Hypercholesterolemia-induced erectile dysfunction: endothelial nitric oxide synthase (eNOS) uncoupling in the mouse penis by NAD(P)H oxidase.

INTRODUCTION: Hypercholesterolemia induces erectile dysfunction (ED) mostly by increasing oxidative stress and impairing endothelial function in the penis, but the mechanisms regulating reactive oxygen species (ROS) production in the penis are not understood. AIMS: We evaluated whether hypercholesterolemia activates nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase in the penis, providing an initial source of ROS to induce endothelial nitric oxide synthase (eNOS) uncoupling and endothelial dysfunction resulting in ED. METHODS: Low-density-lipoprotein receptor (LDLR)-null mice were fed Western diet for 4 weeks to induce early-stage hyperlipidemia. Wild type (WT) mice fed regular chow served as controls. Mice received NAD(P)H oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Erectile function was assessed in response to cavernous nerve electrical stimulation. Markers of endothelial function (phospho [P]-vasodilator-stimulated-protein [VASP]-Ser-239), oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NAD[P]H oxidase subunits p67(phox) , p47(phox) , and gp91(phox) ), P-eNOS-Ser-1177, and eNOS were measured by Western blot in penes. MAIN OUTCOME MEASURES: The main outcome measures are the molecular mechanisms of ROS generation and endothelial dysfunction in hypercholesterolemia-induced ED. RESULTS: Erectile response was significantly (P<0.05) reduced in hypercholesterolemic LDLR-null mice compared with WT mice. Relative to WT mice, hypercholesterolemia increased (P<0.05) protein expressions of NAD(P)H oxidase subunits p67(phox) , p47(phox) and gp91(phox) , eNOS uncoupling, and 4-HNE-modified proteins, and reduced (P<0.05) P-VASP-Ser-239 expression in the penis. Apocynin treatment of LDLR-null mice preserved (P<0.05) maximal intracavernosal pressure, and reversed (P<0.05) the abnormalities in protein expressions of gp67(phox) and gp47(phox) , 4-HNE, P-VASP-Ser-239, and eNOS uncoupling in the penis. Apocynin treatment of WT mice did not affect any of these parameters. Protein expressions of P-eNOS-Ser-1177 and total eNOS were unaffected by hypercholesterolemia. CONCLUSION: Activated NAD(P)H oxidase in the penis is an initial source of oxidative stress resulting in eNOS uncoupling, thus providing a mechanism of eNOS uncoupling and endothelial dysfunction in hypercholesterolemia-induced ED.

Post-translational regulation of endothelial nitric oxide synthase (eNOS) by estrogens in the rat vagina.

INTRODUCTION: Estrogens control vaginal blood flow during female sexual arousal mostly through nitric oxide (NO). Although vascular effects of estrogens are attributed to an increase in endothelial NO production, the mechanisms of endothelial NO synthase (eNOS) regulation by estrogens in the vagina are largely unknown. AIMS: Our hypothesis was that estrogens regulate eNOS post-translationally in the vagina, providing a mechanism to affect NO bioavailability without changes in eNOS protein expression. METHODS: We measured eNOS phosphorylation and eNOS interaction with caveolin-1 and heat shock protein 90 (HSP90) in the distal and proximal vagina of female rats at diestrus, 7 days after ovariectomy and 2 days after replacement of ovariectomized rats with estradiol-17beta (15 microg). MAIN OUTCOME MEASURES: Molecular mechanisms of eNOS regulation by estrogen in the rat vagina. RESULTS: We localized phospho-eNOS (Ser-1177) immunohistochemically to the endothelium lining blood vessels and vaginal sinusoids. Estrogen withdrawal decreased phosphorylation of eNOS on its positive regulatory site (Ser-1177) and increased eNOS binding to its negative regulator caveolin-1 (without affecting eNOS/HSP90 interaction), and they were both normalized by estradiol replacement. Protein expressions of phosphorylated Akt (protein kinase B) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) were not affected by estrogen status, suggesting that the effect of estrogens on eNOS (Ser-1177) phosphorylation was not mediated by activated AKT or ERK1/2. eNOS phosphorylation on its negative regulatory site (Ser-114) was increased in the vagina by estrogen withdrawal and normalized by estradiol replacement, implying that the maintenance of low phosphorylation of eNOS on this site by estradiol may limit eNOS interaction with caveolin-1 and preserve the enzyme's activity. Total eNOS, inducible NOS, caveolin-1, and HSP90 protein expressions were not affected by ovariectomy or estradiol replacement in the distal or proximal vagina. CONCLUSIONS: These results define novel estrogen signaling mechanisms in the vagina which involve eNOS phosphorylation and eNOS-caveolin-1 interaction.

