Male patients who suffer from painful inflammatory disease of the lower urinary tract, often chronic prostatitis/chronic pelvic pain syndrome (CPPS), often lack satisfactory treatment options. The situation is worsened by the negative effects that CPPS has on urinary bladder function [1]. Considering the relatively large number of male patients suffering from CPPS who do not respond positively to treatment, new and more effective pharmacological options are needed. For this, potential common pathophysiological changes in urinary bladder and prostate need to be investigated. In particular, more effort is required to investigate the potential association between inflammatory pathways in pelvic organs.
The aim of the present study was to investigate which effects CPPS has on bladder function and pathophysiology. For this purpose, a broad methodological approach was used.

A total of 24 adult male Sprague-Dawley rats (300-450 g) were used in the study, which followed national guidelines for the care and use of laboratory animals and was approved by the local ethics committee (permit number:1794/2018). 
The animals were randomly divided into four groups. In the first three groups, zymosan (Sigma-Aldrich, St Louis, MO, USA; 0.1 mg in 10 µl saline) was injected into the dorsal lobe of the prostate to create a functional model for CPPS. In the fourth group, rats were injected with vehicle (10 µl saline, serving as control). Metabolic cage experiments were performed 7, 14 and 21 days after zymosan injection (termed as group Z7, Z14 and Z21, respectively) and after 14 days in the control group. The obtained parameters included the total number of micturitions, total urine volume produced and total water intake. From these parameters it was possible to calculate micturition frequency and volume per micturition. 
Immediately following the metabolic cage experiment, cystometry was performed. Briefly, the femoral artery and vein were catheterized to allow for blood pressure monitoring and drug administration, respectively. Via a careful incision in the bladder dome a pressure sensing catheter and cannula were placed in the urinary bladder and subsequently fixed with a ligature. Bladder pressure, residual urine volume, bladder capacity, compliance, voiding time and non-voiding contractions (NVCs) were measured during cystometry. Bladder compliance was calculated by dividing the volume change (ΔV) by the change in intravesical pressure (ΔP) that occurred during bladder filling (ΔV/ΔP). Saline was infused via the cannula to induce simulated micturition cycles. This was repeated five times both before and after concentration-response series of the cholinergic agonist methacholine (1, 2 and 5 µg*kg-1, i.v.) and the purinergic agonist ATP (5, 10 and 100 µg*kg-1, i.v.). 
Following cystometry, the prostate and urinary bladder were excised and weighed, then fixed in paraformaldehyde and subsequently examined histopathologically for possible inflammatory changes. For this blinded assessment, the tissues were stained with haematoxylin-eosin and given an inflammatory score (0-3) by an independent pathologist. Two-way ANOVA followed by Tukey’s correction for multiple comparisons was used to compare data between groups. Statistical analysis was performed using GraphPad Prism version 8.3 (GraphPad Software Inc., San Diego, USA). The level of statistical significance was set at p < 0.05.

Compared to controls, the volume/micturition was lower in all zymosan-treated CPPS groups (Figure 1a; 0.91±0.23, 0.92±0.09, 0.78±0.11 and 1.62±0.28 ml in Z7, Z14, Z21 and control groups, respectively; control vs Z7, p=0.065; control vs Z14, p=0.066; control vs Z21, p=0.014). The number of micturitions per hour were significantly higher in all CPPS groups, as compared to controls (Figure 1b; 0.95±0.09, 1.08±0.13, 1.18±0.10 and 0.40±0.03 in Z7, Z14, Z21 and control groups, respectively; control vs Z7, p=0.0029; control vs Z14, p=0.0005; control vs Z21, p<0.0001). Similarly, the total number of micturitions during the entire metabolic cage time period were higher in all CPPS groups, as compared to controls (Figure 1c; 15.25±1.44, 17.25±2.06, 18.83±1.62 and 6.40± 0.51 in Z7, Z14, Z21 and control groups, respectively; control vs Z7, p=0.0029; control vs Z14, p=0.0005; control vs Z21, p<0.0001).
Cystometry revealed a significant increase in the number of NVCs in all CPPS groups, as compared to controls (Figure 2a; control vs Z7, p=0.046; control vs Z14, p=0.033; control vs Z21, p=0.014). Similarly, lower bladder compliance was seen in the CPPS groups, as compared to controls. This decrease tended to worsen as the time after zymosan injection proceeded.
Rats with chemically induced CPPS displayed significantly longer voiding times compared to controls. There was also a trend towards longer voiding time as a result of increased time after zymosan injection (Z21 & Z14 > Z7). 
There was a tendency towards decreased methacholine-induced contractions in all CPPS groups, as compared to controls (Figure 2b; at 5 µg*kg-1; Z14 vs control, p=0.0023; Z21 vs control, p<0.0001). ATP-induced bladder contractions tended to be lower in the CPPS groups, as compared to controls (Figure 2c; at 100 µg*kg-1; Z7 vs control, p=0.0007; Z14 vs control, p=0.0016; Z21 vs control, p=0.0003). 
There were no significant differences between the groups regarding total bladder or prostate weight. However, all zymosan-treated prostate tissues displayed signs of inflammation (avg score 2.2 as compared to 1.0 in controls). Interestingly, while excised bladders from control animals did not show signs of inflammation (avg score 0.5), bladders from zymosan-treated animals did (avg score 1.5).

In the present study, we created an animal model for CPPS without benign prostate hyperplasia (BPH) to investigate potential effects of chronic prostate inflammation on bladder function. To the best of our knowledge, the current study represents the first study in an animal model for CPPS to demonstrate the effects of chronic prostate inflammation on bladder function over time. Further, this is the first study that simultaneously studies basic bladder function (micturition parameters), afferent (cystometry) and efferent (drug) effects. Induction of prostatitis by zymosan injection did not cause any increase in total prostate weight, confirming it to be a good model for CPPS without BPH. Metabolic cage data showed that zymosan-induced prostatic inflammation could provoke urinary frequency. Likewise, cystometry showed a significant increase of NVCs and a trend towards worsened bladder compliance over time in the animals with CPPS.  In addition, we observed that CPPS caused significantly increased voiding times. Taken together, these findings indicate cross-sensitization between prostate and bladder.
Our findings revealed incidences of reduced cholinergic contractile bladder responses in animals with CPPS. Similar findings were seen regarding ATP-induced contractions. These findings are in accordance with previous studies that investigated the possible effects of non-bacterial urinary bladder inflammation on ATP and acetylcholine evoked contractile responses [2]. Based on our findings, and how they correlate with previous findings, we hypothesize that CPPS causes functional changes in the urothelium in a similar way as what is observed during cystitis. It is possible that the negative effects of CPPS on bladder function are conveyed via common dorsal root pathways, which has been suggested in a previous study [3].

The current findings demonstrate a potential prostate-to-bladder cross-sensitization which causes bladder dysfunction and evident signs of inflammation. The underlying mechanisms of this cross-talk remain to be unravelled. Future clinical studies are required to verify the outcomes of the current study and enable advancement of patient care.

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Funahashi Y, Takahashi R et al. Bladder overactivity and afferent hyperexcitability induced by prostate-to-bladder cross-sensitization in rats with prostatic inflammation. J Physiol. 2019; doi: 10.1113/JP277452