Hypothesis / aims of study
The circadian clock programs daily rhythms and coordinates multiple behavioral and physiological processes, including micturition. Partial bladder outlet obstruction (pBOO) in mice produces bladder hypertrophy and hyperactive voiding on the cystometrogram. However, the cystometrogram cannot replicate physiological conditions completely even when performed under the conscious condition. In our previous study, using a metabolic cage, long-term pBOO led to disruption of the circadian rhythms (the day/night cycle) in mice, similar to those observed in humans as nocturia. In clinical practice, alpha-1 blockers relieve the nocturia of benign prostatic hypertrophy (BPH) patients. However, the mechanism of this improvement has not yet been completely clarified. In the present study, the effects of chronic administration of an alpha-1 blocker on the circadian rhythm of micturition behavior of long-term pBOO in mice were investigated. To clarify the mechanism, several growth factors and cytokines were examined.
Study design, materials and methods
A total of 22 female C57/BL6 mice were used. All mice had free access to food and water and were kept under a 12-h light/dark cycle. The mice were divided randomly into three groups: the normal group fed a standard chow diet (intact, n = 7); one pBOO group that underwent tying of the proximal urethra as in the procedure for pBOO and was fed a standard chow diet (pBOO, n = 7); and another pBOO group that underwent the same procedure and received chronic administration of an alpha-1 blocker with a naftopidil mixed diet (pBOO + NAF, n = 7). The content of naftopidil in the chow was estimated by the amount of feeding a day and the average body weight of mice and was calculated as almost equal to 20 mg/kg/day. Naftopidil was then administered for 6 months. Micturition behavior was evaluated 6 months after pBOO surgery. The automated voided stain on paper (aVSOP) method, which is a precise micturition recording system for mice, was used. Urine stains were counted and traced, ranging from 10 to 800 μl. The parameters evaluated were voided volume and time per void, total urination frequency (daytime and night time), and total urine volume. Bladders were harvested with RNAlater (QIAGEN, Venlo, The Netherlands) and flash frozen in liquid nitrogen after being weighed. Quantitative assessment of gene expression was performed using the qRT-PCR procedure. RNA was extracted from the urinary bladder using an RNeasy Fibrous Tissue Mini Kit (QIAGEN) according to the manufacturer’s protocol. Purified RNA was reverse transcribed to cDNA using a SuperScript VILO cDNA Synthesis Kit (Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocol. QRT-PCR was performed using Quant Studio 7 Flex (Applied Biosystems, Waltham, MA, USA) with Taqman (Applied Biosystems) and THUNDERBIRD (TOYOBO, Osaka, Japan). RPL 19 was used as a housekeeping gene. The relative expression level of each mRNA was then normalized to the expression level of RPL 19, using the 2-ΔΔCT method. The expression levels of mRNA for collagen I, collagen III, 5-HT2A-2C, tissue inhibitor metalloprotease (TIMP) 1, TIMP2, matrix metalloprotease (MMP) 2, MMP9, and IL-1b were assessed. Data are presented as means ± standard error. For qRT-PCR, the mean ± deviation is presented. Statistical significance was tested between the 2 age groups using Student’s t-test, and that between periods was tested by the paired t-test. The differences in mRNA-expression levels were checked using the values of ΔΔCT by the Tukey test. All animals were maintained in a pathogen-free environment, under experimental protocol guidelines approved by University’s Committee for Animal Investigations.
There was a tendency for an increase in the body weight of pBOO mice compared to intact mice (27.4 ± 4.6g vs. 24.3 ± 1.5 g) 6 months after the pBOO operation. There was no significant difference in body weight between pBOO and pBOO + NAF (26.0 ± 1.5 g). Wet bladder weight was significantly increased in pBOO and pBOO + NAF compared to intact mice (50.0 ± 12.6 mg, 63.4 ± 9.4 mg, and 22.7 ± 1.3 mg, respectively). There was no significant difference in the number of micturitions 6 months after surgery in the intact, pBOO, and pBOO + NAF groups. However, an increase in day time micturition frequency and a decrease in average voided volume in the day time were observed in pBOO mice compared to intact mice (p < 0.05) (circadian rhythm disturbance). Day time micturition frequency was significantly decreased in the pBOO + NAF group compared to the pBOO group (p < 0.05) (Figure 1). The mRNA-expression levels of collagen I, collagen III, 5-HT2A, TIMP1, and TIMP2 were significantly increased, and that of MMP9 was significantly decreased in pBOO mice compared to intact mice (p < 0.05). In addition, mRNA-expression levels of 5-HT2A and TIMP2 were significantly decreased in the pBOO + NAF group compared to the pBOO group (p < 0.05) (Figure 2).
Interpretation of results
To the best of our knowledge, this is the first study to show that long-term administration of an alpha-1 blocker reverses the micturition pattern in pBOO mice. Moreover, certain background mechanisms were clarified. Mice are nocturnal animals and also show a rhythm in micturition frequency, which is decreased during daytime, their sleep phase. However, long-term pBOO leads to disruption of one of the circadian rhythms (the day/night cycle) in mice. pBOO is known to induce morphological and functional changes in the bladder that can be attributed to ischemia, hypoxia, or mechanical stress. Our group previously reported that IL-1b-mRNA levels were significantly higher compared to a sham group after short-term pBOO (3 weeks) (ref.1). In the current study, the level of IL-1b was not significantly increased with long-term pBOO. A group of enzymes called MMPs and their specific inhibitors, TIMPs, were critical factors in tissue fibrosis. Both TIMP1 and TIMP2 were increased after long-term pBOO, and then naftopidil reversed TIMP2 expression.
Naftopidil has alpha-1-adrenoceptor antagonism as a common pharmacological property, in addition to behaving as a 5-HT2 antagonist. In the current study, pBOO increased 5-HT2A, which was reversed by long-term administration of naftopidil. This phenomenon could be a background mechanism relieving storage symptoms in BPH patients. However, long-term administration of naftopidil could not decrease the expression levels of collagen I and collagen III mRNA expression levels. This is an understandable result. The long-term effect of alpha-1 blocker treatment could not induce morphological changes, but it did induce a functional change. Although the long-term effect may be suppression of fibronectin and laminin expression levels, this need to be clarified in the future.