Endothelial nitric oxide synthase regulation in female genital tract structures.

INTRODUCTION: Female sexual arousal disorder (FSAD) is a major component of female sexual dysfunctions, affecting 25-70% of women. The mechanisms of FSAD are poorly understood. Estrogen contributes to the control of genital blood flow during the sexual response. Vascular effects of estrogen are mostly attributed to its regulation of endothelial nitric oxide (NO) production. However, the role of endothelial NO synthase (eNOS) and the mechanisms that regulate eNOS in female genital tract structures are largely unknown. AIM: To review available evidence of the mechanisms of eNOS regulation in female genital tract structures. METHODS: This article reviews the literature that relates to the role of NO and eNOS in female sexual arousal and its modulation by estrogen. MAIN OUTCOME MEASURES: Association between female sexual arousal, NO, and eNOS. RESULTS: The NO/cyclic guanosine monophosphate pathway is believed to have a primary role in the regulation of clitoral and vaginal blood flow, and smooth muscle relaxation during sexual arousal. Estrogen is critical for maintaining vaginal and clitoral blood flow and vaginal transudate production. Estrogen regulates eNOS by genomic mechanisms, involving augmented mRNA transcription and protein synthesis, and by non-genomic mechanisms, which occur without alterations in gene expression. However, limited studies have evaluated the physiological role of endothelial NO and the molecular mechanisms of eNOS regulation in the female genital tract. CONCLUSIONS: The effects of estrogen on increasing genital blood flow and smooth muscle relaxation have been attributed mostly to regulation of eNOS. However, the exact mechanisms of eNOS regulation in female genital tract structures and the molecular basis for the eNOS defect with aging and vascular diseases warrant further investigation.

Noncontact stimulation of the cavernous nerves in the rat prostate using a tunable-wavelength thulium fiber laser.

BACKGROUND AND PURPOSE: Laser nerve stimulation has recently been studied in neuroscience as an alternative to electrical stimulation. Its advantages include noncontact stimulation, better spatial selectivity, and elimination of electrical stimulation artifacts. This study explored laser stimulation of the rat cavernous nerves as a potential alternative to electrical nerve mapping during nerve-sparing radical prostatectomy. MATERIALS AND METHODS: The cavernous nerves were surgically exposed in 10 male rats. A thulium fiber laser stimulated the nerves, with a wavelength of 1870 nm, pulse energy of 7.5 mJ, radiant exposure of 1 J/cm2, pulse duration of 2.5 msec, pulse rate of 10 Hz, and 1-mm laser spot diameter, for a stimulation time of 60 sec. RESULTS AND CONCLUSION: A significant increase in the intracavernosal pressure was detected on laser stimulation, with pressure returning to baseline values after stimulation ceased. This study demonstrates the feasibility of noncontact stimulation of the cavernous nerves using near-infrared laser radiation.

Elevated RhoA/Rho-kinase activity in the aged rat penis: mechanism for age-associated erectile dysfunction.

