Sandrine Compagnie

ORIGINAL RESEARCH—BASIC SCIENCE: Erectile Dysfunction in Hypercholesterolemic Atherosclerotic Apolipoprotein E Knockout Mice

INTRODUCTION Erectile dysfunction (ED) and cardiovascular diseases share the same risk factors. Although the use of hypercholesterolemic rabbit models has proven to be useful to illustrate the link between ED and hypercholesterolemia, the cost of daily maintenance of the animals and necessity for important amounts of drug have limited their use. AIM We aimed to develop a new model of atherosclerosis-associated ED in a well-known experimental model of atherosclerosis, the apolipoprotein E knockout (ApoE KO) mouse. METHODS Erectile function was evaluated by recording frequency-dependent increases in intracavernous pressure following electrical stimulation of the cavernous nerve in 26-, 32-, and 38-week-old ApoE KO mice fed a Western-type diet and age-matched C57BL6/J anesthetized mice. Atherosclerotic lesions were evaluated by planimetry in oil red O-stained aortas. RESULTS We found that in contrast to C57BL6/J mice, ApoE mice displayed atherosclerotic lesions covering 22% of the aortic luminal surface at 26 weeks of age and increasing to 27% and 35% at 32 weeks and 38 weeks of age, respectively. The amplitude of erectile responses to electrical stimulation of the cavernous nerve was markedly impaired in 26-week-old ApoE KO mice as compared with age-matched C57BL6/J mice. Impairment in erectile function persisted in ApoE KO mice 32 and 38 weeks of age. CONCLUSIONS The ApoE KO mouse, a well-characterized model to study disorders associated with hypercholesterolemia and atherosclerosis in cardiovascular research, could therefore be suitable for investigation of disease-modifying effects of new therapeutic strategies aiming to target both atherosclerosis and ED.

Distinct mechanisms implicated in atherosclerosis-induced erectile dysfunction in rabbits

Ageing and atherosclerosis (ATH) are well-known risk factors for erectile dysfunction (ED). To identify the mechanisms implicated in ATH-induced ED, independently of its ageing-associated component, we studied (i) erectile responses in vivo, and, (ii) endothelium-dependent and independent relaxations of corporal strips from young adult (YAD, n=6), adult (AD, n=6), and cholesterol-fed (ATH, n=8) New-Zealand white rabbits. Measurement of Intima/Media (I/M) ratio on iliac arteries from ATH rabbits determined those with moderate (Mod ATH, 0.5+/-0.3) or severe (Sev ATH, 1.5+/-0.4, P<0.05 Mann-Whitney) atherosclerotic lesions. Erectile responses were reduced in AD compared with YAD rabbits (at 6 V to 10 Hz: 51.6+/-4.6% vs. 57.5+/-1.4%); they were similar in AD and mod ATH rabbits (48.1+/-4.6%) but drastically impaired in Sev ATH rabbits (34.8+/-5.4%, P<0.05, two-way analysis of variance (ANOVA)). Corporal endothelium-dependent and -independent relaxations were comparable in YAD and AD rabbits (maximal relaxation to acetylcholine: 51.3+/-9.5 vs. 56.1+/-9.3%) but decreased in ATH rabbits (37.1+/-1.6%, P<0.001, two-way ANOVA). These results suggest that the mechanisms implicated in ATH-induced ED are distinct from the ageing-related process in rabbits. Thus, future therapeutic targets to treat or prevent ATH-induced ED may include the reduction of the atherosclerotic plaque size or progression, as well as an improvement of the smooth muscle and endothelial reactivity of the corpus cavernosum.

Minimal effective dose of dysport and botox in a rat model of neurogenic detrusor overactivity.

