Effect of Intravesical Liposome-Based Nerve Growth Factor Antisense Therapy on Bladder Overactivity and Nociception in a Rat Model of Cystitis Induced by Hydrogen Peroxide

Abstract

The aim of this study was to evaluate whether liposome-based local suppression of nerve growth factor (NGF) in the bladder has effects on bladder hypersensitivity in a rat cystitis model induced by intravesical instillation of hydrogen peroxide (HP). HP (1.5%) was intravesically administered to adult female Sprague-Dawley rats. Liposomes complexed with NGF antisense oligonucleotide (OND) labeled with TYE563 fluorescent tag were intravesically instilled on day 2. Red fluorescence from the TYE 563 tag was observed with fluorescent microscopy on day 3. Four separate groups of rats were used in the following experiments: (a) sham-liposome group, (b) sham-OND group, (c) cystitis-liposome group, and (d) cystitis-OND group. Saline or 1.5% HP was intravesically administered on day 0. Empty liposomes or liposomes-antisense OND were instilled into the bladder on day 2. The following experiments were conducted to evaluate the effect of NGF antisense treatment on day 7: (a) continuous cystometry was performed in an awake condition; (b) pain behavior induced by instillation of resiniferatoxin into the bladder, including licking behavior (lower abdominal licking) and freezing behavior (motionless head-turning toward lower abdomen), was observed; (c) immunohistochemical staining of the bladder and L6 DRG for NGF was performed; (d) the expression of several genes in the bladder was analyzed by reverse transcription polymerase chain reaction (RT-PCR); and (e) after Fast Blue was injected into the bladder wall, Fast Blue-positive or -negative cells in DRG neurons were separately collected by using a laser-capture microdissection method 7 days later. RT-PCR was performed to evaluate gene expressions in captured neuronal cells. The expression of TYE563 was identified only in the urothelial layer. In cystometric investigation, intercontraction intervals (ICI) were significantly (p = 0.001) shorter in the cystitis-liposome group in comparison to the sham-liposome group. ICI was significantly (p = 0.007) longer in the cystitis-OND group compared to the cystitis-liposome group. Comparisons of the sham-liposome and the sham-OND groups showed no significant difference in ICI (p = 0.56). Licking events did not significantly differ among the four groups. In contrast, the cystitis-liposome group showed significantly more freezing events than the sham-liposome group did (p = 0.002). A significant reduction in the number of freezing events was observed in the cystitis-OND group compared to the cystitis-liposome group (p = 0.04). Immunofluorescence staining demonstrated that NGF expression in the mucosa (p = 0.02) and L6 DRG (p = 0.01) was significantly higher in the cystitis-liposome group than it was in the sham-liposome group. The expression of NGF was significantly lower in the mucosa (p = 0.002) and L6 DRG (p = 0.01) in the cystitis-OND group compared to the cystitis-liposome group. RT-PCR showed that the expression of NGF and TRPV1 mRNA in the mucosa was significantly higher in the cystitis-liposome group than it was in the sham-liposome group (p = 0.001 and 0.03, respectively). On the other hand, these gene expressions were significantly lower in the cystitis-OND group than they were in the cystitis-liposome group (p = 0.007 and 0.02, respectively). The cystitis-liposome group showed significantly higher expression of TRPA1, P2X3, and BDNF mRNA in labeled bladder afferent neurons than the sham-liposome group did (p = 0.03, 0.01, and 0.001, respectively). These gene expressions were significantly lower in the cystitis-OND group compared to the cystitis-liposome group (p = 0.04, 0.006, and 0.03, respectively). The study indicated that intravesical application of liposome-NGF antisense OND significantly improved bladder hypersensitivity induced by chemical cystitis in rats. Intravesical treatment with liposome-OND conjugates could be a novel local therapy of hypersensitive bladder disorders such as bladder pain syndrome/interstitial cystitis.

Keywords: antisense; cystitis; liposome; nerve growth factor; nociception; rat.

Figure 1.

Results of cystometric analyses in an awake condition. Representative traces of cystometry (A) and the comparison of intercontraction intervals (B).

Figure 2.

Resiniferatoxin (RTx)-induced nociceptive behavior. RTx (3 μM) was administered into the bladder through a temporary indwelled urethral catheter and kept in the bladder for 1 min. The number of licking events (A) and freezing events (B) was counted for a 15 min period with 5 s intervals.

Figure 3.

Nerve growth factor (NGF) immunofluorescence in the bladder. Photomicrographs of bladder sections (I) from a sham-liposome rat (A), a sham-oligonucleotide (OND) rat (B), a cystitis-liposome rat (C), and a cystitis-OND rat (D). The percentage area of positive immunoreactivity above the threshold in the urothelium (II) and the detrusor (III). D, detrusor; U, urothelium. Scale bars: 200 μm.

Figure 4.

NGF immunofluorescence in L6 DRG. Photomicrographs of L6 DRG sections (I) from a sham-liposome rat (A), a sham-OND rat (B), a cystitis-liposome rat (C), and a cystitis-OND rat (D). The percentage ratio of the number of NGF-positive cells relative to the total cell count in L6 DRG sections (II). Arrows indicate NGF-positive neurons. Scale bars: 100 μm.

Figure 5.

Expression levels of NGF mRNA (A) and TRPV1 mRNA (B) in the bladder mucosa, and NGF mRNA (C) and TRPV1 mRNA (D) in the detrusor.

Figure 6.

Expression levels of TRPV1 mRNA (A), TRPA1 mRNA (B), P2X3 mRNA (C), and BDNF mRNA (D) in bladder afferent neurons obtained from L6 DRG.