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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 and in spontaneously hypertensive rats

AJ Bechara1, G Cao2, AR Casabe´1, SV Romano1 and JE Toblli2*

1Sexual Dysfunction Section, 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 (CSM), vascular smooth muscle; TGFb1 in clitoral vessel wall; higher wall/ 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. fibers from clitoral and vaginal tissue in SHR showed important fibrosis at perineurium. SHR showed positive correlation between systolic 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 ; hypertension; spontaneously hypertensive rats

Introduction play an important role during the process of sexual excitement in females.3,4 The functions or effects of 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 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– 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 especially under the fourth diminished , pain and discomfort decade of life, causes a well-known vessel remodel- with intercourse, decreased arousal, and difficulty ing in the majority of the along the human 2 in achieving . body. Convincing evidence supports that female Compared to male , 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 , understood. Clitoral 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 , 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 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 , 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 –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 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 in 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. *Po0.01.

Values were expressed as mean 7s.d. All the statistical analyses were performed using absolute Table 2 Parameters after 6 months values and processed through GraphPad Prism, 7 version 2.0 (GraphPad Software, Inc. San Diego, Mean s.d. G1 WKY (n=10) G2 SHR (n=10)P California, USA). Assumption test to determine the 7 7 Gaussian distribution was performed by the Kolmo- Rat body weight (g) 226.1 3.7 200.5 2.7 * SBP (mmHg) 118.871.2 180.775.2 * gorov and Smirnov method. For parameters with Glycemia (mmol/l) 6.270.4 6.170.6 NS Gaussian distribution, all the comparisons between Triglycerides (mmol/l) 0.4770.03 0.4970.02 NS groups were carried out using unpaired t-test. Cholesterol (mmol/l) 0.9770.06 0.9870.06 NS Serum creatinine (mmol/l) 56.572.6 60.173.5 NS Statistical analysis for those parameters like histo- + [Na ]s (mmol/l) 144.171.1 14571.7 NS logical data with non-Gaussian distribution was [K+] (mmol/l) 5.270.2 5.170.1 NS performed by Mann–Whitney test. In order to s evaluate the correlation between blood pressure SBPFsystolic blood pressure, NSFnot significant. and histological parameters, the Spearman correla- *Po0.01. tion test was performed. A value of Po0.05 was considered to be significant. percentage of collagen types III and I were also increased in the interstitium from clitoris and vagina in SHR (Figure 3a and b). Results In the SHR group, a highly significant positive correlation between: (1) SBP and cavernous a-SMA percentage; (2) SBP and interstitial fibrosis in At the beginning of the study, there were no clitoris; and (3) SBP and collagen types I and III, significant differences between SHR and WKY rats in clitoris as well as in vagina, was observed regarding metabolic parameters, however, SHR (Table 4). presented lower body weight and higher blood An interesting finding was a substantial increase pressure than WKY rats, as illustrated in Table 1. in the surrounding at perineurium As expected, after 6 months SHR showed a in clitoris (Po0.01) and vagina (Po0.01) in the SHR marked elevation of SBP relative to WKY rats group in comparison with WKY rats (Figure 4a and b (Table 2). On the other hand, at the same time, no and Table 3). significant changes in serum creatinine, glucose, cholesterol and triglycerides, as well as serum sodium and potassium were observed between the groups (Table 1). Discussion Microscopy examination of the clitoris in SHR revealed a significant increased amount of a-smooth muscle actin in arteries and in the cavernous spaces In the present study, SHR showed significant as illustrated in Figure 1a and b, and Table 3. In morphologic changes in clitoris and vagina in addition, wall/lumen ratio was markedly increased comparison with WKY rats. These differences were not only in clitoral vessels, but also in vascular characterized by an increase in CSM in clitoris, structures from the vagina in comparison with increased vascular smooth muscle with a high wall/ WKY rats (Table 3). Furthermore, the TGFb1 expres- lumen ratio and remarkable interstitial fibrosis sion in vessel wall from clitoris was also increased expressed by a higher amount in interstitial collagen in SHR relative to WKY rats (Table 3). Fibrosis types III and I, in both clitoris and vagina. In was significantly higher in SHR when it was addition, nerve fibers from clitoral and vaginal compared to WKY rats as shown in Table 3 and tissue in SHR showed a notable fibrosis at perineur- illustrated in Figure 2a and b. Moreover, the ium and endoneurium.

