Bladder overactivity and afferent hyperexcitability induced by prostate-to-bladder cross-sensitization in rats with prostatic inflammation

Abstract

Key points: There is clinical evidence showing that prostatic inflammation contributes to overactive bladder symptoms in male patients; however, little is known about the underlying mechanisms In this study, we investigated the mechanism that prostatic inflammation causes detrusor overactivity by using a rat model of chemically induced prostatic inflammation. We observed a significant number of dorsal root ganglion neurons with dichotomized afferents innervating both prostate and bladder. We also found that prostatic inflammation induces bladder overactivity and urothelial NGF overexpression in the bladder, both dependent on activation of the pelvic nerve, as well as changes in ion channel expression and hyperexcitability of bladder afferent neurons. These results indicate that the prostate-to-bladder cross-sensitization through primary afferent pathways in the pelvic nerve, which contain dichotomized afferents, could be an important mechanism contributing to bladder overactivity and afferent hyperexcitability induced by prostatic inflammation.

Abstract: Prostatic inflammation is reportedly an important factor inducing lower urinary tract symptoms (LUTS) including urinary frequency, urgency and incontinence in patients with benign prostatic hyperplasia (BPH). However, the underlying mechanisms inducing bladder dysfunction after prostatic inflammation are not well clarified. We therefore investigated the effects of prostatic inflammation on bladder activity and afferent function using a rat model of non-bacterial prostatic inflammation. We demonstrated that bladder overactivity, evident as decreased voided volume and shorter intercontraction intervals in cystometry, was observed in rats with prostatic inflammation versus controls. Tissue inflammation, evident as increased myeloperoxidase activity, and IL-1α, IL-1β, and IL-6 levels inside the prostate, but not in the bladder, following intraprostatic formalin injection induced an increase in NGF expression in the bladder urothelium, which depended on activation of the pelvic nerve. A significant proportion (18-19%) of dorsal root ganglion neurons were double labelled by dye tracers injected into either bladder or prostate. In rats with prostatic inflammation, TRPV1, TRPA1 and P2X2 increased, and Kv1.4, a potassium channel α-subunit that can form A-type potassium (K_A ) channels, decreased at mRNA levels in bladder afferent and double-labelled neurons vs. non-labelled neurons, and slow K_A current density decreased in association with hyperexcitability of these neurons. Collectively, non-bacterial inflammation localized in the prostate induces bladder overactivity and enhances bladder afferent function. Thus, prostate-to-bladder afferent cross-sensitization through primary afferents in the pelvic nerve, which contain dichotomized afferents, could underlie storage LUTS in symptomatic BPH with prostatic inflammation.

Keywords: bladder; cross-sensitization; inflammation; prostate.

Figure 1. Histological findings of bladder and prostate sections

Haematoxylin and eosin staining showed that intra‐prostatic formalin injection caused either a low‐grade inflammation (B) or high‐grade inflammation (C) in the prostate compared to the vehicle‐injected prostate (A). There were no inflammatory changes in the bladder of vehicle (D) or formalin‐treated rats (E). Scale bars; 100 μm (A–C); 400 μm (D and E).

Figure 2. Inflammatory markers

Proinflammatory cytokine levels (A, IL‐1α; B, IL‐1β; C, IL‐6) and MPO activity were measured in the prostate and bladder using ELISA. Light grey columns, vehicle‐treated rats. Dark grey columns, formalin‐treated rats. n = 6 in each group. Columns with bars represent mean ± SEM. ^** P < 0.01 between groups. TP, total protein.

Figure 3. Continuous cystometry

Representative traces of cystometry under an awake condition in vehicle‐ (A) or formalin‐injected rats (B). Cystometric parameter analyses showed that intercontraction intervals rats were shorter and the number of non‐voiding contraction during urine storage in formalin‐injected was greater than those in vehicle‐injected rats, indicating bladder overactivity after prostatic inflammation.

Figure 4. Single cystometry in rats with unilateral pelvic nerve transection

Representative traces of cystometry under an awake condition in rats with transection of the right pelvic nerve, which received no additional treatment (A), formalin injection into the contralateral side (left) of the ventral prostate lobe (B) or formalin injection into the ipsilateral side (right) of the ventral prostate lobe (C). D, the comparison of the number of non‐voiding contraction (NVC) per voiding cycle shows that NVC are more frequent (P < 0.05) in pelvic nerve‐transected rats with formalin injection into the left prostate lobe (Rt. PNT + Lt. formalin) compared to rats with Rt. PNT alone or PNT rats with the right lobe formalin injection (Rt. PNT + Rt. formalin) (n = 7–8 rats per group).

