Circadian Rhythms Fluctuate the Treatment Effects of Intravesical Treatments on Rat Urinary Frequency Models

Objectives It is still not clear how the intravesical instillation of drugs affects rat urinary frequency. This study aimed to examine the dynamics of intravesical treatments' treatment effect on rat urinary frequency models by real-time and extended monitoring using a novel continuous urination monitoring system. Methods Nine eleven-week-old female Wistar rats were divided into three groups to receive intravesical instillation of 0.1% acetic acid (AA), 1.0% AA, or phosphate-buffered saline (PBS). Thirty minutes later, these drugs were voided, and rats were moved to a continuous urination monitoring system, UM-100. UM-100 monitored rat urination quantitatively and continuously for 24 hours. Rats were then euthanized, and histopathologic examinations using a damage score validated the severity of bladder inflammation. We used nine additional rats to determine the treatment effect of various drugs against the urinary frequency. These rats were also treated with 1.0% AA in the same way and divided into three groups (n = 3 each) to receive intravesical instillation of lidocaine, silver nitrate (AgNO3), or dimethyl sulfoxide (DMSO), respectively. Thirty minutes later, rats were catheterized again and moved to the UM-100, and their voiding was monitored for 24 hours. Results Intravesical instillation of AA increased the urinary frequency and decreased the mean voided volume (VV) in a concentration-dependent manner, with statistical significance at a concentration of 1.0% (urinary frequency; p=0.0007, mean VV; p=0.0032, respectively) compared with PBS. Histopathological analysis of these models demonstrated a significantly higher damage score of bladder mucosa in both 0.1% AA and 1.0% AA compared with PBS, with the severity in concordance with the clinical severity of urinary frequency (0.1% AA: p < 0.0001, 1.0% AA: p < 0.0001). Moreover, intravesical instillation of lidocaine, AgNO3, and DMSO decreased the urinary frequency. Continuous monitoring with UM-100 also demonstrated that the treatment effect of these intravesically instilled drugs occurred only at night. Conclusions The extended monitoring of rat urination by UM-100 revealed a significant fluctuation in the treatment effect of intravesically instilled drugs between day and night. These findings may help establish novel therapies for urinary frequency.


Introduction
Urinary frequency and lower urinary tract symptoms (LUTSs) are common and negatively impact patient quality of life, with more than 60% prevalence in adult women [1][2][3].Various treatments, including behavioral and drug therapy, have shown efcacy in treating LUTS [2,4].However, not all patients respond to these therapies, and there is an urgent need to develop more efective treatments for urinary frequency.
One promising approach for treating urinary frequency and LUTS is the intravesical administration of drugs.Intravesical instillation can directly deliver a higher concentration of drugs with minimal adverse events compared with whole-body administration.Te intravesical instillation of several drugs/chemicals such as lidocaine, capsaicin, resiniferatoxin, silver nitrate (AgNO 3 ), and dimethyl sulfoxide (DMSO) has already demonstrated some efcacy in treating overactive bladder, interstitial cystitis, bladder pain syndrome, and hemorrhagic cystitis [5][6][7][8][9].However, the reported treatment efect of these drugs to date is less remarkable than expected, and a more efective technique for intravesical instillation of drugs, such as bladder indwelling devices, is needed [10].
Several methods have been tested in animal models, such as dogs and rodents, to examine the mechanisms of LUTS in humans and to establish novel treatments for LUTS.Te voided stain on paper (VSOP) is one of the most common tests to evaluate mouse/rat LUTS noninvasively [11].While this method provides many advantages, some have pointed out its limitations.For example, VSOP is a semiquantitative assay, and the recommended assay duration of the VSOP is limited to 2-4 hours [11].Tus, the VSOP seems unft for evaluating the efect of intravesical treatment, which requires accurate extended monitoring.Moreover, many variables infuence VSOP assay outcomes (e.g., the flter paper used and its odor [11]).Tus, an alternative noninvasive method is required to overcome these limitations.
UM-100 (Melquest, Toyama, Japan) is a novel continuous urination monitoring tool for rodents that can record rat urination quantitatively and automatically every second for over 24 hours, similar to a previously reported human urinary monitoring system [12].Due to its weight-based, real-time, and quantitative measurement method, UM-100 can potentially replace the VSOP and improve the accuracy of the assessment of rat lower urinary function in combination with the VSOP.In this study, we established rat LUTS models by intravesical instillation of acetic acid (AA) and examined the dynamics in the treatment efect of intravesical treatments on rat urinary frequency models by real-time and extended monitoring using UM-100 for the frst time in the world.

