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Animal Models for the Study of Post-Micturition Dribble in Aged Male
Korean J Clin Lab Sci 2022;54:307-315  
Published on December 31, 2022
Copyright © 2022 Korean Society for Clinical Laboratory Science.

Mi-Young Park

Department of Clinical Laboratory Science, Suwon Science College, Hwaseong, Korea
Correspondence to: Mi-Young Park
Department of Clinical Laboratory Science, Suwon Science College, 288, Seja-ro, Jeongnam-myeon, Hwaseong 18516, Korea
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Since attaining middle age, many patients suffer from the post-micturition dribble (PMD) syndrome but do not receive proper treatment. This is because the etiology and treatment mechanisms of PMD differ from conventional lower urinary tract symptoms. Several pathophysiologies have been proposed, but the mechanisms we observed are the weakening of the bulbocavernosus muscle and dilation of the urethra due to atrophy of the cavernosal tissue. This study investigates the mechanism of PMD by observing anatomical changes in the corpus cavernosum and urethra. SD male rats were categorized by age into the young rats group (8 weeks old) and the old rats group (24 months or more). The ratio area of the bulbous urethra and corpus cavernosum to the total penis was statistically significant between both groups (P<0.05). Significant differences were obtained between the two groups for restored smooth muscle contents and several other parameters related to PMD mechanisms (nNOS, α-SMA) (P<0.05). The changes in size and parameters of the urethra and corpus cavernosum in young and old rats were confirmed. The results from this study are expected to contribute to the study of PMD in the future.
Keywords : Post micturition dribble, Urethra, Lower urinary tract symptoms

In general, urologists tend to think that lower urinary tract symptoms (LUTS) make it easy to recall voiding and storage symptoms [1]. The effect of international prostate symptom score (IPSS), which is often used for LUTS treatment, is thought to have a lot of influence, but poorly treated post micturition dribble (PMD) is likely to escape the attention of urologists [2]. PMD is a term used to describe the involuntary loss of urine immediately after an individual finishes passing urine, usually after leaving the toilet in men or after rising from the toilet in women [3]. PMD is one of the most bothersome symptoms of middle-aged male LUTS, however the etiology and treatment mechanism of PMD differ from those of conventional lower urinary tract symptoms, so proper treatment is not done yet [1, 2]. Factors associated with PMD include weakening of the bulbocavernosus muscle, an abnormal urethro-corporocavernosal reflex, reduced compliance of the bulbar urethra, weakening of the external urethral sphincter, and bladder neck obstruction [4]. Several pathophysiology has been proposed, so the mechanisms we noted are the weakening of the bulbocavernouos muscle and the dilation of urethra due to atrophy of cavernosal tissue [1, 3].

Although clinical trial results have already been published, there are no preclinical studies on the evidence. So, we want to develop a disease animal model for PMD and compare the effects of senile change of penis and urethra. So the purpose of this study is to investigate the mechanism of PMD by observing the anatomical changes of the corpus cavernosum and urethra.


1. Experimental animal and study design

This study was performed in accordance with the Guide for Care and Use of Laboratory Animals.

All animal experiments in this study were approved by the Institutional Animal Care and Use Committee of the Catholic University of Korea (CUMC-2016-0218-01).

Experimental animals were obtained from Orient Bio Co. (Seongnam, Korea) 24 months old male Sprague-Dawley rats were chosen for the old rat group and 8-week-old male Sprague-Dawley rats were chosen as a young rat group. All rats were sacrificed and then whole urethra from prostate to urethral orifice and corpus cavernosum were collected for histochemical and Western blot analysis.

2. Measurement of diameter of urethra using confocal laser scanning microscope analysis

Sections were viewed using an Carl Zeiss LSM900 w/AiryscanII Confocal microscope with ×20 objectives. Digital images were obtained using a Zeiss LSM 900 confocal microscope (Zeiss, Oberkochen, Germany), smooth muscle ratio and urethral luminal area was calculated using AxioVision Rel. 4.8 Software (Carl Zeiss Kore, Seoul, South Korea).