Epidemiologic studies have shown that aging accounts significantly for the prevalence of erectile dysfunction (ED). The pathophysiology of ED during aging and its underlying molecular mechanisms are largely unknown. We hypothesized that increased RhoA/Rho-kinase signaling is a major factor in the pathogenesis of age-associated ED and the mechanism involves increased penile smooth muscle contractility through inhibition of myosin light chain phosphatase. Male Fischer 344 young (4 month old) and aged (20-22 month old) rats underwent erectile function testing in vivo by measuring intracavernosal pressure (ICP) and mean arterial blood pressure (MAP) upon electrical stimulation of the cavernous nerve. The data demonstrated that erectile function was significantly lower in aged rats than that in young rats at all voltages tested (P<0.05). Western blot analysis results showed that there were no significant changes in protein expressions of RhoA, Rho-kinase-alpha and -beta isoforms, and myosin light chain phosphatase target subunit (MYPT1); however, membrane-bound RhoA and phosphorylated MYPT1 were increased in aged rat penes by 95 +/- 15 and 56 +/- 8% (P<0.05), respectively, indicating enhanced RhoA and Rho-kinase activity. Inhibition of Rho-kinase with Y27632 maximally increased ICP/MAP to 0.72 +/- 0.05 in aged rats vs. 0.47 +/- 0.06 in young rats (P<0.05). Gene transfer of adeno-associated virus (AAV) encoding dominant negative RhoA (T19NRhoA) to penes of aged and young rats for 7 days markedly improved erectile function in aged rats when compared with that in young rats (P<0.05). These observations were also supported by Rho-kinase activity assay results showing that basal Rho-kinase activity in aged rat penes receiving AAV vehicle treatment was twofold greater than that in young rat penes receiving AAV vehicle treatment, while it was reduced to a level similar to that in young rat penes after gene therapy of T19NRhoA (P<0.05). Taken together, these data suggest that impaired erectile function during the aging process involves increased RhoA/Rho-kinase signaling, and this pathway may be exploited for the treatment of age-associated ED.

Temperature-controlled optical stimulation of the rat prostate cavernous nerves.

Optical nerve stimulation (ONS) may be useful as a diagnostic tool for intraoperative identification and preservation of the prostate cavernous nerves (CN), responsible for erectile function, during prostate cancer surgery. Successful ONS requires elevating the nerve temperature to within a narrow range (~42 to 47°C) for nerve activation without thermal damage to the nerve. This preliminary study explores a prototype temperature-controlled optical nerve stimulation (TC-ONS) system for maintaining a constant (±1°C) nerve temperature during short-term ONS of the rat prostate CNs. A 150-mW, 1455-nm diode laser was operated in continuous-wave mode, with and without temperature control, during stimulation of the rat CNs for 15 to 30 s through a fiber optic probe with a 1-mm-diameter spot. A microcontroller opened and closed an in-line mechanical shutter in response to an infrared sensor, with a predetermined temperature set point. With TC-ONS, higher laser power settings were used to rapidly and safely elevate the CNs to a temperature necessary for a fast intracavernous pressure response, while also preventing excessive temperatures that would otherwise cause thermal damage to the nerve. With further development, TC-ONS may provide a rapid, stable, and safe method for intraoperative identification and preservation of the prostate CNs.

Continuous-wave infrared subsurface optical stimulation of the rat prostate cavernous nerves using a 1490-nm diode laser.

OBJECTIVE: To optimize the infrared laser wavelength and optical nerve stimulation (ONS) parameters for both deep and rapid subsurface cavernous nerve (CN) stimulation in a rat model, in vivo. MATERIALS AND METHODS: A 150-mW, 1490-nm diode laser providing an optical penetration depth (OPD) of 518 µm in water was operated in continuous-wave mode during stimulation of the CNs in 8 rats for 15 seconds irradiation time through a custom-built, single-mode fiber optic probe capable of producing a collimated, 1-mm diameter laser beam. Successful ONS was judged by an intracavernous pressure response in the rat penis. Subsurface ONS at 1490 nm was also compared with previous studies using 1455 nm and 1550 nm near-infrared diode laser wavelengths. RESULTS: Subsurface ONS of the rat CN was successful through fascia layers with a thickness up to 380 µm using an incident laser power of ∼50 mW. Intracavernous pressure response times as short as 4.6 ± 0.2 seconds were recorded using higher laser powers below the nerve damage threshold. CONCLUSION: The 1490-nm diode laser represents a compact, low cost, high power, and high quality infrared light source for use in ONS. This wavelength provides deeper penetration than 1455-nm diode laser and more rapid and efficient nerve stimulation than 1550-nm diode laser.

Subsurface near-infrared laser stimulation of the periprostatic cavernous nerves.

Successful identification and preservation of the cavernous nerves (CN), which are responsible for sexual function and vulnerable to damage during prostate cancer surgery, will require subsurface detection of the CN's beneath a thin fascia layer. This study explores the feasibility of optical nerve stimulation (ONS) in the rat with a fascia layer placed over the CN. Two near-infrared diode lasers with wavelengths of 1455 and 1550 nm were operated in continuous-wave mode for stimulation of the CN in 8 rats, in vivo. Successful ONS was confirmed by an intracavernous pressure (ICP) response in the rat penis at 1455 nm through fascia with a thickness up to 110 µm and at 1550 nm through fascia with a thickness up to 450 µm. Higher incident laser power was required to produce an ICP response as fascia thickness was increased. Also, weaker and slower ICP responses were observed as fascia thickness was increased. Subsurface ONS of the rat CN at a depth of 450 µm using a 1550 nm laser is feasible as an intermediate step towards developing ONS as an intra-operative diagnostic tool for identification and preservation of the cavernous nerves during prostate cancer surgery.

