Tomohiko Oguchi

Bladder afferent hyperexcitability in bladder pain syndrome/interstitial cystitis

Bladder pain syndrome/interstitial cystitis is a disease with lower urinary tract symptoms, such as bladder pain and urinary frequency, which results in seriously impaired quality of life of patients. The extreme pain and urinary frequency are often difficult to treat. Although the etiology of bladder pain syndrome/interstitial cystitis is still not known, there is increasing evidence showing that afferent hyperexcitability as a result of neurogenic bladder inflammation and urothelial dysfunction is important to the pathophysiological basis of symptom development. Further investigation of the pathophysiology will lead to the effective treatment of patients with bladder pain syndrome/interstitial cystitis.

Glycine Transporter Type 2 (GlyT2) Inhibitor Ameliorates Bladder Overactivity and Nociceptive Behavior in Rats

BACKGROUND Glycine is a major inhibitory neurotransmitter in the spinal cord, the concentration of which is regulated by two types of glycine transporters (GlyTs): GlyT1 and GlyT2. We hypothesized that the inhibition of GlyTs could ameliorate bladder overactivity and/or pain sensation in the lower urinary tract. OBJECTIVE Investigate the effects of GlyT inhibitors on bladder overactivity and pain behavior in rats. DESIGN, SETTING, AND PARTICIPANTS Cystometry was performed under urethane anesthesia in cyclophosphamide (CYP)-treated rats. In behavioral studies using conscious rats, nociceptive responses were induced by intravesical administration of resiniferatoxin (3μM). Selective GlyT1 or GlyT2 inhibitors were administered intrathecally to evaluate their effects. MEASUREMENTS Cystometric parameters, nociceptive behaviors (licking and freezing), and messenger RNA (mRNA) levels of GlyTs and glycine receptor (GlyR) subunits in the dorsal spinal cord (L6-S1) were measured. RESULTS AND LIMITATIONS During cystometry in CYP-treated rats, significant increases in intercontraction interval and micturition pressure threshold were elicited by ALX-1393, a selective GlyT2 inhibitor, but not by sarcosine, a GlyT1 inhibitor. These effects were completely reversed by strychnine, a GlyR antagonist. ALX-1393 also significantly suppressed nociceptive behaviors in a dose-dependent manner. In sham rats, GlyT2 mRNA was expressed at a much higher level (23-fold) in the dorsal spinal cord than GlyT1 mRNA. In CYP-treated rats, mRNA levels of GlyT2 and the GlyR α1 and β subunits were significantly reduced. CONCLUSIONS These results indicate that GlyT2 plays a major role in the clearance of extracellular glycine in the spinal cord and that GlyT2 inhibition leads to amelioration of CYP-induced bladder overactivity and pain behavior. GlyT2 may be a novel therapeutic target for the treatment of overactive bladder and/or bladder hypersensitive disorders such as bladder pain syndrome/interstitial cystitis.

Herpes Simplex Virus Vector Mediated Gene Therapy of Tumor Necrosis Factor-α Blockade for Bladder Overactivity and Nociception in Rats

PURPOSE We examined the effects of tumor necrosis factor-α blockade on bladder overactivity and nociception using replication defective HSV vectors expressing tumor necrosis factor-α soluble receptor. MATERIALS AND METHODS HSV vectors expressing tumor necrosis factor-α soluble receptor or β-galactosidase/green fluorescent protein as the control were injected into the bladder wall of female Sprague-Dawley® rats. Green fluorescent protein was observed with fluorescent microscopy in the bladder and L6 dorsal root ganglia. mRNA and protein expression of tumor necrosis factor-α, and interleukin-1β and 6 as well as myeloperoxidase activity in the bladder were determined by quantitative reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay 4 hours after intravesical resiniferatoxin administration. c-Fos positive neurons were counted in the L6 spinal dorsal horn. Cystometry and behavioral analyses were also performed. RESULTS Green fluorescent protein expression was confirmed in the bladder and L6 dorsal root ganglia. Resiniferatoxin administration significantly increased tumor necrosis factor-α mRNA and protein levels in the bladder in controls. Tumor necrosis factor-α mRNA was also increased in the tumor necrosis factor-α soluble receptor group, although tumor necrosis factor-α protein up-regulation was suppressed. The up-regulation of interleukin-1β and 6 mRNA and protein levels, and the myeloperoxidase activity seen in controls were suppressed in the tumor necrosis factor-α soluble receptor group. c-Fos positive cells in the L6 spinal dorsal horn were less prominent in the tumor necrosis factor-α soluble receptor group than in controls. On cystometry the significant decrease in intercontraction intervals after resiniferatoxin infusion detected in controls was not seen in the tumor necrosis factor-α soluble receptor group. On behavioral analyses freezing behavior was significantly decreased in the tumor necrosis factor-α soluble receptor group without affecting licking behavior. CONCLUSIONS HSV vector mediated tumor necrosis factor-α blockade gene therapy in the bladder and bladder afferent pathways decreases the bladder pain and overactivity induced by nociceptive bladder stimuli.

Pelvic organ cross-sensitization to enhance bladder and urethral pain behaviors in rats with experimental colitis.