Figure 5. Fluorescent microscopic images of DRG sections

Representative fluorescent microscopic images of DRG neuron labelled with Fast Blue (A) and DiI (B) injected into the bladder wall and the ventral lobes of prostate, respectively. C, a merged image of A and B showed that some neurons were labelled with both dyes, as indicated by arrowheads. Scale bars; 100 μm. The number of neurons dye‐labelled with Fast Blue (white column), DiI (grey column) and both (hatched column) in sections of Th13 to S2 DRG from vehicle‐injected (D) and formalin‐injected rats (E) was quantified (6 DRG from 3 rats per groups). Columns represent the mean number of dye‐labelled neurons per section.

Figure 6. Action potential characteristics of capsaicin‐sensitive double‐labelled and Fast Blue‐labelled afferent neurons

Representative recordings of action potentials in double‐labelled, dichotomized afferent neurons from vehicle‐injected (A) and formalin‐injected rats (B), and Fast Blue‐labelled bladder afferent neurons from vehicle‐injected (C) and formalin‐injected rats (D). The thresholds for eliciting action potentials (panels a of A–D) in formalin‐injected rats was lower than those in vehicle‐injected control rats (Aa vs. Ba or Ca vs. Da). The number of action potentials during 800 ms membrane depolarization (panels b of A–D) in formalin‐injected rats was greater than that in control rats (Ab vs. Bb or Cb vs. Db). ^*Firing thresholds of action potentials.

Figure 7. Changes of K⁺ currents in capsaicin‐sensitive bladder afferent neurons

A, representative recordings in a capsaicin‐sensitive bladder afferent neuron from a control rat showing superimposed outward K⁺ currents evoked by depolarizing voltage pulses to 0 mV from holding potentials (HPs) of –120 and –40 mV and sustained delayed rectifier‐type K⁺ (sustained K_DR) currents evoked by depolarization from –40 mV HP. B, representative recordings in a capsaicin‐sensitive bladder afferent neuron from a control rat showing slow decaying A‐type K⁺ (slow K_A) currents obtained by subtracting K⁺ currents evoked by depolarization from HP of –40 to 0 mV from the K⁺ currents evoked by depolarization from HP of –120 mV to 0 mV. C, I–V relationships of slow K_A currents showing that the density of K_A currents in capsaicin‐sensitive bladder afferent neurons from formalin‐injected rats (n = 9 cells from 9 rats) was significantly reduced compared to that from vehicle‐injected rats (n = 9 cells from 9 rats). D, I–V relationships of sustained K_DR currents showing that the density of K_DR currents was not significantly different between two groups (n = 10 cells from 8 rats each). ^* P < 0.05 compared to vehicle‐injected rats (Student's unpaired t test).

Figure 8. Relative mRNA levels in the afferent neurons

Fast Blue (FB)‐, DiI‐, and double‐labelled neurons in DRG sections were dissected by LCM, and mRNA levels of TRPV1 (A), TRPA1 (B), Kv1.4 (C), P2X2 (D) and P2X3 (E) were measured by qRT‐PCR and expressed relative to the levels expressed in non‐labelled neurons. Light grey columns, vehicle‐treated rats (n = 10). Dark grey columns, formalin‐treated rats (n = 11). Columns with bars represent means ± SEM. ^* P < 0.05, ^** P < 0.01 compared to non‐labelled neurons.

Figure 9. NGF expression in the bladder

NGF expression was markedly enhanced in formalin‐injected rats, especially in the urothelium (C, D), compared to the vehicle‐injected controls (A, B). Scale bars; 200 μm (A, C) and 50 μm (B, D). Western blotting in bladder tissue extracts showed upregulation of NGF expression in the formalin‐injected compared to vehicle‐injected rats (E). F, in rats with the right pelvic nerve transection (PNT), formalin injection into the left ventral lobe of prostate induced NGF upregulation in the bladder mucosa (Rt. PNT +Lt. formalin) compared to rats with PNT alone (Rt. PNT alone); however, increased NGF expression was not seen when formalin was injected into the right ventral lobe of prostate (Rt. PNT + Rt. formalin).