Cyclophosphamide (CYP) is an alkylating agent used for a wide range of benign and malignant disease. CYP treatments are known to trigger hemorrhagic cystitis in patients and animal models. This can be explained by a known mechanism that Acrolein, a metabolite of CYP, is stored in the urinary bladder during the storage phase, damaging urothelial components. In both clinical and biomedical research, a significant effort has been made to avoid CYP/Acrolein-induced cystitis, while still maintaining the benefits of using an alkylating agent for therapy. As a result, lower doses of CYP are currently given to targeted patients in clinic compared to past treatments. Given that the majority of previous studies aimed to understand the mechanism of Acrolein-induced acute cystitis, little is known about the effects of repeated low-dose CYP/Acrolein on the histopathology and physiology of urinary bladder. Therefore, we aimed to establish a murine model recapitulating in vivo changes in urinary bladder among patients that receive a low-dose of CYP. As a substantial number of patients undergoing CYP treatment are known to undergo repeated periodic exposures, our murine model is designed with repeated treatment at intervals, which are known to be sufficient for reversal of a one time dose of CYP/Acrolein-induced voiding dysfunction.

The study protocol was approved by the Institutional Committee and all experiments were performed in accordance with relevant guidelines and regulations. To establish a murine model of repeated low-dose of Acrolein-induced bladder dysfunction, three doses of 10 ug Acrolein in 15 ul were transurethrally given to adult female C57BL/6 mice on day 0, 7 and 14 (N=9). Body weight was measured and voiding behavior was determined by filter paper assay (FPA) on day 1, 8, 15 and 30 post instillation (pi). Thus, mice in the Acrolein group have a seven day treatment interval in between the three doses of 10 ug Acrolein, and fourteen days of a treatment free period between the last dose of Acrolein treatment and test/tissue harvest. Histopathology of urinary bladder harvested from day 1 and 30 pi was determined by hematoxylin and eosin (H&E) stained formalin-fixed paraffin-embedded (FFPE) tissue slides sectioned with 5 um thickness. A murine model of Acrolein-induced acute cystitis was established by one dose of 85 ug Acrolein in 15 ul on day 0 (N=4). FPA was performed on 4, 24, 48 and 72 hours pi. For both murine models, 15 ul of PBS transurethral instillation was given to control groups (N=8 for repeated low-dose Acrolein model, N=4 for acute cystitis model). To further analyze data from FPA, cut-off points for voiding frequency and voided volume were established based on 90th, 75th, 50th, 25th and 10th percentiles of mice studied.

In a murine model of repeated low-dose of Acrolein-induced bladder dysfunction, mice that had both one and three time transurethral instillation of 10 ug Acrolein showed lamina propria edema (Figure C-J) determined by hematoxylin and eosin (H&E) stained urinary bladder from day 1 and day 30 pi. In this model, the first dose of 10 ug Acrolein did not trigger voiding dysfunction but the second dose triggered transient voiding dysfunction with high volume low frequency (HVLF) voiding (Figure K-M). Body weights were not different between PBS and Acrolein group (Figure B). In contrast, 85 ug of one time Acrolein-instilled mice showed low volume high frequency (LVHF) voiding, a typical pattern of voiding dysfunction previously shown in acute inflammation of lower urinary tract.

Repeated low-doses of 10 ug Acrolein-induced lamina propria edema which was not reversed after two weeks of a treatment free period. Furthermore, mice who received repeated low-doses of 10 ug Acrolein showed HVLF voiding, which is different from the phenotype of voiding dysfunction shown in 85 ug of one time Acrolein-instilled mice, LVHF voiding. Interestingly, mice that received repeated low-doses of 10 ug Acrolein at day 30 do not show altered voiding behavior while their urinary bladder maintained histopathological abnormality. This may represent a patient population that has asymptomatic urinary disease progression. Our study suggests that urinary symptoms among human subjects that have a prolonged low dose exposure to Acrolein may differ from previously reported urinary symptoms among patients that receive a high dose of Acrolein for their therapy.

Repeated low-dose of Acrolein triggered irreversible histopathological changes in mouse urinary bladder and transient voiding dysfunction.