Ethical Consideration.
All animal experiments were conducted following the national guidelines and the relevant national laws on the protection of animals.Experimental protocols required for the animal studies were approved by the Animal Experiment Committee at Okayama University.Te approval number is OKU-2021235.

Void Monitoring
Using UM-100.UM-100 (Melquest, Toyama, Japan) is a continuous monitoring system for rodent voiding behavior [13].It comprises a metabolic cage manufactured by Techniplast (Buguggiate, Italy), a urine bag, a weight sensor that can measure weight variations in 10 mg increments, a central control unit, and a computer with software for data storage and analysis of the data.Te weight sensor under the metabolic cage detects urine drops, and UM-100 records each weight variation every second.UM-100 defnes the beginning of a voiding event as a series of weight changes and the termination of the voiding event as no additional weight change in fve seconds after that (Figures 1(a) and 1(b)).Te metabolic cage, urine collecting bags, and weight sensors can be expanded up to 4 units, and UM-100 can monitor up to 4 rodents simultaneously.After the examination, the mean voided volume (VV) and urinary frequency were calculated.
We used nine rats to establish rat urinary frequency models.After the adaptation period for 24 hours in the cage, all rats were catheterized and divided into three groups (n � 3 each) to receive intravesical instillation of 300 μL of 0.1% AA, 1.0% AA, or phosphate-bufered saline (PBS) as a negative control, respectively [14,15].Tirty minutes later, the rats were catheterized again and moved to the UM-100.Urination was monitored for 24 hours.When we focused on the circadian rhythm, the data were analyzed with the monitoring period fractionated into two, i.e., daytime and nighttime, which were 12 hours each; daytime was from 8 AM to 8 PM, while nighttime was from 8 PM to 8 AM.When we focused on the elapsed time, we analyzed the data into three periods as follows: 0-8 hours, 8-16 hours, and 16-24 hours.Before and during the experiment, rats had access to food and drinking water ad libitum.After the experiment, rats were euthanized, and their bladders were subjected to histopathological analysis to validate the severity of infammation (Figure 2(a)).We used an additional nine rats to determine the treatment efect of various drugs against urinary frequency.Tese rats were also treated with 1.0% AA in the same way and subsequently divided into three groups (n � 3 each) to receive intravesical instillation of drugs: 0.1% AgNO 3 , 4% lidocaine, or DMSO, respectively [16][17][18].Tirty minutes later, rats were catheterized again and moved to the UM-100, and their voiding was monitored for 24 hours, as described above (Figure 3(a)).

Histopathological Analysis.
Fixed tissues were processed routinely using standard techniques, subsequently embedded in parafn, and sectioned into fve μm-thick sections stained with hematoxylin and eosin (HE).Histological changes in the bladder mucosa were assessed at 200x magnifcation under a BZ-X700 microscope (Keyence).In each specimen (n � 3 per treatment group), edema of the bladder mucosa in two randomly selected felds was evaluated using the following damage scoring system: 0 � normal (no edema), 1 � mild (no change in connective tissue thickness), 2 � moderate (connective tissue thickness increased by < 2fold), and 3 � severe (connective tissue thickness increased by >2 folds) as previously reported in rabbit cystitis models [19].

Statistical Analysis.
All quantitative data were presented as bar graphs with the means ± standard error of the mean (SEM).Te relationships between continuous variables among groups were compared using one-way ANOVA.For multiple tests, Bonferroni corrections were used.All tests      Figure 3: Intravesical instillation of drugs can relieve the urinary frequency of rat urinary frequency models at night.An additional nine rats were used to determine the treatment efect of various drugs against urinary frequency.Tese rats were treated with 1.0% AA in the same way as Figure 2 and subsequently divided into three groups (n � 3 each) to receive intravesical instillation of drugs (0.1% AgNO 3 , 4% lidocaine, or DMSO).Tirty minutes later, rats were catheterized again and moved to the UM-100, and their voiding was monitored for 24 hours.2(b)).Te elapsed time from the initiation of the experiment slightly, but not signifcantly, afected the voiding behavior in rat models (Figure 2(c)).Te mean VV increased as time passed from the beginning of the experiment.Also, AA afected the voiding behavior of rats throughout the experiment, with a similar tendency in all three time periods mentioned above (Figure 2(d)).Moreover, histopathological analysis of these models demonstrated increased stromal edema (black arrows) in an AA concentration-dependent manner, with the severity in concordance with the clinical severity of urinary frequency (Figure 4(a)).When we quantifed the bladder infammation using the damage scoring system (e.g., PBS � 0, 0.1% AA � 2, and 1.0% AA � 3 in Figure 4(a)), the histopathological severity of the bladder mucosa was signifcantly higher in both 0.1% AA and 1.0% AA compared with PBS (PBS vs. 0.1% AA: p < 0.0001, PBS vs. 1.0%AA: p < 0.0001) (Figure 4(b)).Intravesical instillation of 1.0% AA induced severer bladder infammation than 0.1% AA.However, the diferences were not statistically signifcant (p � 0.3634) (Figure 4(b)).Intravesical instillation of drugs can relieve urinary frequency in rat urinary frequency models, especially at night.

