Introduction

Female sexual dysfunction is a health problem affecting 30–50% of women in the United States,¹ and probably worldwide. However, there is a lack of effective therapies to treat this disease. Extensive research on male erectile dysfunction has been performed, yielding therapeutic successes, but the study of female sexual physiology and the development of treatments for female sexual dysfunction lag far behind.

Blood flow into the vagina and clitoris increases during the sexual arousal phase which, combined with enhanced capillary permeability, promotes a neurogenic transudate leading to vaginal lubrication. Thus, blood flow into the vagina and clitoris is a measurable physiological response to sexual stimulation, and is therefore important for the study of female sexuality. The increase of female genital blood flow in response to pelvic nerve stimulation has been reported recently. Studies have been carried out in rabbits and rats as newly developed animal models for the study of the female sexual response.^2,3,4 Although the neurogenic control of the vascular events in these tissues has not been thoroughly investigated, it is known that the nitric oxide (NO)/cGMP pathway mediates the neurogenic relaxation of rabbit clitoral corpus cavernosum and is involved in the neurogenic relaxation of rabbit vagina.^5,6,7

Vardenafil is a potent and selective type 5 phosphodiesterase (PDE5) inhibitor that enhances NO-mediated relaxation of human corpus cavernosum and NO-induced rabbit penile erection, and it has been reported to potentiate erectile function in patients.^8,9 Furthermore, vardenafil is currently under clinical investigation for the treatment of erectile dysfunction (ED). The aim of this work was to evaluate the effects of vardenafil on blood flow responses to pelvic nerve stimulation in the vagina and clitoris in a female dog model.

Methods

Sexually mature female beagle dogs (>12 months old) were anesthetized with thiopental (25 mg/kg) and the anesthesia was maintained with halothane (1.7% in air mixture). Left carotid and femoral arteries were catheterized to register continuously arterial pressure with a pressure transducer. Heart rate was calculated from the carotid blood pressure signal. Blood pressure and heart rate parameters were registered in a PowerLab (ADInstruments, Castle Hill, Australia) data acquisition system. Both cephalic veins were catheterized for the intravenous administration of drug or vehicle and for continuous saline infusion.

An abdominal midline incision was made, and the pelvic nerve was dissected and surrounded with a subminiature bipolar electrode (Harvard, Apparatus, Harvard, MA, USA). The clitoris was carefully exposed and a flow needle probe (P4s, Moor Instruments, Devon, UK) was inserted into the corpus cavernosum, approximately at 1 cm from glans. Another flow needle probe was placed inside the vaginal wall. The point of insertion of this probe was at 1.5–2 cm from introitus in the left lateral part of the vagina, entering approximately 0.5 cm into the tissue. Signal from the probes was processed by a laser Doppler blood flow monitor (floLAB, Moor Instruments) and registered in the PowerLab data acquisition system. Pelvic nerve electrical stimulation (PNES) was applied by means of a constant current stimulator (Cibertec CS-9, Madrid, Spain) connected to the bipolar electrode. Intensity of the current was 30 mA with a pulse duration of 1 ms for 1 min. Frequency–response curves at 5, 10 and 20 Hz were performed.

After a stabilization period, PNES was applied at 5, 10 and 20 Hz. This first stimulation was to check the existence of an increment of vaginal wall and clitoris blood flow to PNES. After 20 min, PNES was repeated and the responses were compared to those obtained with check PNES. If response curves were similar, this latter PNES was considered as the control PNES for the comparisons. If response curves were different, another PNES was applied 20 min later. This process was repeated until two similar consecutive responses to PNES were obtained. In a few cases, we only performed two PNES applications before drug or vehicle administration, because the first two consecutive frequency–response curves were similar. Also in a few cases, we had to perform four PNES applications to obtain two consecutive similar responses. However, in the majority of cases the second and third consecutive PNES applications produced similar responses allowing for a satisfactory control PNES after three frequency–response curves. Then, an intravenous injection of either vehicle (saline) or vardenafil (1 mg/kg) was administered, and 20 min later another PNES was applied. Two additional response curves to PNES were obtained at 50 and 80 min after vehicle or drug administration.

