Effect of Onabotulinum Toxin A on Substance P and Receptor Neurokinin 1 in the Rat Ventral Prostate

Abstract

Introduction: The objective of this work is to examine if sensory innervation impacts lower urinary tract symptoms (LUTS). Onabotulinum toxin A (BoNTA) has been used for the treatment of overactive and neurogenic bladder and as a treatment for LUTS secondary to benign prostatic hyperplasia (BPH). The mechanism of how BoNTA impacts LUTS/BPH is unclear. In rats, BoNTA injection causes prostate denervation, apoptosis and atrophy. In clinical trials reduced prostate size and LUTS are observed inconsistently, suggesting a neurologic component. We will examine if BoNTA treatment inhibits substance P production in sensory nerve fibers in the rat prostate.

Methods: Twenty Sprague Dawley rats were divided into four groups including 1X PBS (control, n=6), 2.5 units Onabotulinum toxin A (BoNTA, n=6), 5 units BoNTA (n=6) injected into both lobes of the ventral prostate (VP) and sham surgery (n=2). Rats were Euthanized after one week. Substance P and its receptor neurokinin 1 localization and quantification were performed by counting the number of stained neurons and nerve bundles, by semi-quantitative immunohistochemical analysis and by western analysis.

Results: Substance P was localized in neuronal axons and bundles in the stroma of the VP but not in the epithelium. Receptor neurokinin 1 was identified in neuronal bundles of the stroma and in columnar epithelium of the VP ducts. Substance P decreased ~90% after BoNTA treatment (p=0.0001) while receptor neurokinin 1 did not change by IHC (p=0.213) or Western (p=0.3675).

Conclusions: BoNTA treatment decreases substance P in the rat VP.

Keywords: BoNTA; LUTS/BPH; Prostate; Substance P.

Figure 1

Optimization of injections into the Sprague Dawley VP. (A) Methylene blue (100-10μl) was used to determine the optimal volume for injection that filled both lobes of the VP but did not leak into other tissues. Two injections of 10μl into each VP lobe were optimal. Arrows indicate the VP lobes. (B) Fluorogold injected into both lobes of the VP was used to determine dispersion of fluid within the VP. Fluorogold was observed evenly dispersed in sectioned VP 7 days after injection. (C) H&E staining of VP injected with 10 μl PBS shows that the injection did not damage normal VP morphology since normal columnar epithelium and stroma were observed.

Figure 2

Localization of substance P and its receptor neurokinin 1 in normal Sprague Dawley VP. (A) Substance P is localized in neurons and nerve bundles in the stroma but was not identified in the epithelium. 400× magnification. (B) Receptor neurokinin 1 was identified in neurons and nerve bundles in the stroma and in columnar epithelium of the prostatic ducts. 250X magnification. Arrows indicate substance P and receptor neurokinin 1 proteins.

Figure 3

Quantification of substance P was performed by counting the number of substance P and neuron specific enolase stained axons and nerve bundles at 100X magnification in sham, PBS, 2.5 units BoNTA and 5 units BoNTA injected Sprague Dawley VP. (A) Immunohistochemical analysis was performed for substance P (green) and neuron specific enolase (red) and the photos were merged prior to quantification by counting. (B) Substance P stained nerve axons and bundles were significantly decreased with 2.5 and 5 units BoNTA treatment relative to sham and PBS controls (p<0.0001).

Figure 4

(A) Representative photos of receptor neurokinin 1 immunohistochemical analysis used for quantification by Image J. No difference in receptor neurokinin 1 was observed between groups by Image J (B, p=0.213) and by Western (C, p=0.3675) analysis.