There is increasing evidence indicating the positive correlation between prostatic inflammation and lower urinary tract symptoms (LUTS) in male with benign prostatic hyperplasia (BPH).  Also, in animal models, prostatic inflammation (PI) reportedly induces bladder overactivity via prostate-to-bladder cross-organ afferent sensitization through activation of the pelvic nerve [1].  Previous reports further demonstrated that nerve growth factor (NGF) is an important mediator to induce bladder afferent hyperexcitability, which contributes to lower urinary tract dysfunction (LUTD) [2] and that local suppression of NGF in the bladder improves bladder overactivity in rat models of cystitis or colitis [3]. The present study therefore examined the effect of liposomes conjugated with antisense oligonucleotide (OND) targeting NGF on local overexpression of NGF and bladder overactivity in rats with PI.

Male Sprague-Dawley rats were divided into three groups: (1) Control group; intact rats, (2) PI-NS group; rats with PI and intravesical instillation of Normal Saline (NS), (3) PI-OND group; rats with PI and intravesical instillation of liposome-conjugated NGF antisense. PI was induced by intraprostatic 5% formalin injection (50 μl per each ventral lobe). On day 0, PI was induced, and on day 14, 0.5ml of 12μM of phosphorothioated NGF antisense OND complexed with liposomes (PI-OND group) or NS (PI-NS group) was instilled into the bladder directly using a 30G needle punctured through the bladder dome after draining urine, and kept for 60 minutes under isoflurane anesthesia. Then, on day 28, we evaluated awake cystometry (CMG) and harvested tissues for the histological analysis as well as measurements of protein levels of NGF by ELISA methods and mRNA expressions of NGF and TRPV1 in the bladder, inflammation markers (IL-β and IL-18) in the prostate, and mRNA expressions of C-fiber afferent markers (TRPV1 and TRPA1) and the A-type K+ channel α-subunit (Kv 1.4) in L6-S1 dorsal root ganglia (DRG) by RT-PCR.

In CMG, PI-OND group had significant longer Intercontraction intervals (ICI) than PI-NS group while there were no significant differences between PI-OND and Control groups (Fig. 1A, 1B). Other CMG parameters were not significantly different among groups. mRNA expression levels of TRPV1 and TRPA1 in L6-S1 DRG of PI-OND group were significantly lower than those of PI-NS group, while there were no significant differences in their expressions between PI-OND and Control groups (Fig. 1C). mRNA expression levels of Kv 1.4 subunit in L6-S1 DRG in PI-OND group were significantly lower than those in PI-NS group while there were no significant differences between PI-OND and Control groups (Fig. 1C). Both mRNA and protein expressions of NGF in the bladder mucosa in PI-OND group were significantly lower than those in PI-NS group, while there were no significant differences in their expressions between PI-OND and Control groups (Fig. 2A). In addition, in the prostate tissue, mRNA expression levels of NGF as well as IL1β & IL-18 in PI-OND group were significantly lower than those in PI-NS group (Fig. 2B).

Intravesical instillation of liposome-conjugated NGF antisense OND reduced local NGF suppression in both bladder and prostate, which was increased after prostatic inflammation. Also, the intravesical NGF antisense treatment reduced PI-induced bladder overactivity evident as longer ICI in association with the reduction of TRPV1 and TRPA1 mRNA expressions in L6-S1 DRG, which contain bladder and prostate afferent neurons. Moreover, mRNA expressions of Kv1.4 in L6-S1 DRG, which is an α-subunit of A-type K+ channels and reportedly decreased in hyperexcitable bladder afferent neurons from LUTD animal models [1], was reduced after prostatic inflammation, but improved after local NGF suppression in PI-OND group.

These results indicate that NGF locally expressed in the bladder is an important mediator to induce bladder overactivity with upregulation of C-fiber afferent markers and downregulation of an A-type K+ channel subunit in L6-S1 DRG following prostatic inflammation and that liposome-based, local NGF-targeting therapy could be effective for not only bladder overactivity and afferent sensitization, but also prostatic inflammation.  Thus, local blockade of NGF in the bladder could be a therapeutic modality for male LUTS due to BPH with prostatic inflammation.

Funahashi, Y., Takahashi, R., Mizoguchi, S., Suzuki T., Takaoka, E., Ni, J., Wang, Z., DeFranco, D.B., de Groat, W.C., Tyagi, P., Yoshimura, N.: Bladder overactivity and afferent hyperexcitability induced by prostate-to-bladder cross-sensitization in rats with prostatic inflammation.  Journal of Physiology (London), 597: 2063-2078, 2019
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