Menopause is a condition that almost all women experience. Decreased secretion of estrogen influences the whole body and causes many changes. Overactive bladder symptoms occur in a very early stage in menopause women. Furthermore, menopause women experience increased sensitivity to cold which may cause urinary frequency. Former research has showed that decrease of estrogen shows cold stress related frequency in aged rats, which are correlated with increase of TRPM8 channels in the skin. However, in this study, decrease of estrogen did not show increase of cold stress related frequency or increase of TRPM8 channels in young rats. From these results, we estimated that decrease of estrogen against bladder function and skin temperature channels may be an indirect functional mechanism which are correlated with aging such as vessel damage. The purpose of this study was to assess the relation of blood flow and decreased estrogen against cold stress related frequency, including difference in expression of temperature channels in the skin, using an ovariectomy model.

A total of 28 female Spontaneously Hypertensive rats (160-180g) at postnatal week 10 were used for the experiments. The rats were randomly divided into sham operation group (Sham; n=10) and ovariectomy group (OVX; n=18), which received bilateral ovariectomy to induce surgical menopause. All rats underwent operation under anesthesia (sevoflulane 3%). Four weeks later, the rats were anesthetized and the urinary bladder was exposed and incised at the center of the dome. A polyethylene catheter was inserted and the free end was funneld subcutaneously and exteriorized t the back of the neck. Three days after cannulation, the rats received cystometography (CMG) under cold stress un-anesthetized. CMG was first performed in room temperature (RT, median 25 celsius) for 20 minutes. The rats were then put into low temperature (LT, median 4 celsius) for 20 minutes. After LT, the rats were put into RT again for 20 minutes. Voiding interval and micturition volume were measured to see the change rate between RT and LT. Secondly, 10 female Spontaneously Hypertensive rats at postnatal week 14 which were ovariectomized 4 weeks prior, received surgical transplantation of an osmotic pump under the skin and were randomly divided into estrogen replaced group (EST; n=5), which estrogen including corn oil was placed in the pump, and control group (CTL; n=5), which corn oil was placed. 4 weeks after transplantation, all rats received CMG under cold stress. Before CMG, all rats underwent measurement of blood flow of the bladder using a laser calculation device (Omega Zone). After CMG, skin from the lower back and the whole bladder were harvested for RT-PCR and immunohistology to see the effect of decrease and replacement of estrogen against neurochemical receptors.

Cold stress CMG showed increased frequency in OVX rats compared to Sham rats. EST rats showed improvement of frequency compared to CTR rats (Fig 1). RT-PCR showed increase of TRPV1 channels in the bladder in OVX rats, but improvement in EST rats. Blood flow of the whole bladder measured by laser calculation did not show any difference between OVX and Sham rats. However, HIF-1 alpha in the bladder mucosa, which were histologically measured showed an increase in OVX rats. TRPM8 channels in the skin showed an increase in OVX rats compared to Sham rats, but an improvement in EST rats (Fig 2).

From this study, decrease of estrogen showed increase of TRPM8 channels in the skin and cold stress related frequency in young rats. The mechanism of decreased estrogen against these results may be an indirect mechanical function via vessel damage. Furthermore, decrease of estrogen causes impairment of blood flow in the bladder mucosa, but not in the muscle layer in an early stage, which may cause difference in expression of neurochemical receptors. Early expression of overactive bladder symptoms with decreased estrogen maybe due to micro ischemia of the bladder mucosa.

Decrease of estrogen influences vessel damage which increases TRPM8 channels in the skin and will provoke cold stress related frequency. Decrease of estrogen against bladder function may be influenced by an very early stage of vessel damage which maybe an option for treatment of overactive bladder in menopause women.