Four-week repeated oral dose toxicity study of zinc maltol in rats.

Zinc (Zn) is one of the trace elements, and Zn deficiency causes many adverse effects. Zn complexes are used for Zn supplementation, but there are few toxicity reports. Zn maltol (ZM) was orally administered for 4 weeks to male rats at a dose of 0, 200, 600, or 1000 mg/kg to assess its toxicity. As a ligand group, maltol was administered at a dose of 800 mg/kg/day. General conditions, ophthalmology, hematology, blood biochemistry, urinalysis, organ weights, necropsy, histopathology, and plasma Zn concentration were investigated. Plasma Zn concentration increased with dose levels of ZM. The following toxicities were observed at 1000 mg/kg. Pancreatitis was observed with histopathological lesions and increases in white blood cell parameters and creatine kinase. Anemia was observed with changes in red blood cell parameters and extramedullary hematopoiesis in the spleen. Decreases in the trabecula and growth plate in the femur were observed. On the other hand, no toxicities were observed in the ligand group. In conclusion, these toxicities induced by ZM have been reported as Zn-related toxicities. It was considered that these results will be helpful for a creation and development of new Zn complexes as well as supplements.

Pathophysiological Role of Transient Receptor Potential Ankyrin 1 in a Mouse Long-Lasting Cystitis Model Induced by an Intravesical Injection of Hydrogen Peroxide.

Chronic inflammatory bladder disorders, such as interstitial cystitis/bladder pain syndrome, are associated with poor quality of life. The exact pathological processes remain unclear, but accumulating evidence suggests that reactive oxidative species (ROS) are involved in urinary bladder disorders. Transient receptor potential ankyrin 1 (TRPA1), the most sensitive TRP channel to ROS, was shown to be responsible for urinary bladder abnormalities and hyperalgesia in an acute cystitis model. However, the roles of TRPA1 in chronic inflammatory bladder are not fully understood. We previously established a novel mouse cystitis model induced by intravesical injection of hydrogen peroxide (H2O2), resulting in long-lasting frequent urination, bladder inflammation, pain-related behavior, and histopathological changes. In the present study, we investigated the pathophysiological role of TRPA1 in the H2O2-induced long-lasting cystitis mouse model. Under anesthesia, 1.5% H2O2 solution was introduced transurethrally into the bladder of female wild-type (WT) and TRPA1-knockout mice and maintained for 30 min. This increased the number of voids in WT mice at 1 and 7 days after injection, but reduced the number in TRPA1-knockout mice at 1 day but not 7 days after injection. Spontaneous locomotor activities (increase in freezing time and decrease in distance moved) were reduced at 3 h after injection in WT mice, whereas the spontaneous visceral pain-related behaviors were attenuated in TRPA1-knockout mice. Furthermore, upregulation of c-fos mRNA in the spinal cord at 1 day after injection was observed in WT but not TRPA1-knockout mice. However, there was no difference in histopathological changes in the urinary bladder, such as edematous thickening in the submucosa, between WT and TRPA1-knockout mice at 1 or 7 days after injection. Finally, Trpa1 mRNA levels in the L5-S1 dorsal root ganglion were not altered, but levels in the urinary bladder were drastically increased at 1 and 7 days after injection. Taken together, these results suggest that TRPA1 contributes to acute bladder hyperactivity such as frequent urination and bladder pain, but does not appear to play a major role in the pathological processes of long-lasting cystitis.

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.

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.

A rat long-lasting cystitis model induced by intravesical injection of hydrogen peroxide.

Novel longer lasting inflammatory bladder animal models are needed to better understand the pathophysiology of chronic cystitis. We previously developed a relatively long-lasting mouse cystitis model by intravesical injection of hydrogen peroxide (H2O2). To further evaluate its pathophysiology, in this study, we established and analyzed a rat cystitis model. Under anesthesia, 1.5% H2O2 solution was introduced transurethrally into the bladder of female rats, and kept for 30 min. The H2O2 injection significantly increased the number of micturition events up to day 14 and decreased urine volume per micturition, with the smallest volumes on day 3, compared with the vehicle-treated group. Cystometric analysis on day 7 revealed that intercontraction intervals were significantly shortened without affecting the baseline, threshold, or maximum pressures. Intravesical resiniferatoxin-evoked nociceptive behaviors, such as freezing, were significantly enhanced on days 7 and 14. Furthermore, histopathology revealed hemorrhage, edema, infiltration of neutrophils into the lamina propria, and urothelial denudation in the early phase (day 1). These damages were gradually repaired, while hyperplasia of the urothelium, vascularization, increases in fibroblast counts, and infiltration of mast cells and eosinophils were observed through the later phase (days 7 and 14). These results suggest that intravesical H2O2 injection induces relatively long-lasting cystitis with enhanced bladder activity and pain sensation in rats. This approach thus provides a novel rat long-lasting cystitis model that allows us to analyze detailed symptoms and pathophysiology of H2O2-induced cystitis model than the mouse model and may be used to investigate the pathophysiology and treatment of chronic bladder hypersensitive disorders, such as bladder pain syndrome/interstitial cystitis.

Long-lasting pain-related behaviors in mouse chronic cystitis model induced by a single intravesical injection of hydrogen peroxide.

We previously established a long-lasting cystitis model by an intravesical injection of hydrogen peroxide (H2O2) into mice. In this study, we assessed the pain-related behaviors in the cystitis model. An intravesical injection of 1.5% H2O2 transiently decreased spontaneous locomotor activity at 3 h after injection, indicative of acute spontaneous pain. In contrast, licking response to a bladder distention was slowly observed as licks to the lower abdomen at 7 and 14 days after injection, which was attenuated by amitriptyline and morphine, but not by oxybutynin. These results suggest that H2O2-induced chronic cystitis model shows delayed and long-lasting painful pathological condition.

Novel mouse model of chronic inflammatory and overactive bladder by a single intravesical injection of hydrogen peroxide.

There is so far no generally accepted animal model of chronic cystitis by which potential therapies can be evaluated. In this study, we aimed to establish a new mouse model of cystitis based on the proinflammatory effects of reactive oxygen species. A single intravesical injection of 1.5% hydrogen peroxide (H2O2) significantly increased the numbers of voids by 1 day after injection in female mice, which lasted up to 7 days. The H2O2 injection rapidly increased the bladder weight by 3 h in parallel with the histological damage and hyperpermeability of urothelial barrier. Although the urothelial dysfunction was recovered to normal by 7 days, increase in bladder weight, edematous thickening of the submucosa, and vascular hyperpermeability were apparent even 7 days after injection. During the time course, massive infiltration of neutrophils and increased expression of inflammatory cytokines were observed in the bladder. An intraperitoneal administration of oxybutynin, amitriptyline, indomethacin, or morphine attenuated the H2O2-induced frequent urination. These findings suggest that an intravesical injection of H2O2 induces relatively long-lasting inflammatory and overactive bladder, compared with existing cystitis models. The intravesical H2O2 injection model may be a simple and useful tool in the pathological study and drug discovery for chronic cystitis.