential promoter binding area for cyclooxygenase (COX)-2 activation. A–D: J82 cells were transfected with different mutants of COX-2 promoter plasmids [pXC 918 (B), pXC 250 (C), and pXC 80 (D)] and stimulated with urine for 4 hours. E: The effect of NF-κB inhibitor (BAY ) on COX-2 expression after urine stimulation. J82 cells were transfected with COX-2 promoter plasmid (pXC 918) for 24 hours. The cells were pretreated with or without BAY for 30 minutes and then stimulated with urine. F: The effect of COX-2 inhibitor on COX-2 expression. J82 cells were transfected with pXC 918 for 24 hours. The cells were then treated with urine obtained from rats treated with saline, ketamine, or ketamine + COX-2 inhibitor. Luciferase activity and protein concentration in cell lysates were determined and normalized to the saline group. Data are expressed as means ± SEM. n = 10. ∗P < 0.05, ∗∗P < 0.01 versus saline group; ††P < 0.01 versus ketamine group. AP2, activator protein 2; CRE, cAMP-response element; GAS, gamma activating site; NF-AT, nuclear factor of activated T cells; SP1, specificity protein 1.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2015 American Society for Investigative Pathology Terms and Conditions

10. Figure 9
A model illustrates ketamine and its metabolites mediated COX-2 gene (PTGS2) activation. Stimulation of cells by ketamine and its primary metabolite (norketamine) induces nuclear translocation of NF-κB that regulates COX-2 gene activity. NF-κB inhibitor (BAY ) can inhibit NF-κB translocation. The activated NF-κB is then translocated into the nucleus, where it binds to specific sequences of DNA and thereafter transcribes downstream COX-2 gene expression. AP2, activator protein 2; CRE, cAMP-response element; GAS, gamma activating site; P, phosphorylation; SP1, specificity protein 1.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2015 American Society for Investigative Pathology Terms and Conditions