3. Measurement of erectile function

A 23-gauge butterfly needle containing 250 U/mL heparin solution was carefully inserted into the proximal corpus cavernosum and the other end of the PE-50 tube was connected to a pressure transducer (Grass model S48 K; Astro-Med Inc., West Warwick, RI, USA), to measure the ICP [5]. A bipolar stainless-steel electrical stimulator was used to stimulate the major pelvic ganglion at 10 V for 100 s and 2.4 mA with a pulse width of 2.5 ms. The maximum ICP value of three stimulations was used for the statistical analysis in each rat. ICP was normalized to MAP, which was recorded using a BD Intramedic PE-50 tubing (BD, Franklin Lakes, NJ, USA) inserted into the left carotid artery at the same time [6].

4. Histochemistry

The collected tissue samples were fixed in 4% parafor-maldehyde for 24 h at 4℃ before creating a paraffin block. The following primary antibodies were used: nNOS (diluted 1:200; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), α-SMA (diluted 1:500; Abcam, Cambridge, UK), and 6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Inc., Burlingame, CA, USA) was used to stain nuclei. Digital images were obtained using a Zeiss LSM 800 Meta confocal microscope (Zeiss, Oberkochen, Germany), and the mean intensity was calculated using ZEN 2012 (Zeiss).

5. Masson’s trichrome staining

Each set of sections was processed with Masson’s trichrome staining for urethral muscle fiber and collagen components. Urethral tissues were immersed in warm (60℃) Bouin solution for 30 minutes, rinsed, then stained with Weigert Hematoxylin for 10 minutes, and then rinsed until only nuclei remained stained. The sections were then stained with Biebrich Scarlet-Acid Fuchsin for 3 minutes, rinsed, and immersed in phosphomolybdic acid for 40 minutes. Next, the sections was stained with Aniline Blue for 5 minutes, rinsed in distilled water for 3 minutes, immersed in 1% acetic acid for 1 minutes, Finally, the sections were dehydrated through increasing concentrations of ethanol, and mounted.

6. Western blotting

The collected tissue was homogenized using ice-cold RIPA buffer (Cell Signaling Technology) containing ethylene diamine tetra acetic acid-free protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche Diagnostics GmbH, Basel, Switzerland) and particulate mass was removed by centrifugation (15,000×g) for 15 min at 4℃. Supernatants were analyzed by SDS-PAGE. The following primary antibodies were used: p-AKT (diluted 1:200; Cell Signaling Technology), AKT (diluted 1:200; Cell Signaling Technology), Bax (diluted 1:200; Cell Signaling Technology), Bcl-2 (diluted 1:200; Cell Signaling Technology), Cleaved PARP (diluted 1:200; Cell Signaling Technology), PARP (diluted 1:200; Cell Signaling Technology), and β-actin (diluted 1:1000; Abcam) were used.

7. Cyclic guanosine monophosphate assay

Tissues were treated with 350 mL of 0.1 M HCl, after which silica beads were added (BioSec Enviro, Inc., Guelph, ON, Canada). The resulting samples were processed through a homogenizer (Precellys 24; Bertin Technologies, Montigny-le-Bretonneux, France) and spun by centrifugation at 12,000×g for 10 minutes at 4℃, after which their supernatants were extracted. A cyclic guanosine monophosphate (cGMP) direct immu-noassay kit (K372-100; BioVision, Mountain View, CA, USA) was used to measure corporal cGMP levels.

8. Statistical analysis

All data are presented as means±standard error (SD) and were analyzed using SPSS version 22.0 (IBM, Armonk, NY, USA). Student’s t-tests, one-way ANOVA, and 2×2 factorial ANOVA were used, as appropriate, to evaluate differences among groups. P<0.05 was consi-dered statistically significant.


