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Morphological Modifications in Clitoris and Vagina in Spontaneously Hypertensive Rats

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Morphological Modifications in Clitoris and Vagina in Spontaneously Hypertensive Rats International Journal of Impotence Research (2003) 15, 166–172 & 2003 Nature Publishing Group All rights reserved 0955-9930/03 $25.00 www.nature.com/ijir Morphological modifications in clitoris and vagina in spontaneously hypertensive rats AJ Bechara1, G Cao2, AR Casabe´1, SV Romano1 and JE Toblli2* 1Sexual Dysfunction Section, Urology Division, Hospital Durand, Buenos Aires, Argentina; and 2Laboratory of Experimental Medicine, Hospital Alema´n, CONICET, Buenos Aires, Argentina We evaluated possible morphological alteration in clitoris and vagina from spontaneous hypertensive rats (SHR) and normotensive WKY rats. Clitoris and vagina were processed by Masson’s trichrome, anti-a-smooth-muscle actin, anticollagen type I (COL I) and type III (COL III), and anti-TGFb1. SHR presented higher amount of clitoral cavernous smooth muscle (CSM), vascular smooth muscle; TGFb1 in clitoral vessel wall; higher wall/lumen ratio in both vaginal and clitoral vessels; and remarkable interstitial fibrosis, expressed by a higher amount in interstitial COL I and III in both clitoris and vagina, compared to WKY rats. Nerve fibers from clitoral and vaginal tissue in SHR showed important fibrosis at perineurium. SHR showed positive correlation between systolic blood pressure (SBP) and clitoral CSM; SBP and fibrosis in clitoris; and SBP and COL I and III in clitoris, respectively. Similar findings were observed between SBP and COL I and III in vagina. In conclusion, SHR present morphologic changes in clitoral vessels as well as in clitoral cavernous space, which have a high positive correlation with the high blood pressure level. Moreover, the increase in extracellular matrix affects not only the clitoral and vaginal interstitium but also the nerve structures from both clitoris and vagina. International Journal of Impotence Research (2003) 15, 166–172. doi:10.1038/sj.ijir.3900982 Keywords: female sexual dysfunction; hypertension; spontaneously hypertensive rats Introduction play an important role during the process of sexual excitement in females.3,4 The functions or effects of hormones on NO in the Female sexual dysfunction (FSD) is a highly clitoris have not been elucidated, although it was prevalent multicausal and multidimensional disor- confirmed to be present in the female vagina and der, which results in significant personal distress, clitoris.5,6 Anatomic and functional abnormalities adversely impacting quality of life and interpersonal have been reported in genital organs in females in relationships. Current epidemiological data reveal some experimental models, especially regarding a that up to 43% of women have some type of sexual decrease in the muscle–collagen ratio.7 1 dysfunction. Approximately 10 million American Arterial hypertension, which is also highly pre- women aged 50–74 y self-report complaints of valent in both sexes especially under the fourth diminished vaginal lubrication, pain and discomfort decade of life, causes a well-known vessel remodel- with intercourse, decreased arousal, and difficulty ing in the majority of the arteries along the human 2 in achieving orgasm. body. Convincing evidence supports that female Compared to male erectile dysfunction, the sexual response is characterized by an increase in pathophysiology and treatment of FSD is not totally clitoral and vagina blood flow.8 On the other hand, understood. Clitoral erection and vaginal congestion structural and functional abnormalities have been that occur in response to sexual stimuli are based on reported in the genitals of hypertensive subjects.9 relaxation of smooth muscle, and similar to penile Since adequate vasorelaxation should take place to erection in males, nitric oxide (NO) is considered to achieve an appropriated vaginal lubrication and clitoral engorgement, morphological alterations could be responsible for FSD in patients with high blood pressure. *Correspondence: Dr JE Toblli, MD, PhD, Laboratory of In previous studies, we have demonstrated Experimental Medicine, Hospital Alema´n, Av. Pueyrredon that male spontaneously hypertensive rats (SHR) 1640; Buenos Aires (1118), Argentina. present morphologic changes in vessels as well as in E-mail: [email protected] cavernous spaces of the erectile tissue, which has a Morphological changes in clitoris and vagina in SHR AJ Bechara et al 167 high positive correlation with the high blood Ltd., Crumlin, N. Ireland). Serum cholesterol, pressure level. Moreover, we also reported that the triglycerides and electrolytes were assessed accord- increase in extracellular matrix expansion seems to ing to standard methods. affect not only the interstitium but also the neural structures of the penis from these animals.10 In this regard, the aim of the present study was to evaluate Tissue processing and examination possible morphological alteration in clitoris and vagina from SHR. Clitoris and vagina were carefully dissected out from each animal. Clitoris were cut