Data analysis

The effects of vehicle and vardenafil on blood pressure and heart rate were determined by unpaired Student's t-test. Blood flow recordings were obtained in arbitrary flow units (flU). Increases of vaginal and clitorial blood flow induced by PNES were measured as the area under the curve (AUC) of the response, taking into account amplitude and duration of the response. The AUC was calculated by using the PowerLab software (ADInstruments) that integrates the values of the blood flow increase over the baseline for each selected individual response. Data were expressed as means.e.m. of the percentage of the control response (control PNES) before the treatment was applied. n indicates the number of female dogs used for the experiments (only one complete experiment was performed in each animal). Comparison of vehicle and vardenafil effects on vagina and clitoris blood flow increases after PNES was performed by a two-factor analysis of variance (ANOVA) test.

Results

Effects of pelvic nerve stimulation on blood flow into the vagina and clitoris

Basal blood flow levels were 57.79.9 flU in the vagina (n=11) and 42.47.9 flU in the clitoris (n=11). Application of PNES caused consistent increases of blood flow into the vagina and clitoris in anesthetized female dogs. The amplitude and duration of responses were dependent on the frequency applied but were quite variable between the different animals. The mean increments of blood flow induced by the 20-Hz stimulation were 130.2 40.5 flU in vagina (n=11) and 69.3520.77 flU in clitoris (n=11). Nevertheless, vagina and clitoris blood flow responses to PNES were similar when repeated in the same animal (Figure 1).

Figure 1.

Representative tracings showing the effects of PNES on vagina (a, b) and clitoris (c, d) blood flow in an anesthetized female dog measured by laser Doppler method. Repeated stimulation after a lapse of 20 min (b, d) in the same animal yields similar responses. Application of PNES at each specific frequency is indicated by horizontal bars.

Full figure and legend (39K)

Effects of vardenafil on blood flow in the vagina and clitoris

Intravenous vardenafil (1 mg/kg) significantly enhanced the PNES-mediated increase in blood flow in the clitoris and vagina in anesthetized female dogs. Vardenafil not only enhanced the amplitude of the blood flow increase by PNES but also modified the shape of the response, augmenting the duration of the response (Figure 2). The increased vaginal blood flow caused by PNES was significantly potentiated by vardenafil 20 min after i.v. administration (Figure 3a). This enhancement of blood flow responses in the vagina was still present 50 min (Figure 3b) and 80 min (Figure 3c) after i.v. administration of vardenafil. Similarly, the potentiation of clitoris blood flow responses to PNES induced by vardenafil was observed 20 min after the treatment (Figure 4a), and the effect remained 50 min (Figure 4b) and 80 min (Figure 4c) after the administration of vardenafil.

Figure 2.

Representative example of the enhancement induced by vardenafil (1 mg/kg) on vagina (a, b) and clitoris (c, d) blood flow responses to PNES in anesthetized female dogs. The responses were obtained by stimulating the pelvic nerve at 10 Hz frequency during the period indicated by the horizontal bars (1 min) in control conditions (a, c) and after 20 min of intravenous vardenafil administration (b, d). Note that, in addition to the increased amplitude of the response in the presence of vardenafil, the duration of the rise of blood flow was enhanced, augmenting the area delimited by the blood flow values above the baseline.

Full figure and legend (40K)

Figure 3.

Effects of vardenafil (1 mg/kg) on vaginal blood flow increases in response to PNES (5, 10 and 20 Hz) in female anesthetized dogs. Responses were determined at 20 (a), 50 (b) and 80 (c) min after treatment administration. Data are expressed as means.e.m. of the percentage of control responses previously obtained in the same animal in absence of the drug. The responses were measured as the AUC of the blood flow increase to PNES at each frequency. n indicates the number of animals used for the experiments. ^***P<0.005, ^**P<0.01 and ^*P<0.05 vs vehicle-treated group by a two-factor ANOVA test.

Full figure and legend (47K)

Figure 4.

Effects of vardenafil (1 mg/kg) on clitoral blood flow increases in response to PNES (5, 10 and 20 Hz) in female anesthetized dogs. Responses were determined at 20 (a), 50 (b) and 80 (c) min after treatment administration. Data are expressed as means.e.m. of the percentage of control responses previously obtained in the same animal in absence of the drug. The responses were measured as the AUC of the blood flow increase to PNES at each frequency. n indicates the number of animals used for the experiments. ^**P<0.01 and ^*P<0.05 vs vehicle-treated group by a two-factor ANOVA test.