International Journal of Impotence Research Morphological changes in clitoris and vagina in SHR AJ Bechara et al 169 a Table 3 Morphometric and immunohistochemical parameters after 6 months

G1 WKY G2 SHR Mean 7 s.d. (n=10) (n=10) P

Clitoris vessels wall/lumen ratio 0.5770.07 1.7470.29 * Vagina vessels wall/lumen ratio 0.6170.15 1.6370.22 * Vasculara a-smooth muscle actin 972 16.173.6 * (%) from clitoris Cavernousa a-smooth muscle 774.4 2076.9 * actin (%) from clitoris Fibrosis in clitoris (%) 38.672.7 60.774.9 * Collagen type I in 4.570.5 8.271.8 * clitorisa (%) Collagen type I in 7.670.6 10.371.2 * vaginaa (%) Collagen type III in 11.37 0.1 27.571.7 * clitorisa (%) b Collagen type III in 14.871.1 25.071.3 * vaginaa (%) TGFb1 in vessel wall 1.470.5 2.771.4 * (%) in clitorisa Perineural fibrosis (%) 16.473.7 33.775.4 * in clitorisb Perineural fibrosis (%) 14.572.5 29.273.1 * in vaginab

F TGFb1 transforming growth factor b1. No. of vessels from 16 to 80 mm of diameter. *Po0.01. aCorrespond to the percentage of area with positive staining with respect to a confined area of tissue of 2.314 Â 104 mm2. bCorrespond to the percentage of area with fibrosis with respect to a confined area of tissue of 2.314 Â 104 mm2.

Figure 1 (a) Clitoral section from SHR. Note an increase in the thickness of the CSM layer (in brown) from cavernous space (a- SMA, magnification  400). (b) Clitoral section from WKY rat. ment and depend on increased Normal thickness of the CSM layer (in brown) from cavernous blood flow. Furthermore, nerve-stimulated changes space (a-SMA, magnification  400). in blood flow were found to be significantly less in the atherosclerotic group. Such altered blood flow Experiments in women using photoplethysmogra- responses were also associated with other dimin- phy, heat-clearance and 133Xenon washout demon- ished physiological changes in vaginal wall pres- strated that normal female sexual response was sure, vaginal length and clitoral intracavernosal characterized by an increase in clitoral and vaginal pressure.8 blood flow.12 Consequently, in spite of the multi- Common risk factors associated with arterial causal origin of FSD, in some special circumstances insufficiency include arterial hypertension, such as high blood pressure, FSD may have its mellitus, hyperlipidemia, smoking and pelvic irra- source in abnormal arterial circulation into the diation. Proliferation of smooth muscle and deposi- vagina and/or clitoris during , tion of lipids in the vessels wall14 characterize usually from atherosclerotic vascular disease or atherosclerotic lesions. In normal vessels, the en- perhaps by the remodeling process in vascular wall, dothelial cells provide a mechanical barrier for which often occurs in arterial hypertension. underlying smooth muscle cells to circulating Vasculogenic FSD may include such clinical substances and blood cells and produce local factors symptoms as delayed vaginal and clitoris engorge- that act in a paracrine fashion to alter vascular tone ment, diminished lubrication in vagina, diminished and growth.15 These factors include angiotensin- clitoral as well as vaginal sensation and orgasm. converting enzyme, angiotensin II, kinines, prosta- There is evidence that traumatic injury to the glandins, endothelin and nitric oxide. iliohypogastric-pudendal arterial bed from pelvic Hypertension affects blood vessels by shear stress, fractures or blunt perineal trauma may also result in which can lead to endothelial abnormalities such as diminished vagina/clitoral blood flow following an altered production and an activity of vasoactive sexual stimulation and mimicry vasculogenic substances, enhanced endothelial proliferation and FSD.13 Using pelvic nerve stimulation in normal intimal permeability, which could be reversible by and atherosclerotic New Zealand White female antihypertensive therapy.16 All of these factors lead , it was determined that both vaginal engorge- to endothelial dysfunction, which ultimately results

