TRPV1 in experimental autoimmune prostatitis

Abstract

Background: Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a disorder that is characterized by persistent pelvic pain in men of any age. Although several studies suggest that the transient receptor potential vanilloid 1 (TRPV1) channel is involved in various pathways of chronic pain, the TRPV1 channel has not been implicated in chronic pelvic pain associated with CP/CPPS.

Methods: Male C57BL/6J (B6) and TRPV1 knockout (TRPV1 KO) mice (5-7 weeks old) were used to study the development of pelvic allodynia in a murine model of CP/CPPS called experimental autoimmune prostatitis (EAP). The prostate lobes, dorsal root ganglia (DRG), and spinal cord were excised at day 20. The prostate lobes were assessed for inflammation, TRPV1 expression, and mast cell activity. DRG and spinal cord, between the L6-S4 regions, were analyzed to determine the levels of phosphorylated ERK1/2 (p-ERK 1/2). To examine the therapeutic potential of TRPV1, B6 mice with EAP received intraurethral infusion of a TRPV1 antagonist at day 20 (repeated every 2 days) and pelvic pain was evaluated at days 20, 25, 30, and 35.

Results: Our data showed that B6 mice with EAP developed pelvic tactile allodynia at days 7, 14, and 20. In contrast, TRPV1 KO mice with EAP do not develop pelvic tactile allodynia at any time point. Although we observed no change in the levels of TRPV1 protein expression in the prostate from B6 mice with EAP, there was evidence of significant inflammation and elevated mast cell activation. Interestingly, the prostate from TRPV1 KO mice with EAP showed a lack of mast cell activation despite evidence of prostate inflammation. Next, we observed a significant increase of p-ERK1/2 in the DRG and spinal cord from B6 mice with EAP; however, p-ERK1/2 expression was unaltered in TRPV1 KO mice with EAP. Finally, we confirmed that intraurethral administration of a TRPV1 antagonist peptide reduced pelvic tactile allodynia in B6 mice with EAP after day 20.

Conclusions: We demonstrated that in a murine model of CP/CPPS, the TRPV1 channel is key to persistent pelvic tactile allodynia and blocking TRPV1 in the prostate may be a promising strategy to quell chronic pelvic pain.

Keywords: CPPS; inflammation; mast cells; pelvic pain; vanilloid 1.

FIGURE 1.

TRPV1 is required for pelvic pain. (A-B) Graphs show response frequencies measured with von Frey filaments applied to C57BL/6J (B6) mice and TRPV1 KO mice at days 0, 7, 14, and 20. (A) B6 mice with EAP (n=10) develop increased pelvic tactile allodynia compared to naive (n=10) mice at days 7, 14, and 20; (B) however, mice lacking the TRPV1 channel with EAP (TRPV1 KO-EAP; n=7) do not show a change in pelvic tactile allodynia at any time point, compared to respective control (TRPV1 KO-Naive; n=8). Data represents the mean ± SEM and was generated from three independent experiments. (*) denotes P<0.05.

FIGURE 2.

Absence of TRPV1 does not change prostate inflammation. (A,C) Representative images and (B,D) relative quantification of prostate inflammation in the dorsal and lateral prostate (DLP), ventral prostate (VP), and anterior prostate (AP) lobes stained with H&E. (A-B) The DLP lobes excised from mice with (Ab) EAP showed a significant increase in inflammation compared to (Ac) control cohorts. (C-D) Likewise, (Cb) the DLP lobes excised from TRPV1 KO mice with EAP (TRPV1 KO-EAP) demonstrated robust inflammation and leukocyte infiltration (red arrows), compared to respective (Ca) controls (TRPV1 KO-Naive). (A-D) In contrast, the (Ac,d; Cc,d) VP and (Ae,f; Ce,f) AP lobes lacked significant inflammation in all mice. Scale bars indicate 100 microns. Data represents the mean ± SEM. (*) denotes P<0.05.

FIGURE 3.

TRPV1 expression is unaltered in the prostate of mice with EAP. (A-B) Protein analysis and immunohistochemistry of the DLP lobes showed no differences in expression of TRPV1 between B6 mice with EAP and respective controls. (A) Representative western blots and (B) densitometry data comparing TRPV1 expression in B6 mice with EAP (n=3) to control (n=3) cohorts. Data represents the mean ± SEM.

FIGURE 4.

Active mast cells increase after prostate inflammation and the absence of TRPV1 hinders activity. (A-D) Panels show representative images of the prostate stained with toluidine blue and excised from B6 and TRPV1 KO mice with or without EAP; graphs depict quantification of mast cell numbers in the prostate for each group. (A) Although B6 with EAP showed no significant changes in total number of mast cells (white arrows) compared to control at day 20, (B) B6 mice with EAP showed an increased number of active mast cells (red arrows) compared to controls. (C-D) Prostates from mice with EAP that lack TRPV1 channels (TRPV1 KO-EAP) showed no changes in mast cell numbers or activity, compared to respective controls (TRPV1 KO-Naive). Scale bars indicate 100 microns. Data represents the mean ± SEM. (*) denotes P<0.05.

FIGURE 5.

ERK1/2 phosphorylation is linked to TRPV1 expression. (A) Western blot analysis and quantification of ERK1/2 phosphorylation in the lumbosacral region of the DRG (n=3) and (B) spinal cord (n=3) from mice with EAP revealed a significant increase in p-ERK1/2 expression compared to control littermates at day 20. However, (C) DRG and (D) spinal cords obtained from TRPV1 deficient mice with EAP showed no changed in ERK1/2 phosphorylation compared to control cohorts. Densitometry data represents the mean ± SEM. (*) denotes P<0.05.

FIGURE 6.

Pelvic tactile allodynia is diminished by TRPV1 antagonist peptide. (A) Schematic shows mice treatment regime. Mice (B6) with or without EAP received intraurethral (IU) instillation (10µl) of either sterile 0.9% saline or a TRPV1 antagonist peptide (L-R₄W_2; 1mM) from day 20 (indicated by black arrows) to 35. (B) After initial treatment at day 20, mice with EAP that received L-R₄W₂ (EAP+Antagonist; n=3) treatment showed a similar response frequency to mice without EAP that received either saline (Naive+Saline; n=3) or L-R₄W₂ (Naive+Antagonist; n=3) at days 25, 30, and 35. However, mice with EAP that received intraurethral saline (EAP+Saline; n=3) showed increased response frequency between days 7-35. Data represents the mean ± SEM from 3 independent experiments. (*) denotes P<0.05.