BACKGROUND Two botulinum toxins A have been evaluated for the treatment of refractory neurogenic detrusor overactivity (NDO) in humans: Dysport (abobotulinumtoxinA) and Botox (onabotulinumtoxinA). However, these two distinct commercialized products have different potency units and are not interchangeable. OBJECTIVE Assessment of the dose response and determination of minimal effective dose (MED) for Dysport and Botox in spinal cord-injured (SCI) rats with NDO. DESIGN, SETTING, AND PARTICIPANTS Female, adult, Sprague-Dawley rats (n=98) underwent T8-T9 spinal cord transection. Nineteen days after spinal cord injury, rats received intradetrusor injections (25μl injected, eight sites) of vehicle (V); Dysport 2, 5, 7.5, 10, and 12.5 U; and Botox 0.8, 2, 5, 7.5, and 10 U. Two days after injection, continuous cystometry was performed in conscious rats. MEASUREMENTS Voiding contractions (VC) were assessed by duration of VC, intercontraction interval, voided volume, maximal pressure, pressure threshold change, and intravesical baseline pressure (BP), while nonvoiding contractions (NVC) were evaluated by amplitude, frequency, and volume threshold to elicit NVC. MEDs for Dysport and Botox were determined by analysis of variance step-down trend test. RESULTS AND LIMITATIONS MEDs for Dysport and Botox were 10 U and 7.5 U, respectively. Regarding VC, only BP significantly decreased after 10 U Dysport and 7.5 U Botox compared to V (from 3.7±0.6 to 1.5±0.1 and 1.4±0.3mm Hg, respectively; p<0.01 and p<0.001, respectively). Dysport (10 U) and Botox (7.5 U) significantly inhibited NVC by decreasing their amplitude (from 7.4±1.1 to 5.8±0.5 and 5.4±0.6mm Hg, respectively; p<0.05); frequency (from 2.2±0.4 to 1.5±0.2 and 1.3±0.3 NVC per minute, respectively; p<0.01); and increasing volume threshold to elicit NVC (from 29.8±3.7 to 47.6±6.9 and 47.7±6.3%, respectively; p<0.05 and p<0.001, respectively). CONCLUSIONS This is the first preclinical dose-ranging study with Dysport and Botox under standardized conditions showing similar inhibiting effects on NDO, albeit at different MEDs. It highlights the importance of distinguishing each preparation for predicted outcomes and doses to be used. Further studies in patients with NDO are warranted to confirm these experimental results.

Inhibition of ejaculation by the non-peptide oxytocin receptor antagonist GSK557296: a multi-level site of action

Oxytocin (OT) plays a major role in the control of male sexual responses. Notably, blockade of OT receptors has been reported to inhibit ejaculation in animals. The study aimed to investigate the action of a highly selective, non‐peptide OT antagonist GSK557296 in a model of pharmacologically induced ejaculation in anaesthetized rats. The site of action was assessed by investigating different delivery routes for this compound.

Impact of a Long-Term Sildenafil Treatment on Pressor Response in Conscious Rats With Insulin Resistance and Hypertriglyceridemia

BACKGROUND Insulin resistance constitutes a risk factor for endothelial dysfunction and subsequent cardiovascular diseases including hypertension. Daily treatment with phosphodiesterase type 5 (PDE5) inhibitors has beneficial effects on endothelial function in men with increased cardiovascular risk. Endothelium-dependent vasomotor function is ultimately linked to blood pressure (BP) regulation. We postulated that sildenafil would ameliorate BP and biological markers of endothelial function in fructose-fed rats (FFRs). METHODS Wistar rats were fed a standard chow or a 60% fructose-enriched diet containing 12% fat for 8 weeks (FFR). From week 6 through 8, sildenafil (twice a day subcutaneously, 20 mg/kg) was administered followed by a 1-week washout period. At the end of the washout period, BP was recorded using radiotelemetry following cumulative infusion of norepinephrine (from 50 to 400 ng/kg/min). RESULTS FFR displayed both an impaired glucose tolerance and elevated triglyceridemia. The latter was corrected by sildenafil treatment. Resting BP was similar in all rats, whereas pressor responses were significantly enhanced in FFR (maximal increase in mean BP to norepinephrine: 25.6 +/- 3.8 vs. 40.8 +/- 4.0 mm Hg, P < 0.05) and normalized by sildenafil treatment (24.9 +/- 5.3 mm Hg, not significant vs. control). Urinary levels of 8-isoprostanes and thromboxane B(2) were increased in FFR and corrected by sildenafil treatment. CONCLUSION Thus, chronic treatment with sildenafil normalized BP regulation in an experimental model of insulin resistance and hypertriglyceridemia while restoring normal excretion of urinary biological markers of oxidative stress and cyclooxygenase-derived vasoconstrictors. The modulation of ROS and cyclooxygenase-derived vasoconstrictors generation by a chronic treatment with sildenafil may represent an added benefit beyond PDE5 inhibition.