Neural cross-sensitization has been postulated as a mechanism underlying overlaps of chronic pelvic pain disorders such as bladder pain syndrome/interstitial cystitis (BPS/IC) and irritable bowel syndrome (IBS). Animals with experimental colitis have been used to study the underlying mechanisms for overlapped pelvic pain symptoms, and shown to exhibit bladder overactivity evidenced by frequent voiding; however, it has not directly been evaluated whether pain sensation derived from the lower urinary tract is enhanced in colitis models. Also, the cross-sensitization between the colon and urethra has not been studied previously. In the present study, we therefore investigated pain behaviors induced by nociceptive stimuli in the lower urinary tract and the involvement of C-fiber afferent pathways using rats with colitis induced by intracolonic application of 2,4,6-trinitrobenzenesulfonic acid (TNBS). In TNBS-induced colitis rats at 10 days, intravesical application of resiniferatoxin (RTx) induced a significantly greater number of episodes of both licking and freezing behaviors, which were reduced by capsaicin-sensitive C-fiber afferent desensitization. Histochemical studies using fluorescent dye tracers injected into the colon, bladder or urethra showed that dichotomized afferent neurons comprised 6.9-14.5% of L1, L6 and S1 dorsal root ganglion (DRG) neurons innervating the colon or the lower urinary tract. Transient receptor potential vanilloid 1 (TRPV1) mRNA expression was significantly increased in, the bladder, urethra and S1 DRG in colitis rats. An increase in myeloperoxidase (MPO) activity was found in the colon, but not in the bladder or urethra after intracolonic TNBS treatment. These results indicate that TNBS-induced colitis increased pain sensitivity in the bladder and urethra via activation of C-fiber afferent pathways due to colon-to-bladder and colon-to-urethral cross-sensitization, suggesting the contribution of pelvic organ cross-sensitization mechanisms to overlapped pain symptoms in BPS/IC and IBS.

Gene therapy for lower urinary tract dysfunction

Lower urinary tract dysfunction is caused by functional and pathophysiological alterations of the peripheral organs, including the urothelium and detrusor smooth muscle, as well as peripheral and central nervous systems. Recent research in this field has increased our understanding of the mechanisms of lower urinary tract dysfunction, and new drugs have been developed, leading to increased treatment options and changing medical care for lower urinary tract symptoms. Nevertheless, clinicians still often experience refractory and treatment‐resistant cases against conventional therapeutic modalities. For such cases, gene therapy targeting for the lower urinary tract and its afferent pathway is anticipated to offer a new therapeutic approach. Therefore, in this article, we review the possibility and current status of gene therapy for lower urinary tract dysfunction.

40 ENHANCEMENT OF GLYCINE EFFECTS ON BLADDER OVERACTIVITY AND NOCICEPTION BY HERPES SIMPLEX VIRUS (HSV) VECTOR-MEDIATED GLYCINE RECEPTOR GENE THERAPY

INTRODUCTION AND OBJECTIVES: Bladder pain syndrome/ interstitial cystitis (BPS/IC) is a serious disease whose main symptoms are bladder pain and frequent urination. We examined whether the effects of glycine, an inhibitory neurotransmitter, on pain behavior and bladder overactivity are enhanced by gene transfer of glycine receptors (GlyR) using replication-deficient HSV vectors. METHODS: Female SD rats were used, and 20 l of viral suspension of either GlyR (6.8 10 pfu/ml) or control vector (E1G6) (7.1 10 pfu/ml) was injected to the bladder wall. GlyR vectors also expressed Red Fluorescent Protein (RFP). (1) The bladder and L6 dorsal root ganglia (DRG) were harvested 2 weeks after viral injection. (2) In an awake condition, nociceptive behaviors such as lower abdominal licking (licking) and motionless head-turning (freezing) induced by 3 M resiniferatoxin (RTx) intravesical administration (0.3ml for 1min) through a urethral catheter were scored every 5 sec for 15 min among control virus and GlyR groups (n 7-8). (3) Rats were divided into 4 groups, which were treated with control vector, GlyR, control vector with glycine, or GlyR with glycine. In the glycine administration groups, an osmotic pump filled with 0.9% glycine was placed in the abdomen to administer glycine continuously. Two weeks later, cystometry (n 7-10) was performed under urethane anesthesia. After 2 hours of saline infusion, 10nM RTx was infused to induce bladder overactivity. Intercontraction intervals (ICI) were recorded and compared. RESULTS: (1) RFP positive cells were observed in both L6 DRG and bladder of GlyR injected rats. (2) GlyR-treated rats showed a significant reduction in licking (p 0.05) and freezing behavior (p 0.01) compared to E1G6 rats. (3) Virus (control or GlyR) injection without glycine administration did not alter the ICI reduction rate after RTx intravesical infusion in cystometry. Both control virus and GlyR groups given with glycine also showed a significant reduction in ICI after RTx, with a smaller reduction rate (p 0.05) in GlyR virus-injected rats with glycine. CONCLUSIONS: The GlyR vector intensified the therapeutic effect of glycine on bladder overactivity and pain in rats with RTxinduced cystitis, suggesting that glycine administration with HSV-mediated glycine receptor gene therapy might be effective for treating BPS/IC. In addition, systemic administration of inhibitory transmitters such as glycine in combination with local HSV gene therapy of their receptors could enhance the drug-receptor interaction in target organs.