It has been known for >60 years that chemotherapy with cyclophosphamide can give rise to hemorrhagic cystitis in humans. This knowledge was used to establish an animal model for cyclophosphamide-induced cystitis to study bladder inflammation, functional changes and effects of interventions. Apart from inflammation per se, bladder overactivity is perhaps the most studied adverse effect of cyclophosphamide. However, a description of the time course for the development of bladder inflammation in relation to functional changes is lacking. Instead, numerous variants of the animal model have been used. In an effort to improve rationale, this study aimed to investigate the correlation of bladder inflammation and bladder overactivity over time in a rat model of cyclophosphamide-induced cystitis.

Thirty-six male Sprague-Dawley rats were included in the study. Each rat received a single intraperitoneal injection with either saline (n=12), serving as control, or cyclophosphamide (n=24; 100 mg/kg). After 0, 24, 48 or 72 hours the animals were placed in a metabolic cage for 16h, measuring micturition parameters, water consumption and urine production. Subsequently, the animals were euthanized and their bladders were excised and used for immunohistochemical evaluation of inflammation (H&E stain and inflammatory markers) and expression of functional receptors. Metabolic cage data were analyzed by one-way ANOVA. The H&E stains were evaluated with assistance from ChatGPT (GPT-5.3, OpenAI) to obtain grading of inflammation based on predefined criteria (oedema, urothelial thickness, urothelial disruption/ulceration, inflammatory cells, hemorrhage). Image analysis of protein expression was done by generating Python code in ChatGPT, prompting for measurement of total immunofluorescence as normalized by tissue area, and running the analysis code in the built-in Windows 11 command prompt. Immunohistochemical data were analyzed by non-parametric Kruskal-Wallis test.

Induction of bladder inflammation resulted in a significant increase in micturition frequency which persisted for 72h (Fig 1). No significant differences were observed regarding water intake or total urine production. The H&E staining showed signs of inflammation throughout the studied time period (0-96h; Fig 2). Expression of the inflammatory marker IL-6 gradually increased after cyclophosphamide injection and was significantly increased at later time points (48-96h). The expression of antioxidant enzymes was either unaltered (glutathione peroxidase) or significantly decreased in the urothelium (superoxide dismutase). The expression of functional receptors (muscarinic M3, β3 & α1 adrenoceptor, P2X3 purinoceptor) was unaltered at all time points.

A single injection with cyclophosphamide rapidly led to induction of bladder inflammation, accompanied by an increase in micturition frequency. Interestingly, bladder function recovered before the inflammation was resolved. The pro-inflammatory cytokine IL-6 was increased only at later time points. However, despite being a common marker for acute inflammation, IL-6 is a secondary (downstream) cytokine, mainly released from immune cells in response to primary cytokines such as TNF-α and IL-1β. It is currently demonstrated that bladder function is affected prior to detectable changes in downstream cytokine signaling and is restored before the inflammation is resolved. Considering that inflammation can suppress antioxidant enzyme expression, the data on the expression of superoxide dismutase and glutathione peroxidase are somewhat expected. Previous studies in animal models of cyclophosphamide-induced cystitis have occasionally demonstrated alterations in expression of functional receptors. However, in this study, using an AI based analysis method, we could not detect such changes.

The current study describes how induction of rat bladder inflammation with cyclophosphamide correlates with functional changes. Tissue damage and invasion of inflammatory cells occur rapidly, leading to functional changes. As normal bladder function returns, the inflammation still persists, albeit in a later-stage form. However, more data would improve the resolution of the time course description. Future studies should therefore examine additional inflammatory markers, urodynamic changes in vivo and alterations in key signaling molecules.