Effect of Dapoxetine on Ejaculatory Performance and Related Brain Neuronal Activity in Rapid Ejaculator Rats

INTRODUCTION A brain network specifically activated when ejaculation occurs has been described in rats. Increasing serotonin (5-hydroxytryptamine [5-HT]) tone impairs ejaculation and chronic 5-HT selective serotonin reuptake inhibitors (SSRIs) are known to inhibit ejaculation. However, efficacy of acute treatment with SSRI varies from one compound to another. The SSRI dapoxetine has been reported to delay ejaculation when given on demand to men with premature ejaculation (PE), although the mechanism of action is unclear. Effects of acute SSRIs on activity of the brain ejaculation circuit in relation with ejaculation have never been examined. AIM To test the effects of acute administration of the short half-life SSRI dapoxetine on ejaculatory performance and activity in brain ejaculation circuit in rapid ejaculator rats taken as PE model. METHODS Standard copulatory test was used to attribute one sexual category (sluggish, middle, or rapid) to male rats on the basis of their ejaculatory performance. Parameters of sexual, including ejaculatory, behavior, and Fos level of expression in discrete brain areas were assessed in the three sexual categories and in rapid category following acute oral treatment with dapoxetine. MAIN OUTCOME MEASURES Ejaculation frequency (EF) and latency (EL) were measured as primary end points of ejaculatory behavior. Density of Fos-immunopositive cells in specific brain areas of brain stem, hypothalamus, and thalamus was determined as marker of neuronal activity. RESULTS EL and Fos level of expression in hypothalamic and thalamic structures were found related. Dapoxetine acute oral administration (300 mg/kg) to rapid ejaculator rats resulted in (i) diminution of ejaculatory performance (lengthened EL and decreased EF); and (ii) modulation of Fos level of expression in hypothalamic and thalamic nuclei of the brain ejaculatory circuit. CONCLUSION Acute treatment with dapoxetine, which reduced ejaculatory performance in rapid ejaculator rats, was also accompanied with changes in neuronal activity in components of the brain ejaculatory network.

Does Reduction of Number of Intradetrusor Injection Sites of aboBoNTA (Dysport®) Impact Efficacy and Safety in a Rat Model of Neurogenic Detrusor Overactivity?

Intradetrusor injections of Botulinum toxin A—currently onabotulinumtoxinA—is registered as a second-line treatment to treat neurogenic detrusor overactivity (NDO). The common clinical practice is 30 × 1 mL injections in the detrusor; however, protocols remain variable and standardization is warranted. The effect of reducing the number of injection sites of Dysport® abobotulinumtoxinA (aboBoNTA) was assessed in the spinal cord-injured rat (SCI). Nineteen days post-spinalization, female rats received intradetrusor injections of saline or aboBoNTA 22.5 U distributed among four or eight sites. Two days after injection, continuous cystometry was performed in conscious rats. Efficacy of aboBoNTA 22.5 U was assessed versus aggregated saline groups on clinically-relevant parameters: maximal pressure, bladder capacity, compliance, voiding efficiency, as well as amplitude, frequency, and volume threshold for nonvoiding contractions (NVC). AboBoNTA 22.5 U significantly decreased maximal pressure, without affecting voiding efficiency. Injected in four sites, aboBoNTA significantly increased bladder capacity and compliance while only the latter when in eight sites. AboBoNTA significantly reduced NVC frequency and amplitude. This preclinical investigation showed similar inhibiting effects of aboBoNTA despite the number of sites reduction. Further studies are warranted to optimize dosing schemes to improve the risk-benefit ratio of BoNTA-based treatment modalities for NDO and further idiopathic overactive bladder.