Full figure and legend (48K)

Discussion

The sexual response in females is associated with increased vaginal lubrication, vaginal wall engorgement, and increased clitoral length and diameter. These processes result from increased blood flow into and concomitant relaxation of the vaginal wall and the cavernosal smooth muscle in the clitoris.¹⁰ Thus, the increase of blood flow into the clitoris and vagina is a physiological response important for the evaluation of the female sexual function. Indeed, several techniques are used to assess sexual function by measuring indices of genital blood flow in women.^11,12,13

The increase of genital blood flow in response to pelvic nerve stimulation has been reported in rabbit and rat models.^2,3,4 In our canine model, the electrical stimulation of the pelvic nerve resulted in consistent increases of vaginal and clitoral blood flow measured by the laser Doppler method. These responses, although variable between different animals, remained unaltered in each animal throughout the period of observation. This allowed us to express the results as percentage of change from control responses in the same animal, reducing the variability and the number of animals required. Blood flow was measured in arbitrary flow units because of the fact that we used needle probes (0.5 mm diameter) and it was hard to define the exact area of tissue giving the blood flow signal. As a result of this it would have been rather speculative to give a value of perfusion in milliliters of blood per gram of tissue. Indeed, the use of arbitrary flow units has been previously reported.⁴ The determination of the responses by calculating the AUC of blood flow increases gave us substantial information because such measurements consider the amplitude, the duration and the shape of the responses, which are all parameters that can be modified by the treatment.

The neurogenic mediators of the female sexual response are not yet well characterized. Recent studies reported the existence of an adrenergic regulation of blood supply to female genitalia, which promotes smooth muscle contraction and low blood flow levels, analogous to the male genitalia.^4,14 Inhibitory nonadrenergic, noncholinergic (NANC) neurotransmission in vagina and clitoris has been reported but is not yet fully characterized. Some evidence suggests that vasoactive intestinal polypeptide (VIP) acts as NANC neurotransmitter.^15,16 Nevertheless, the role of NO in neurogenic relaxation and blood flow control in the clitoris and vagina is strongly supported. Nitrergic innervation has been found in human vagina and clitoris.^17,18 Furthermore, the NO/cGMP pathway has been reported to mediate the neurogenic relaxation of the rabbit clitoral corpus cavernosum and vagina.^5,6 In the latter tissue, an additional unidentified neurotransmitter is also involved.⁷

The results of the present study support the hypothesis that NO/cGMP plays a significant role in the regulation of vaginal and clitoral blood flow changes in response to neurogenic stimulation in female dogs. The marked potentiation of vaginal and clitoral blood flow responses to pelvic nerve stimulation when PDE5 is inhibited by vardenafil suggest that the NO/cGMP pathway plays a key role in facilitating blood supply to genitalia in response to sexual stimulation in female dogs. Consistent with these observations, PDE5 inhibition has been shown to increase cGMP concentrations in human clitoral corpus cavernosum,¹⁹ to potentiate neurogenic relaxation of rabbit clitoral smooth muscle^5,6 and to potentiate the increase of rabbit genital blood flow in response to pelvic nerve stimulation.²⁰ In this study, vardenafil was able to potentiate the vascular sexual response in clitoris and vagina 20 min after intravenous administration, with the effect lasting at least 80 min afterwards, raising the possibility of a wide therapeutic window.

The efficacy of PDE5 inhibitors in treating ED in men raised the possibility of treating female sexual dysfunction with the same compounds.^9,21,22,23 Although the preliminary results were not satisfactory in postmenopausal women treated with the PDE5 inhibitor sildenafil,²⁴ maybe because of the poor expression of NOS in the vaginal wall of these women,¹⁷ additional encouraging results have been obtained with this drug in selected cohorts.^25,26,27

In conclusion, further research efforts are needed to augment the knowledge of the physiology and pathophysiology of female sexual function, including the development of animal models for the evaluation of new therapeutic approaches to treat sexual dysfunction. The results obtained in this study demonstrate that vardenafil enhances the sexual vascular response in anesthetized female dogs. This fact supports the hypothesis that vardenafil could be a useful therapeutic agent for the treatment of certain forms of female sexual dysfunction in which the local vascular response is impaired.

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Acknowledgements

We thank Carlos Correa and Ana Isabel Ortiz for veterinary animal care and preparation. This work was partially supported by a grant from Bayer AG.