1. Measurement of diameter and area of bulbous urethra and corpus cavernosum

The diameter of bulbous urethra was statistically signi-ficantly greater in old rats than in young rats (P<0.001). Corpus cavernosum was also statistically significantly greater in old rats than in young rats (P<0.001). The ratio area of the bulbous urethra to total penis were significantly lower in the young rat group, than in old rat group (P<0.01) Young rat group had significantly higher (P<0.01) corpus cavernosum ratio than the old rat group (Table 1).

Measurement of diameter and area of bulbous urethra and corpus cavernosum

Bulbous urethra Corpus cavernosum Urethra/
total penis
Corpus cavernosum/total penis
Young (µm2) 191049.66±1235.56 3286741±6781.73 2.42±1.54% 46.10±4.29%
Old (µm2) 538470±199.11 5259875±2175.63 4.22±0.16% 39.69±1.75%
P-value <0.001 <0.001 <0.001 <0.01

2. Analysis of erectile function

All rats were tested for erectile function by measuring intracavernous pressure (ICP) and mean arterial pressure (MAP) under anesthesia. Representative images of ICP results are shown in Figure 1. The old group showed statistically significantly lower ICP values than young group. In a quantitative analysis (Figure 1B), the ICP of the young group was 0.82±0.11, and the old group was 0.28±0.07. The mean ratio was significantly lower in the old group than in the young group (P<0.05), These results showed that ICP/MAP ratio was significantly higher in the young groups than in the old group (P<0.05).

Fig. 1. Comparison of erectile function for each study group. (A) Representative images of intracavernous pressure (ICP) in response to electrical stimulation of the cavernosal nerve. (B) Ratio of ICP to mean MAP in each group. Each bar shows the mean value (standard devi-ation). *P<0.05 compared with the old group.

3. Smooth muscle content in the urethra and corpus cavernosum

Masson’s trichrome staining was performed to visualize the urethra present at the site. The smooth muscle and collagen contents in the urethra were observed by Masson’s trichrome staining. Figure 2A illustrates images of transverse sections of the proximal and the distal urethra, which gradually decreased in size (Figure 2B). In the young distal urethra the lumen is crescent‐shaped due to the presence of the smooth muscle protrusion. As shown in Figure 2, the smooth muscle contents were higher in the young group than in the old group (P<0.05).

Fig. 2. Histological analysis of the urethra and corpus cavernosum. Repre-sentative images of Masson’s Trichrome staining in the urethra. (A) Red indicates smooth muscle, and green represents collagen. Original magnification: ×200. (B) Smooth muscle ratio for each group. Each bar shows the mean value (standard deviation). *P<0.05 compared with the old group.

4. Alpha smooth muscle actin expression in urethra

In the urethra, smooth muscle positive areas were analyzed by immunohistochemical staining (Figure 3A). As shown in Figure 3, α-smooth muscle actin (α-SMA) expression levels were elevated in the urethra, indicating that smooth muscle higher in young rat urethra tissues (P<0.05). Staining showed nuclear localization with DAPI, with cell nuclei stained blue.

Fig. 3. Representative images of immunofluorescence staining in the urethra. (A) Representative images of α-smooth muscle actin (α-SMA) staining for each group. Green indicates s α-SMA, and blue represents cell nuclei. Original magnification: ×200. (B) Mean intensity of α-SMA for each group. *P<0.05 compared with the old group.

5. Alpha smooth muscle actin expression in corpus cavernosum

In the corpus cavernosum, smooth muscle positive areas were analyzed by immunohistochemical staining (Figure 4A). As shown in Figure 4, α-SMA expression levels were elevated in the corpus cavernosum, indicating that smooth muscle higher in young rat tissues (P<0.05).