An NMDA antagonist in the MPOA impairs copulation and stimulus sensitization in male rats.

Systemic injections of an NMDA antagonist have been shown to impair mating in male rats. One site where glutamate and its NMDA receptors may contribute to mating is the medial preoptic area (MPOA), which is vital for male sexual behavior. Glutamate is released in the MPOA during copulation, and especially at the time of ejaculation. We report here that the NMDA antagonist MK-801, microinjected into the MPOA, impaired copulatory behavior in sexually naïve as well as experienced males. In rats tested both as naïve and after sexual experience, drug treatment produced more profound impairment in naïve males. In addition, MK-801, microinjected into the MPOA before each of 7 noncopulatory exposures to receptive female rats, resulted in copulatory impairments on a drug-free test on Day 8, relative to aCSF-treated rats; their behavior was similar to that of males that had not been preexposed to females. Therefore, NMDA receptors in the MPOA contribute to the control of copulation and stimulus sensitization. Glutamate, acting via NMDA receptors, regulates many neural functions, including neuronal plasticity. This is the first demonstration that a similar mechanism in the MPOA sensitizes male rats to the stimuli from a receptive female, and thereby enhances their behavior.

Continuous-wave laser stimulation of the rat prostate cavernous nerves using a compact and inexpensive all single mode optical fiber system.

BACKGROUND AND PURPOSE: Laser stimulation of the rat cavernous nerve (CN) recently has been demonstrated as an alternative to electrical stimulation for potential application in nerve mapping during nerve-sparing radical prostatectomy. Advantages include noncontact stimulation and improved spatial selectivity. Previous studies, however, have used large and/or expensive laser sources for stimulation. This study demonstrates the feasibility of optical stimulation of the rat CN, in vivo, using a compact, inexpensive all-single-mode fiberoptic system. MATERIALS AND METHODS: A 1455-nm wavelength infrared diode laser beam was coupled into a 9-µm-core single-mode fiber for delivery through a 10F laparoscopic probe and used for laser stimulation of the CN in a total of eight rats, in vivo. RESULTS: Laser stimulation of the CN was observed at threshold temperatures of 41°C, with intracavernous pressure response times as short as 4 s, and magnitudes up to 50 mm Hg, compared with baselines of 10 mm Hg. CONCLUSION: This novel, all-single-mode-fiber laser nerve stimulation system introduces several advantages including: (1) lower cost laser; (2) more robust fiberoptic design, eliminating alignment and cleaning of bulk optical components; and (3) improved Gaussian spatial beam profile for simplified alignment of the laser beam with the nerve. With further development, laser nerve stimulation may be useful for identification and preservation of the CN during prostate cancer surgery.

Continuous-wave infrared optical nerve stimulation for potential diagnostic applications.

Optical nerve stimulation using infrared laser radiation has recently been developed as a potential alternative to electrical nerve stimulation. However, recent studies have focused primarily on pulsed delivery of the laser radiation and at relatively low pulse rates. The objective of this study is to demonstrate faster optical stimulation of the prostate cavernous nerves using continuous-wave (cw) infrared laser radiation for potential diagnostic applications. A thulium fiber laser (λ=1870 nm) is used for noncontact optical stimulation of the rat prostate cavernous nerves in vivo. Optical nerve stimulation, as measured by an intracavernous pressure (ICP) response in the penis, is achieved with the laser operating in either cw mode, or with a 5-ms pulse duration at 10, 20, 30, 40, 50, and 100 Hz. Successful optical stimulation is observed to be primarily dependent on a threshold nerve temperature (42 to 45 °C), rather than an incident fluence, as previously reported. cw optical nerve stimulation provides a significantly faster ICP response time using a lower power (and also less expensive) laser than pulsed stimulation. cw optical nerve stimulation may therefore represent an alternative mode of stimulation for intraoperative diagnostic applications where a rapid response is critical, such as identification of the cavernous nerves during prostate cancer surgery.