International Journal of Impotence Research Morphological changes in clitoris and vagina in SHR AJ Bechara et al 170 a a

b b

Figure 3 (a) Clitoral section from SHR. Note an important Figure 2 (a) Clitoral section from SHR. Significant increase in extracellular matrix (in blue), with small number of cavernous amount in collagen type III fibers (in brown). Similar pattern was observed in samples from vagina (Collagen type III, magnification spaces (Masson’s trichrome, Â 100). (b) Clitoral section from WKY Â 400). (b) Clitoral section from WKY rat. Scanty amount in rat. Normal amount of extracellular matrix (in blue), with collagen type III fibers (in brown) (Collagen type III, magnification adequate proportion of cavernous spaces (Masson’s trichrome, Â 400). Â 100).

in smooth muscle cell proliferation and collagen these results could be expected according to the deposition in the vessel wall. proliferation related to the animal’s age, there was a TGFb1, one of the most relevant fibrotic cytokine, significant difference in relation to the match-ages is markedly increased in plasma of hypertensive WKY rats. Furthermore, no other risk factor like patients,17 and it has a critical role in the arterial dyslipidemia or hyperglycemia was mediated since remodeling process by stimulating vascular smooth SHR as well as WKY rats showed similar and normal muscle cells proliferation through angiotensin II metabolic parameters values at the time of the pathway.18–20 experiment as illustrated in Table 1. Collagen type In the present study, SHR showed a vascular III is initially produced in fibrotic process. This form smooth muscle proliferative pattern in clitoral and of collagen disappears through the time and it is vaginal arteries, as well as an increase in the smooth followed by an increase in collagen type I expansion muscle layer in the cavernous spaces. In addition, in the extracellular matrix.21 Interestingly, in the these findings had a high significant correlation present study SHRs showed higher proportion in with blood pressure levels. It is worthy noting that collagen type III than collagen type I in clitoris as in the present study TGFb1 was highly expressed in well as in vagina. A possible explanation for these clitoral vessel wall from SHR, suggesting that the findings is the fact that these rodents, although remodeling process in this tissue is triggered in hypertensive, are still young. This observation may these animals. Moreover, the higher proportion in have therapeutic implications since pharmacologi- fibrosis along with an increased collagen type III and cal intervention could reverse this process espe- I deposition in the interstitium from vagina and cially in early stages. clitoris, seen in the SHR group, suggest a tight Data presented in our study, suggest that smooth relation between morphologic changes and the muscle cells hyperplasia could produce some degree of arterial hypertension. Although some of degree of impediment to achieve the complete

International Journal of Impotence Research Morphological changes in clitoris and vagina in SHR AJ Bechara et al 171 Table 4 Spearman rank correlation in SHR (n=10) relaxation mechanism for clitoral engorgement and, therefore, an adequate normal response to sexual Variable rPstimulus. Summarizing, our study provides evidence that SBP with cavernous a-smooth muscle 0.8739 0.0016 SHRs present morphological changes in clitoral actin (%) from clitoris SBP with fibrosis in clitoris (%) 0.8124 0.0058 vessels as well as in clitoral cavernous space, which SBP with collagen type I in clitoris (%) 0.9279 0.0003 have a high positive correlation with the high blood SBP with collagen type III in clitoris (%) 0.8411 0.0037 pressure level. Moreover, the increase in extracel- SBP with collagen type I in vagina (%) 0.6875 0.0347 lular matrix expansion affects not only the clitoral SBP with collagen type III in vagina (%) 0.7531 0.0149 and vaginal interstitium but also the nerve struc- F tures from tissues. SBP systolic blood pressure. Finally, it is worth mentioning that all these findings were also observed with similar pattern in erectile tissue from male SHR.10 Therefore, we think that the present data could contribute to emphasize the concept that permanent high blood pressure produces a deleterious effect on genital structures, independent from gender.

Acknowledgements

We gratefully acknowledge the valuable contribu- tion of Ana Uceda and Mariana Feldman who helped in the preparation of laboratory experiments and Jaquelina Mastantuono who thoroughly re- viewed the style of this manuscript.

References

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International Journal of Impotence Research