Fig. 4. Alpha smooth muscle actin expression in corpus cavernosum. Repre-sentative images of immunofluorescence staining in the corpus cavernosum. (A) Representative images of α-SMA staining for each group. Green indicates s α-SMA, and blue represents cell nuclei. Original magnification: ×200. (B) Mean intensity of α-SMA for each group. *P<0.05 compared with the old group.

6. In the dorsal penile nerve fibers and nNOS were analyzed by immunohistochemical staining

Representative images of nNOS staining in the copus carvernosum is shown nNOS was lower in the old group than in the young group (Figure 5A). Immunostaining for neuronal nitric oxide synthase (nNOS) (red) and immu-nostaining staining using nerve marker β-III tubulin (green) in the dorsal penile nerve. In a quantitative analysis, intensities were higher in the young group than in the old group (P<0.05).

Fig. 5. Neuronal nitric oxide synthase (nNOS) expression in the dorsal penile nerve. Representative images of nNOS in the dorsal penile nerve. (A) Red indi-cates nNOS and green indicates the penis dorsal nerve. Original magnifi-cation: ×400. (B) Mean intensity of nNOS for each group. *P<0.05 compared with the old group.

7. PI3K/AKT Signaling Pathway in the urethra and corpus cavernosum

As shown in Figure 6, in the young group, p-AKT/AKT levels were higher (P<0.05), indicating PI3K/AKT pathway activation compared with that in the Old group. Bcl-2 and Bax are downstream effector proteins in the PI3k/AKT pathway. Bcl-2 levels were higher and Bax levels were lower in the young group than in the Old group (P<0.05). We also observed lower cleaved PARP expression in the young group, further supporting the reduction in apoptosis in the urethra. Figure 6C shows that in young group, NO and cGMP level was higher than old groups.

Fig. 6. Protein expression in the urethra and cyclic guanosine mono-phosphate (cGMP) levels in the corpus cavernosum. (A) All groups were com-pared with respect to p-AKT, AKT, Bax, Cleaved PARP, and Bcl-2 expression in the corpus cavernosum by western blotting. (B) Relative density of bands in the western blot analysis for each group. *P<0.05 p-AKT/AKT compared with the Old group, **P<0.05. Cleaved PARP/PARP compared with the Old group, #P<0.05 Bax compared with the old group, ##P<0.05 Bcl-2 compared with the old group. (C) cGMP levels in the corpus cavernosum. Each bar shows the mean values. *P<0.05 compared with the old group.

PMD is one of the most common symptoms of LUTS and big bothersome disease for men. Compared to this discomfort, the treatment and mechanism are not clear, so it can be regarded as one of the types of intractable diseases [7, 8]. The symptoms are not life-threatening and the response to treatment is insignificant, so research on this has not been conducted much recently [9].

In men, PMD is known to occur when urine collects in the bulbous urethra, but opinions are still controversial on the exact mechanism [10]. One of the purpose considered as a mechanism is that the urine collected in the urethra cannot be sufficiently emptied due to the weakening of the bulbocavernous muscle. It is also known that poor milk back of bulbous urethra occurs after end of voiding due to incomplete external urethral sphincter or bladder neck obstruction [11]. Our new hypothesis in addition to the previously mentioned mechanism, that the decrease in the volume of corpus cavernosum according to the aging process can cause the relative volume increase of the bulbous urethra to promote PMD.

So, we conducted a preclinical study using animals to prove this hypothesis. The factors we mainly wanted to report were the difference in urethral diameter by section and volume change of cavernosal tissue between young and old rats. In the previous studies, there have been many reports of senile changes in cavernosal tissue [12, 13]. Most of the experimental results reported an increase in fibrotic tissue and a decrease in smooth muscle as aging progressed in cavernosum. However, no animal studies have been found related to urethra to study PMD [14, 15]. Among the pathophysiology of PMD, our study was the first study conducted to prove our hypothesis that we confirmed the increase of bulbous urethral volume in old rats and analyzed the mechanism.