Laser stimulation of the cavernous nerves in the rat prostate, in vivo: optimization of wavelength, pulse energy, and pulse repetition rate.

The cavernous nerves on the prostate surface are responsible for erectile function. Improved diagnostic techniques are necessary for identification of the nerves during prostate cancer surgery and preservation of sexual function after surgery. Electrical mapping of the nerves has been used as an intra-operative tool during prostate surgery, but it has proven inconsistent and unreliable. Non-contact optical stimulation of the cavernous nerves in the rat prostate has recently been demonstrated as a potential alternative to electrical nerve stimulation. The purpose of this study is to optimize the laser parameters to provide the maximum intracavernosal pressure response after optical nerve stimulation in the rat prostate. Optimal laser nerve stimulation parameters provided comparable response to electrical nerve stimulation. Optical nerve stimulation may represent a potential intra-operative diagnostic technique for use in laparoscopic and robotic nerve-sparing prostate cancer surgery.

Optical coherence tomography of cavernous nerves: a step toward real-time intraoperative imaging during nerve-sparing radical prostatectomy.

OBJECTIVES: To demonstrate the use of optical coherence tomography (OCT) for imaging of the cavernous nerve (CN) and periprostatic tissues. The rates of nerve preservation and postoperative potency after radical prostatectomy might improve with better identification of the CN using emerging intraoperative imaging modalities. OCT is an imaging modality that allows for real-time, high-resolution, cross-sectional imaging of tissues. METHODS: Seven male Sprague-Dawley rats underwent surgery using a midline celiotomy to expose the bladder, prostate, and seminal vesicles. The CNs and major pelvic ganglion were identified. Visual identification of the CN was further confirmed by electrical stimulation with simultaneous intracorporeal pressure measurements. OCT images of the CN, major pelvic ganglion, bladder, prostate, and seminal vesicles were acquired and correlated directly with the histologic findings. Once a baseline technique for the scanning and interpretation of the acquired images was established using the rat model, OCT was used to image ex vivo human prostatectomy specimens. RESULTS: OCT provided unique imaging characteristics, differentiating the CN from the bladder, prostate, seminal vesicles, and periprostatic fat. OCT images of the CN and prostate correlated well with the histologic findings. OCT of ex vivo human prostatectomy specimens revealed findings similar to those with the rat experiments, with, however, less dramatic architecture visualized in part because of the thicker capsule and more dense stroma of human prostates. CONCLUSIONS: The results of our study have shown that OCT provides real-time, high-resolution imaging of the CN in the rat model with excellent correlation to the histologic findings. This study provides a basis for the intraoperative use of this emerging technology during nerve-sparing prostatectomy.

Imaging the cavernous nerves in the rat prostate using optical coherence tomography.

INTRODUCTION: Improvements in identification, imaging, and visualization of the cavernous nerves (CNs) during radical prostatectomy, which are responsible for erectile function, may improve nerve preservation and post-operative potency. Optical coherence tomography (OCT) is capable of real-time, high-resolution, cross-sectional, in vivo tissue imaging. The rat prostate serves as an excellent model for studying the use of OCT for imaging the CNs, as the rat CN is a large, visible, and distinct bundle allowing for easy identification with OCT in addition to histologic confirmation. MATERIALS AND METHODS: Imaging was performed with the Niris OCT system and a handheld 8 Fr probe, capable of acquiring real-time images with 11-microm axial and 25-microm lateral resolution in tissue. Open surgical exposure of the prostate was performed on a total of six male rats, and OCT images of the prostate, CN, pelvic plexus ganglion, seminal vesicle, blood vessels, and periprostatic fat were acquired. CN electrical stimulation with simultaneous intracorporeal pressure measurements was performed to confirm proper identification of the CNs. The prostate and CNs were also processed for histologic analysis and further confirmation. RESULTS: Cross-sectional and longitudinal OCT images of the CNs were acquired and compared with histologic sections. The CN and ganglion could be differentiated from the surrounding prostate gland, seminal vesicle, blood vessels, bladder, and fatty tissue. CONCLUSIONS: We report preliminary results of OCT images of the rat CNs with histologic correlation and erectile stimulation measurements, thus providing interpretation of prostate structures as they appear in OCT images.