Herpes Simplex Virus Vector-Mediated Gene Delivery of Poreless TRPV1 Channels Reduces Bladder Overactivity and Nociception in Rats

Abstract

Increased afferent excitability has been proposed as an important pathophysiology of interstitial cystitis/bladder pain syndrome (IC/BPS) and overactive bladder (OAB). In this study, we investigated whether herpes simplex virus (HSV) vectors encoding poreless TRPV1, in which the segment in C terminus of TRPV1 receptor is deleted, suppress bladder overactivity and pain behavior using a rat model of chemical cystitis. Replication-defective HSV vectors encoding poreless TRPV1 were injected into the bladder wall of adult female Sprague-Dawley rats. Additionally, recombinant HSV virus (vHG) vectors were injected as control. Cystometry (CMG) under urethane anesthesia was performed 1 week after viral injection to evaluate bladder overactivity induced by resiniferatoxin (RTx, a TRPV1 agonist). RTx-induced nociceptive behavior such as licking (lower abdominal licking) and freezing (motionless head-turning) was observed 2 weeks after viral injection. GFP expression in L4/L6/S1 dorsal root ganglia and the bladder as well as c-Fos-positive cells in the L6 spinal cord dorsal horn were also evaluated 2 weeks after viral injection. In CMG, the poreless TRPV1 vector-treated group showed a significantly smaller reduction in intercontraction intervals and voided volume after RTx infusion than the vHG-treated control group. The number of the RTx-induced freezing events was significantly decreased in the poreless TRPV1 group than in the vHG group, whereas there was no significant difference of the number of RTx-induced licking events between groups. The number of c-Fos-positive cells in the DCM and SPN regions of the L6 spinal dorsal horn was significantly smaller in the poreless TRPV1 group than in the vHG group. Our results indicated that HSV vector-mediated gene delivery of poreless TRPV1 had a therapeutic effect on TRPV1-mediated bladder overactivity and pain behavior. Thus, the HSV vector-mediated gene therapy targeting TRPV1 receptors could be a novel modality for the treatment of OAB and/or hypersensitive bladder disorders such as IC/BPS.

Figure 1.

Herpes simplex virus (HSV) vectors construct. The (A) vHG and (B) poreless vectors (vHP) have a deletion of the essential immediately early (IE) genes, ICP4 and ICP27, as well as IE regulatory elements within the promoters of IE genes, ICP22 and ICP47, making their expression dependent on ICP4 and ICP27, and thus they are expressed as early genes only within the complementing cell line used to propagate the vectors. In the genome of the vHG, an HCMV immediate early promoter driving enhanced green fluorescent protein (EGFP) was inserted into both ICP4 loci, while in the poreless TRPV1 genome an HCMV promoter driving poreless TRPV1 was inserted.

Figure 2.

Cystometry under urethane anesthesia in control vHG and poreless vHP groups. Representative traces of cystometry (A) and the effect of resiniferatoxin (RTx), a TRPV1 agonist, on intercontraction interval (ICI) (B) and on voided volume (C). (A) Saline was continuously infused into the bladder at a rate of 0.04 ml/min. After baseline bladder activity was established, 1 μM of RTx was infused at a rate of 0.04 ml/min. (B) The reduction rate of ICI is significantly smaller in the vHP group than in the vHG group (68 ± 4% vs. 55 ± 3%, p = 0.03). The reduction rate was calculated with an equation: (ICI during saline infusion − ICI during RTx infusion)/ICI during saline infusion. (C) The reduction rate of voided volume is significantly smaller in the vHP group than in the vHG group (65 ± 3% vs. 51 ± 5%, p = 0.03). The reduction rate was calculated with an equation: (voided volume during saline infusion − voided volume during RTx infusion)/voided volume during saline infusion.

Figure 3.

Resiniferatoxin (RTx)-induced licking behavior. RTx (3 μM) was administered into the bladder through a temporary indwelling urethral catheter and kept there for 1 min. The number of licking events was counted for a 15 min period with 5 sec intervals. (A) Time-course changes in the number of licking behavior events. (B) Comparison of licking behavior events between vHG and poreless vHP groups. The 15 min observation time was divided into three periods: early (0–5 min), middle (5–10 min), and late (10–15 min). There was no significant (n.s.) difference in the licking events between two groups.

Figure 4.

Resiniferatoxin (RTx)-induced freezing behavior. RTx (3 μM) was administered into the bladder through a temporary indwelling urethral catheter and kept for 1 min. The number of freezing events was counted for 15 min. (A) Time-course changes in the number of freezing behavior events. (B) Comparison of freezing behavior events between vHG and poreless vHP groups. The 15 min observation time was divided into three periods: early (0–5 min), middle (5–10 min), and late (10–15 min). The poreless vHP-injected group showed the significantly lower number of freezing events in all of three 5 min periods than the vHG-injected group.

Figure 5.

HSV vector-mediated green fluorescent protein (GFP) expression in the bladder (A and B), L6 dorsal root ganglia (DRG) (C and D), and L4 DRG (E) from rats treated with vHG control vectors. The photomicrographs (B) and (D) show the magnified portions of (A) and (C) indicated by rectangular boxes, respectively. GFP-positive cells were observed in the bladder (A and B) and the S1/L6 DRG (C and D) in rats treated with vHG control vectors. There were GFP-positive cells neither in the L4 DRG in the rats treated with vHG vectors (E) nor in the L6 DRG in the nontreated rats (data not shown). Scale bars indicate 200 μm (A and C) and 100 μm (B, D, and E). Color images available online at www.liebertpub.com/hum

Figure 6.

c-Fos immunostaining in the L6 spinal cord dorsal horn. (A) The L6 spinal cord dorsal horn was divided into four regions: medial dorsal horn (MDH), lateral dorsal horn (LDH), dorsal commissure (DCM), and sacral parasympathetic nucleus (SPN). The vHG-injected group (B) had more c-Fos-positive cells (black arrows) than the poreless vHP-injected group (C.) (D) There were the significantly lower number of c-Fos-positive cells in the DCM and SPN regions in the poreless vHP-injected group than in the control vector vHG-injected group. Scale bars indicate 200 μm.