Discussion
In this study, we performed quantitative and real-time analysis of rat voiding behavior for as long as 24 hours using UM-100.Te extended monitoring of the voiding behavior of rat urinary frequency models demonstrated signifcant changes in the treatment efect of intravesically instilled drugs according to the circadian rhythm of the rats.
In our data, in accordance with previous reports, the intravesical instillation of AA evoked bladder infammation that resulted in increased urinary frequency in rats, and intravesical instillation of lidocaine and DMSO relieved rat urinary frequency, while the treatment efect of silver nitrate was not as remarkable as other drugs [14,[21][22][23].Tis result refected the treatment efect of these drugs in patients with urinary frequency.Since intravesical instillation of silver nitrate itself often results in damage to the urothelium and scarring of the bladder, it has not been regarded as a promising intravesical instillation drug as lidocaine and DMSO [24].Also, in this study, damage to the urothelium may have hindered the treatment efect of AgNO 3 and failed to show statistical signifcance.
Moreover, our results support those of previous reports that noted a diurnal variation in the voiding behavior of rats and, more importantly, revealed that the treatment efect of intravesically instilled drugs is more signifcant at night [25,26].At night, rats are reportedly more active, have lower bladder capacity, and urinate more often [25].Tus, our results indicate that intravesical instillation of drugs is more efective at night when bladders are more active and, therefore, more sensitive to external stimuli than during the day.Tis theory is consistent with another study, which found that intravesical treatments with b-FGF increased the 6 Veterinary Medicine International urinary frequency only at night [26].Since conventional experimental methods, such as VSOP and cystometry, are recommended to be performed for a relatively short duration, experiments conducted during the day may miss the treatment efect of drug candidates, which might show effcacy at night [11].Moreover, in the VSOP, changing the flter paper or moving to the VSOP cage may wake the rats and afect the analysis during the day.In this study, we achieved extended monitoring of rat urinary behavior for a longer time with minimal external stimuli using UM-100 to reveal the dramatic fuctuation of the treatment efects of intravesical treatments on rat urinary frequency models.Te extended monitoring of the voiding behavior of rat urinary frequency models could be helpful, especially in evaluating the treatment efect of intravesical administration of drugs and intravesical indwelling devices.Since intravesical administration of some medicines demonstrated efcacy in treating urinary frequency or bladder pain, several intravesical indwelling devices have been developed to prolong their treatment efect and were examined in animal models.However, most of those experiments were insufcient to predict efectiveness in humans but remained preliminary, such as plasma concentrations of tested drugs and cystometry under general anesthesia [27,28].Our results indicate that durable monitoring of voiding behavior in conscious and nonrestrained rats has the potential to provide a new perspective on animal experiments to develop a more efective intravesical treatment for LUTS and urinary frequency.
Our study has several limitations.First, we could not directly compare the accuracy of the UM-100 and the VSOP.Second, we could not examine the validity and reproducibility of the UM-100 as reported for the VSOP [29].Also, we could not evaluate the sexual diference in the efect of AA and drugs.In this study, we used female rats for the following two reasons: the prevalence of urinary incontinence or LUTS is higher in women than men and female rats are easier to catheterize than male [30,31].Te results might be diferent if we examined male rats.Moreover, in this study, AA exposure was 30 minutes long in the same way as a previous report using bladder pain rat models [32].A notable loss of urothelial integrity was not observed, and the treatment efect might have been diferent if these experiments had been performed under more severe bladder infammation conditions.Finally, we could not examine the impact of multiple AA instillations to establish chronic cystitis models.Despite these limitations, our study is valuable in performing a quantitative and real-time analysis of rat voiding behavior for as long as hours and demonstrating the signifcant changes in the treatment efect of intravesically instilled drugs according to the circadian rhythm of the rats.