268 RHO-KINASE INHIBITION IMPACTS NEUROGENIC DETRUSOR OVERACTIVITY IN CHRONIC SPINALIZED RATS

Effect of Y-27632 on urodynamic parameters in consc ious SCI rats Spinal cord injury (SCI) severely disrupts normal bladder function by inducing neurogenic detrusor overactivity (NDO). First line SCIinduced NDO treatments i.e. antimuscarinics often associated with intermittent catheterization are somewhat limited by a mild to moderate clinical efficacy and a significant incidence of side effects. Thus the development of new effective drugs for the treatment of NDO is of crucial importance. Rho-kinase has a central role in the regulation of detrusor smooth muscle contraction since components of the rhoA/rho-kinase signalling pathway are involved in the Ca2+-sensitization of the smooth muscle 1. Moreover, in vitro and in vivo data from animal models of overactive bladder (OAB) indicate that rho-kinases are involved in pathophysiological mechanisms responsible for OAB 2,3. Thus, we aimed to evaluate the effects of a rho-kin ase inhibitor (Y-27632) on urodynamic parameters in rats with chronic SCI.

Daily treatment with sildenafil, an inhibitor of cGMP-dependent phosphodiesterase, normalizes the exaggerated hemodynamic response to norepinephrine in fructose-fed rats

Background Enhanced responsiveness of the vascular smooth muscle cell to vasoconstrictor stimuli has been described in insulin-resistant states. Fructose overload leads to the development of the metabolic syndrome and its associated cardiovascular disorders including hypertension. We postulated that chronic treatment with sildenafil would regulate the abnormally increased pressor response to a norepinephrine challenge in fructose-fed rats. Material and methods Wistar rats were fed a standard chow (CONT, n = 12) or a 60% fructose-enriched diet containing 5% fat (% by weight) for 8 weeks (FFR, n = 12). From week 5 through 8, sildenafil (twice a day sc, 20 mg/kg) was administered (FFR+SIL, n = 14), thus reaching clinically relevant plasma concentrations circa 20 nM unbound (Pfizer Inc., data on file), followed by a 1-week wash-out period (W9). On W9, in a sub-group of these conscious rats (n = 5) that had previously been implanted with telemetric devices, AP was recorded following cumulative infusion of noradrenaline (NA 50, 100, 200, 400 ng/kg/min). In all rats, urinary 8-isoprostane (IPT) and TxB2 levels were determined during a 24-hour period. Tissue ET-1 content was measured in segments of aortic and mesenteric artery. Results Fasting glycemia was unchanged in all rats while triglyceridemia were elevated in FFR compared to CONT (2.0 ± 0.4 mM vs 1.2 ± 0.2, p < 0.05) and corrected by sildenafil treatment (1.3 ± 0.2, NS vs CONT). Resting AP was similar in all rats. Pressor responses to NA were exacerbated in FFR (maximal increase from basal MAP: +22.8 ± 1.7 vs +38.0 ± 7.3 mmHg, respectively, p < 0.05) and normalized by sildenafil treatment (+24.9 ± 5.2 mmHg, NS vs CONT). Vascular ET-1 levels were not modified by the fructose nor sildenafil treatment whereas the increased production of urinary IPT and TxB2 in FFR versus CONT (2.07 ± 0.36 ng/ml/24 h vs 0.88 ± 0.13 and 12.86 ± 1.91 pg/ml/24 h vs 7.25 ± 0.59, respectively, p < 0.05) were corrected following sildenafil treatment (0.95 ± 0.14 ng/ ml/24 h and 8.74 ± 1.06 pg/ml/24 h, respectively, NS vs CONT).

APOLIPOPROTEIN E KNOCKOUT MICE AS A NEW MODEL OF HYPERCHORESTOROLAEMIA AND ATHEROSCLEROSIS ASSOCIATED ERECTILE DYSFUNCTION

Erectile dysfunction (ED) and cardiovascular diseases share the same risk factors including hypercholesterolemia and atherosclerosis, but there are few available animal models allowing to study hypercholesterolemia-associated ED. Although the use of hypercholesterolemic rabbit models has proven to be useful to illustrate the link between ED and hypercholesterolemia, the cost of daily maintenance of the animals and necessity for important amounts of drug in view of proof of concept studies to prevent or slow down these disorders have limited their use.