Cyclooxygenase-2 expression on ifosfamide-induced hemorrhagic cystitis in rats

Abstract

Purpose: Hemorrhagic cystitis (HC) is a limiting side effect of chemotherapy with ifosfamide (IFS). In this study, we investigated the participation of cyclooxygenase-2 (COX-2) upon ifosfamide-induced HC.

Methods: Male Wistar rats (150-200 g; six rats per group) were treated with saline, IFS (400 mg/kg, i.p.) and analyzed by changes in bladder wet weight, macroscopic and microscopic parameters, and COX-2 expression. In other groups etoricoxib (selective COX-2 inhibitor), indomethacin (non-selective COX inhibitor), thalidomide (selective TNF-alpha inhibitor), pentoxifyllin (non-selective TNF-alpha inhibitor) were added 1 h before IFS administration. The classical protocol using three doses of Mesna was also evaluated and compared with two extra doses of etoricoxib or indomethacin.

Results: COX-2 was expressed significantly 24 h after IFS administration mainly in myofibroblasts and mast cells evaluated by immunohistochemistry. Treatment 1 h before IFS injection with etoricoxib, indomethacin, thalidomide, and pentoxifylline reduced COX-2 expression and some macroscopic and microscopic parameters in IFS-induced HC. Moreover, addition of etoricoxib or indomethacin with the last two doses of Mesna was more efficient than three doses of Mesna alone when evaluated microscopically.

Conclusions: COX-2 participates in the pathogenesis of IFS-induced HC and the treatment with COX and TNF-alpha inhibitors reduced COX-2 expression. The addition of COX-inhibitors to the last two doses of Mesna represents a new therapeutic strategy of preventing HC.

Fig. 1

Effect of different indomethacin treatments on bladder wet weight in ifosfamide-induced hemorrhagic cystitis. The results are reported as mean ± SEM/100 g body weight (n = 6 per group). Asterisk P < 0.05 when compared to control group (C, treated with saline alone) and Hash P < 0.05 when compared to vehicle (Saline, treated with ifosfamide alone) by ANOVA and Bonferroni’s tests

Fig. 2

Effect of different etoricoxib treatments on bladder wet weight in ifosfamide-induced hemorrhagic cystitis. The results are reported as mean ± SEM/100 g body weight (n = 6 per group). Asterisk P < 0.05 when compared to control group (C, treated with saline alone) and Hash P < 0.05 when compared to vehicle (Saline, treated with ifosfamide alone) by ANOVA and Bonferroni’s tests

Fig. 3

Effect of thalidomide and pentoxyfilline treatments on bladder wet weight in ifosfamide-induced hemorrhagic cystitis. The results are reported as mean ± SEM/100 g body weight (n = 6 per group). Asterisk P < 0.05 when compared to control group (C, treated with saline alone) and Hash p<0.05 when compared to vehicle (Saline, treated with ifosfamide alone) by ANOVA and Bonferroni’s tests

Fig. 4

Effects of the treatment of one dose of Mesna plus two doses of the association (Mesna plus indomethacin—M + I) or (Mesna plus etoricoxib—M + E) on the increase in bladder wet weight of ifosfamide-induced hemorrhagic cystitis when compared to mesna-treated animals only (MMM—three doses of Mesna). The results are reported as mean ± SEM/100 g body weight (n = 6 per group). Asterisk P < 0.05 when compared to control group (C, treated with saline alone) and Hash p<0.05 when compared to vehicle (Saline, treated with ifosfamide alone) by ANOVA and Bonferroni’s tests

Fig. 5

Immunohistochemistry for COX-2 protein. a Bladder of animals treated only with saline. Constitutive COX-2 is seen within urothelium. b Bladder of animals treated with Ifosfamide (400 mg/kg) 24 h before, showing high expression of COX-2 mainly in subepithelial cells underneath urothelium (a and b, ×100, Bar 300). c and d The same as a and b, respectively (×400, Bar 100 μm). e, f, and g Bladder of animals 24 h after treatment with Etoricoxib (6 mg/kg), Indomethacin (2.0 mg/kg) or Thalidomide (45 mg/kg), respectively, and Ifosfamide 1 h later, with almost complete absence of COX-2 marked cells (×400, Bar 100 μm). h Bladders of animals treated with three doses of Mesna and Ifosfamide with some urothelial and subepithelial edema seen along constitutive COX-2 in urothelium. Ur urothelium, L bladder lumen, S subepithelial cells

Fig. 6

Immunohistochemistry for actin, vimentin, CD117, Factor XIIIa, CD68, and CD45 in bladder of animals treated with Ifosfamide and with high positivity for COX-2 protein (Fig. 5b). Several actin and vimentin positive cells are seen below urothelium sharing the same morphology and localization for COX-2 positive subepithelial cells (arrows). Weakly CD 117 positivity is present (arrows). Absence of positivity in subepithelial cells for CD68, Factor XIIIa, and CD45 is also seen (×400, Bar 100 μm). Ur urothelium, L bladder lumen, S subepithelial cells