Hypothesis / aims of study
Cyclophosphamide is a chemotherapeutic agent used for the treatment of breast cancer and lymphoma. Cyclophosphamide is metabolised to acrolein, which is highly toxic and excreted into urine, which can cause severe bladder inflammation with immune alterations (1). The purinergic P2X7 receptor is highly expressed by many immune cells and it is known to regulate the expression and secretion of inflammatory mediators including IL-1β and TNF-α (2). P2X7 receptor activation leads to the formation of a large membrane pore, which results in inflammasome activation and cell apoptosis (3). This study aimed to i) develop an ex-vivo model of urothelial inflammation by perfusing porcine bladders with acrolein, ii) to determine if urothelium inflammation affects the physiological function of the bladder and iii) to determine if pharmacological blockage of P2X7 receptor can protect against acrolein-induced inflammation and damage in the bladder.
Study design, materials and methods
The whole bladders (n=9) from 2-month-old female pigs were placed in 100-ml organ baths containing Krebs-Henseleit solution maintained at 37°C and perfused for 4 hours with carbogenated RPMI culture media (1640 medium Sigma-Aldrich), in the presence or absence of 0.05% acrolein (110221 Sigma-Aldrich) via a fine tube that was inserted into the bladder through the urethral orifice.
After 4 hours of perfusion, each bladder was dissected into 5 x 10 mm strips: intact strips (containing the entire layer of the bladder wall), detrusor strips and mucosal strips. The contractility of each strip in response to acetylcholine (ACh) was measured. Histology staining and immunohistochemistry of caspase-3 (ab2302 Abcam, 1:200), a marker for cell apoptosis, were performed to determine the degree of tissue damage and apoptosis.
To study the role of P2X7 receptor, bladders were pre-treated with the P2X7 receptor antagonist A804598 (Sigma-Aldrich) for 1 hour, prior to 4 hours perfusion with acrolein, to determine the effect of this antagonist on inflammatory responses.
The histology staining showed that in the perfusion control group the structure of the bladder was maintained (Figure 1A). The urothelium, however, was markedly damaged by acrolein perfusion (Figure 1B). Interestingly, pre-treating the bladder with P2X7 receptor antagonist A804598 protected the urothelium from acrolein-induced damage (Figure 1C).
The caspase-3 immunoreactivity (IR), which indicates an apoptosis response, was mainly localised to urothelium in control tissues. In the acrolein treated bladder, increased caspase-3 positive cells were seen in the urothelial and suburothelial regions (Figure 1D). Quantitative analysis of caspase-3-IR staining showed that acrolein-enhanced caspase-3-IR was significantly blocked by the presence of A804598 (10 M), suggesting that A804 598 inhibited apoptosis (Figure 1E).
ACh contracted intact, detrusor and mucosa strips of perfusion control bladder in a concentration dependent manner (Figure 2). In the acrolein-treated bladder, the contractile responses to ACh were significantly diminished in intact strips and completely abolished in mucosal strips but were slightly reduced in detrusor strips. Pre-treating bladders with the P2X7 receptor antagonist A804598 at 10 µM significantly reversed acrolein-induced reduction in response to ACh (Figure 2).
Interpretation of results
In this study, we have established an ex-vivo inflammatory model in the porcine bladder. Acrolein treatment increased apoptosis in the suburothelial layer and the urothelium. Furthermore, acrolein completely abolished the contractility of the mucosal strips to ACh, suggesting that acrolein severely damaged myofibroblasts and other contractile apparatus in the mucosa. An important finding of this study was that the blockage of P2X7 receptor by its antagonist, A804598, remarkably protected the urothelium from structural damage and apoptosis. This may suggest that the P2X7 receptor is involved in the process of cyclophosphamide induced haemorrhagic cystitis in chemotherapy patients.