Some reports have investigated the relationship between androgens and bladder function, and an evaluation of the effect of androgens on blood vessels has indicated that vascular endothelial cell growth is suppressed due to low testosterone along with enhanced calcification of the blood vessel wall 1). However, there are few studies that examined the association between male hormone and histological changes 2). Some reports have compared immature cases with mature cases 3). However, we cannot confirm the clear association between maturity and blood vessels. Therefore, we examined the effects of androgens on BBF and histological changes after castration using a castrated Wistar and SHR rat model. We compared the model complicated with various condition with the castration alone model.

1.Differences in BBF caused by androgen changes: Fluorescent microsphere method
Male Wistar rats were classified into the following groups: control unoperated group (group A), mature group castrated at the age of 8 weeks (group B), and immature group castrated at the age of 4 weeks (group C), and SHR rat castrated at the age of 8 weeks (group D). Each rat was used at the age of 20 weeks. Left carotid artery of the rats were cannulated under pentobarbital anesthesia and a constant quantity of fluorescent microspheres were injected intra-arterially, then bladder was excised and weighted.Left femoral artery was simultaneously cannulated to retrieve reference blood. The absorbance of microsphere in blood and bladder tissue were measured by fluorescence microplate reader and the local blood flow rate was calculated. Blood Flow rate is shown as absorbance rate of bladder weight per 1g×reference blood retrieval rate(ml/min)/absorbance rate of entire microsphere within the reference blood（ml/min/g）.
2.Relationship between androgen changes and bladder function: Examination of bladder irritability 
We assessed androgen and bladder function in all the 4 groups by examining the bladder reaction to irritation. Bladder cystostomy was created under pentobarbital sodium anesthesia. One week later, the rats were placed in metabolic cages, and cystometry was performed without anesthesia or restraint. The bladder was irrigated with normal saline (NS) at room temperature, and 0.25% acetic acid (AA) liquid solution was then injected for 1h. The parameters examined included maximum voiding pressure (cmH2O) and voiding interval (sec).
3.Examination of androgen-related histological changes in bladder and blood vessels
All rats were sacrificed, and the bladder and iliac artery were removed and histologically examined for differences in smooth muscle and quantity of collagen fiber. We used Mallory-stained specimen for the examination of histological change such as denaturation or fibrosis. Sections of stained tissues were observed under light microscope and the images were captured by a Fujix Digital Camera. The images were analyzed using Photograb-2500 for Macintosh SH-25/MO and Macintosh PowerMac G4, and quantified using Image J 1.46 software. The components of smooth muscles and connective tissues were calculated from at least 10 fields from each tissue section.

1.Differences in BBF caused by androgen changes
The mean BBF rates for the experimental rats were 1.47 ± 0.23, 1.22 ± 0.46, 1.23 ± 0.41, and 1.00 ± 0.36 (mL/min/g) for the A, B, C, and D groups, respectively. Compared to Group A, the mean BBF were significantly decreased in Group D (p < 0.01) (Fig. 1). 
2.Relationship between androgen changes and bladder function
No significant difference was noted in the maximum voiding pressure between NS irrigation and AA irrigation among the A, B,C and D groups (39.9 ± 8.1 to 37.5 ± 4.6, 40.5 ± 4.4 to 42.6 ± 5.6, 36.0 ± 7.2 to 42.0 ± 3.4, and 33.9 ± 5.5 to 39.8 ± 3.5 [cmH2O], respectively). 
The voiding intervals in each group were shortened (P < 0.001) following AA irrigation (482.7 ± 69.2 to 201.4 ± 78.4, 596.5 ± 79.7 to 270.9 ± 58.7, 594.0 ± 60.2 to 283.6 ± 163.2, and 373.0 ± 80.8 to 180.0 ± 37.0 [sec]. Further, compared to Group A, the voiding intervals were significantly shorter in Group B and C (p < 0.001), and compared to Group B and C, the voiding intervals were significantly shorter in Group D following NS irrigation (p < 0.001), and compared to Group A, the voiding intervals were significantly shorter in Group B (p < 0.001), compared to Group B, the voiding intervals were significantly shorter in Group C (p < 0.05), and D (p < 0.001), compared to Group C, the voiding intervals were significantly shorter in Group D (p < 0.001) following AA irrigation (p < 0.001) (Fig. 2).
3.Examination of androgen-related histological changes in bladder and blood vessels
The mean bladder smooth muscle/collagen ratios (m/c ratio) were 3.35 ± 0.72, 1.75 ± 0.59, 1.33 ± 0.25, and 0.97 ± 0.24 for A, B, C, and D groups. Compared with the A group, the m/c ratio was lower in the B, C, and D groups (P < 0.001), and compared to Group B and C, m/c ratio was significantly lower in Group D (p < 0.05), and (p < 0.01) (Fig. 3a). The mean m/c ratios at the iliac artery were 2.71 ± 0.89, 2.24 ± 0.68, 0.92 ± 0.46, and 0.56 ± 0.29 for A, B, C, and D groups. Compared to Group A, m/c ratio was significantly lower in Group C and D (p < 0.001), and compared to Group B, m/c ratio was significantly lower in Group C and D (p < 0.001), and compared to Group B, m/c ratio was significantly lower in Group C and D (p < 0.001), and compared to Group C, m/c ratio was significantly lower in Group D (p < 0.001) (Fig. 3b).

Our findings confirmed that castration led to histological changes not only in the bladder but also in the blood vessels. Further, the age at which castration was performed affected the degree of the histological changes. Moreover, decrease of bladder blood flow and progress of fibrosis are found when complications are added.

This is the first report for considering and comparing the age at castration and BBF, bladder function, and histological change by using male Wistar and SHR rat. On analysing the relationship between androgen and bladder function, we observed that the histological changes were associated with bladder function but that the contribution of the blood flow was low. We consider that these effects of castration may constitute the key mechanisms underlying LUTS.

Hak AE, Witteman JC, de Jong FH, Geerlings MI, Hofman A, Pols HA: Low levels of endogenous androgens increase the risk of atherosclerosis in elderly men: the Rotterdam study. J Clin Endocrinol Metab 2002; 87:3632?3639.
Mesut T, Ebru B, Burak Ç, Ozan E, ?Izzet O, et al. The effect of testosterone replacement therapy on bladder functions and histology in orchiectomized mature male rats. Urology 2010;75:886–90.
Magari T, Shibata Y, Arai S, Kashiwagi B, Suzuki K, Suzuki K. Time-dependent effects of castration on the bladder function and histological changes in the bladder and blood vessels. Asian J Androl. 2014 :457-60.