Conclusion
In this study, we achieved a quantitative and real-time analysis of rat voiding behavior for as long as 24 hours using UM-100 and the signifcant changes in the treatment efect of intravesically instilled drugs according to the circadian rhythm of the rats.Tese fndings may help establish novel therapies for urinary frequency.

Figure 1 :
Figure 1: Experimental models with the UM-100.(a) Overview of the UM-100.(b) A representative analysis of data from the UM-100.UM-100 monitored the urination of an 11-week-old female Wistar rat for 24 hours (day and night were 12 hours each).Te weight sensor under the metabolic cage detects urine drops, and UM-100 records each weight variation every second.UM-100 defnes the beginning of a voiding event as a series of the weight changes, and the termination of the voiding event as no additional weight change in fve seconds after that.

Figure 2 :Figure 3
Figure2: Acetic acid evokes bladder infammation and urinary frequency.Nine rats were used to establish rat urinary frequency models.After the adaptation period for 24 hours in the cage, all rats were catheterized and divided into three groups (n � 3 each) to receive intravesical instillation of 300 μL of 0.1% AA, 1.0% AA, or phosphate-bufered saline (PBS) as a negative control.Tirty minutes later, the rats were catheterized again and moved to the UM-100.Urination was monitored for 24 hours.Day and night were 12 hours each.(a) Experimental protocol.(b) Efect of AA on voiding behavior and its diurnal variation.Summary data of the mean voided volume (VV, left) and urinary frequency (right) over 24 hours (upper), day (lower left), and night (lower right) are shown.(c) Voiding behavior of rats in three periods during monitoring.Rats were catheterized, and 300 μL of PBS was intravesically instilled.Tirty minutes later, the rats were catheterized again and moved to the UM-100.Urination was monitored for 24 hours.Summary data of the mean VV (left) and urinary frequency (right) in the indicated periods are shown.(d) Infuence of time course during the experiment on the efect of AA.Summary data of the mean VV (left) and urinary frequency (right) in the indicated periods are shown.One-way ANOVA with the Bonferroni correction was used.Bars, mean; error bars, standard error of the mean; open circles, individual measurements; ns, not signifcant; * , p < 0.05; * * , p < 0.01; * * * , p < 0.001.
Figure3: Intravesical instillation of drugs can relieve the urinary frequency of rat urinary frequency models at night.An additional nine rats were used to determine the treatment efect of various drugs against urinary frequency.Tese rats were treated with 1.0% AA in the same way as Figure2and subsequently divided into three groups (n � 3 each) to receive intravesical instillation of drugs (0.1% AgNO 3 , 4% lidocaine, or DMSO).Tirty minutes later, rats were catheterized again and moved to the UM-100, and their voiding was monitored for 24 hours.Day and night were 12 hours each.(a) Experimental protocol.(b) Efect of intravesical treatment on voiding behavior and its diurnal variation.Summary data of the mean voided volume (VV, left) and the urinary frequency (right) over 24 hours (upper), day (lower left), and night (lower right) are shown.(c) Infuence of time course during the experiment on the efect of various drugs.Summary data of the mean VV (left) and urinary frequency (right) in the indicated periods are shown.One-way ANOVA with the Bonferroni correction was used.Bars, mean; error bars, standard error of the mean; open circles, individual measurements; ns, not signifcant; * , p < 0.05; * * , p < 0.01; * * * , p < 0.001.

Figure 4 :
Figure 4: Histopathological analysis of bladder infammation by AA.After monitoring, rats were euthanized, and their bladders were fxed and processed routinely and embedded in parafn.Five μm-thick sections were stained with hematoxylin-eosin (HE).(a) Representative histopathological images for rats treated with phosphate-bufered saline (PBS, left, no edema), 0.1% acetic acid (AA, middle, moderate connective tissue thickness increase), and 1.0% AA (right, heavy connective tissue thickness increase) are shown.Black arrows indicate stromal edema of the bladder mucosa.(b) Te quantifcation of the bladder infammation using the damage scoring system.Summary data of the damage score are shown.Bars, mean; error bars, standard error of the mean; open circles, individual measurements; ns, not signifcant; * * * * , p < 0.0001.