We identified p-AKT/AKT levels, bax, and bcl-2 levels in urethra and corpus cavernosum. In conclusion, it was confirmed that apoptosis in old rats increased statistically significant in urethra, and corpus cavernosum both. There are already many studies on the mecha-nism of erectile dysfunction due to apoptosis-induced reduction of smooth muscle and increase in collagen in corpus cavernosum [16-20]. Previous study has shown that eNOS elevated the cGMP level, which stimulated penile erection [21]. p-AKT could stimulate endothelial cells to produce eNOS, which is the factor for vasodilatation. It is possible that high levels endothelial cells thereby stimulate vasodilatation and inhibit smooth muscle atrophy [22]. Our results also showed that elevated levels of eNOS in the young group may be related to increased nNOS, higher cGMP levels, and reduced atrophy of the urethra smooth muscle. In the young group related to expression nNOS and cGMP levels, and reduced apoptosis of the corpus caver-nosum, as demonstrated by the increased β-III tubulin and nNOS expression. As a result of our study, this has an effect on the decrease in the volume of the corpus cavernosum, and it is thought that it also affects the increase in the diameter of the bulbous urethra in particular because of the relatively empty space. In addition, a decrease in bulbocavernous muscle contents was also observed, and it was confirmed through the above experiment as one of the known pathophy-siology of PMD.

According to one study [7], there is a literature on the effect of PDE5-inhibitor on not only smooth muscle but also skeletal muscle, so additional interesting research results can be expected on external sphincter which is related to the pathophysiology of PMD. Second, there was a lot of difference in total penis size between young and old rats, it was unreasonable to interpret the results by direct diameter, so the ratio with total size was used. For more accurate results, it would be better to proceed at a time when the penis sizes of young and old rats become similar. Third, since PMD symptoms cannot be fully reproduced using animals, this is only an indirect aspect of the clinical outcome and cannot be directly verified clinical symptoms.

Already reported that the PDE5 inhibitor has an effect on the symptoms of PMD through clinical studies, confirming that our hypothesis is sufficiently possible [7, 11]. It is also believed that more scientific verification will be possible through the expansion of animal experi-ments conducted this time. With these additional experi-ments, we plan to continue our research by helping to improve symptoms of difficult-to-treat PMD and clarifying our understanding of the mechanisms.

According to the recent International continence society (ICS) guideline, the term PMD is thought to be replaced by the term “post voiding incontinence”. The reason that the term of PMD, which has not received attention for a long time, has changed to post voiding incontinence is likely to be due to the recent increase in new issues. Although the pathophysiology of PMD is very diverse, it seems that there is a clear connection with our hypothesis with cavernosal tissue. We observed a decrease in cavernosum volume and an increase in bulbous urethra diameter in old rats, which is thought to be related to apoptosis caused by senile change. We expect our study to be a background that can help patients suffering from PMD.

요 약

중년 이후 많은 환자들이 배뇨 후 요실금을 앓고 있지만 아직 적절한 치료가 이뤄지지 않고 있다. 몇 가지 병태생리학적 기전이 제안되었지만 우리가 주목한 기전은 구해면체근과 해면체 조직의 위축으로 인한 요도의 확장이었다. 본 연구의 목적은 해면체와 요도의 해부학적 변화를 관찰하여 배뇨 후 요실금의 기전을 규명하는 것으로 8주령의 SD 수컷 랫드 그룹과 24개월 이상 된 SD 수컷 랫드 그룹으로 나누었다. 전체 음경에 대한 구부요도 및 해면체 면적의 비율은 두 그룹 간 통계적으로 유의하였다(P<0.05). 평활근 함량과 배뇨 후 요실금의 기전(nNOS, α-SMA)과 관련된 매개변수는 두 그룹 간 유의한 차이가 있었으며(P<0.05), 본 연구의 결과는 배뇨 후 요실금 연구에 기여할 것으로 기대된다.



Conflict of interest


Author’s information (Position)

Jeon SH, Professor; Park MY, Professor.

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