longitudinally Material and methods and, as well as the vaginas, both fixed in phosphate- buffered 10% formaldehyde (pH 7.2) and embedded in paraffin for the LM study. In all, 3 mm sections All the experiments were approved by the Hospital were cut and stained with hematoxylin–eosin Alema´n Ethic Committee and the Teaching and (H&E), periodic acid–Schiff reagent (PAS) and Research Committee and followed the National Masson’s trichrome. Institute of Health Guide for the Care and Use of Laboratory Animals. In all, 12-week-old female SHR and Wistar–Kyoto (WKY) rats (Charles River La- Immunohistochemical staining boratories, Wilmington, MA, USA) were divided into two groups: G1 Control WKY (n ¼ 10) and G2 SHR (n ¼ 10). Animals were housed in individual Paraffin sections were cut at 3 mm, deparaffined and cages at 21721C and a 12 h light/darkness cycle dehydrated. Endogenous peroxidase activity was (07:00–07:00) and were allowed to drink tap water, blocked by treating with 0.5% H2O2 in methanol for and fed standard rat chow (16–18% protein, normal 30 min. Monoclonal antibodies against a-smooth salt content, Cargill-Argentina) ‘ad libitum’. At the muscle actin (a-SMA) (Biogenex, San Roman, beginning of the study and after 6 months, blood California, USA) at a dilution 1:100, TGFb1 (Santa samples were obtained for biochemical determina- Cruz Biotechnology, Inc., Santa Cruz, California, tions. Systolic blood pressure (SBP) was measured USA) at a dilution 1:100 and anticollagen type I (Col monthly by tail cuff plethysmography. Measure- I) and type III (Col III) (Biogenex, San Roman, ments were obtained with the rats restrained in a California, USA) at a dilution 1:100 were used to plastic chamber without anesthesia. A pneumatic evaluate the process of vascular remodeling and pulse transducer positioned on the ventral surface of fibrosis, respectively. Immunostaining was carried the tail distal to the occlusion cuff detected the out with a commercial by modified avidin–biotin– return of the pulse following a slow deflation of the peroxidase complex technique, Vectastain ABC kit cuff. Cuff pressure was determined by a Pneumatic (Universal Elite, Vector Laboratories, California, Pulse Transducer, using a Programmed electro- USA) and counterstained with hematoxylin and 11 sphygmomanometer PE-300 (Narco Bio-Systems, the samples handled as previously described. Austin, Texas, USA), and pulses were recorded on a Physiograph MK-IIIS (Narco Bio-Systems, Austin, Texas, USA). A minimum of three such determina- Morphometric analysis tions were taken at each session, and the SBP registered was the average of the three readings. A total of 10 histological sections, five from clitoris After 6 months, all the rats were killed (pento- and five from vagina were studied in each animal barbital 40 mg/kg body wt, injected intraperitone- using a light microscope Nikon E400 (Nikon Instru- ally). The clitoris and vaginas were rapidly excised, ment Group, Melville, New York, USA). Histomor- and harvested for light microscopy (LM) and phometric evaluation was carried out in order to immunohistochemistry studies. study proliferation of cavernous smooth muscle (CSM) (positive a-SMA immunostaining) from the Biochemical procedures clitoris and vascular smooth muscle in arterial vessels from clitoris. Interstitial and perineural fibrosis from clitoris and vagina were quantified After 14-h fasting, rat blood samples were collected alone with the amount of Col I and Col III in from the tail vein in capillary tubes at the beginning, interstitium from both tissues. The amount of TGFb1 and after 6 months from the inferior cava vein before in vascular structures from clitoris was also deter- the rats being killed. Plasma glucose levels were mined. measured by the glucose oxidase method with an All these parameters were measured by a image Automatic Analyzer (Hitachi 911, Tokyo, Japan). analyzer Image-Pro Plus ver. 3 for windows (Media Aliquots of sera were assayed for creatinine using Cybernetics, LP. Silver Spring, Maryland, USA) on the enzymatic UV method (Randox Laboratories 10 consecutive microscopic fields. Interstitium was International Journal of Impotence Research Morphological changes in clitoris and vagina in SHR AJ Bechara et al 168 defined as tissue excluding muscle or blood vessels. Table 1 Parameters at the beginning of the study Morphometric analysis was performed by an ob- server who was blind to the animal-treatment group Mean 7 s.d. G1 WKY (n=10) G2 SHR (n=10)P at a final magnification of  400, these data were 7 7 averaged. The results were expressed as a fraction Rat body weight (g) 189.1 3 177.9 1.8 * SBP (mmHg) 116.171.7 141.572.6 * area. Arteriolar wall-to-lumen ratio in clitoral and Glycemia (mmol/l) 6.270.7 6.170.6 NS vaginal vessels was also carried out using the same Triglycerides (mmol/l) 0.4770.03 0.4770.03 NS technology. The mean diameter was calculated for Cholesterol (mmol/l) 0.8870.06 0.9570.10 NS Serum creatinine (mmol/l) 54.871.7 53.972.6 NS each group. + [Na ]s (mmol/l) 143.972.1 144.671.4 NS + 7 7 [K ]s (mmol/l) 5.1 0.1 5.0 0.2 NS Statistical method SBPFsystolic blood pressure, NSFnot significant.
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