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* Department of Surgery/Division of Urology,
University of Maryland Medical System, Baltimore, Maryland; and
Department of Urology, The James Buchanan
Brady Urological Institute, The Johns Hopkins Medical Institutions, Baltimore,
Maryland.
| Correspondence to: Michael A. Palese, MD, Department of Urology—Marburg 407, The Johns Hopkins Hospital, 600 N Wolfe St, Baltimore, MD 21287 (e-mail: mpalese{at}msn.com). |
| Received for publication April 8, 2003; accepted for publication May 9, 2003. |
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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30 g) underwent electrical stimulation of the
cavernous nerve in vivo (parameters: 16 Hz frequency, 5 ms duration, 4V
stimulatory voltage) with intracavernosal pressure (ICP) monitoring. A total
of 55 mice (5 WT, 25 CAST, and 25 TEST) were evaluated. CAST and TEST (5.0
mg/pellet, 60-day release) mice were divided into groups of 5 and evaluated at
24 hours, 72 hours, 1 week, 2 weeks, and 4 weeks. Penile tissue was
immunohistochemically stained for 
-actin (marker for smooth muscle
cells) and CD-31 (marker for endothelial cells). Stained slides were analyzed
using Image Pro-plus software. In secondary studies, a Doppler flow meter was
employed to evaluate penile blood flow. ICP measurements (mm Hg) were
significantly decreased in CAST mice at 24 hour-, 72 hour-, 1 week-, 2 week-,
and 4-week time points compared with WT mice (41.9 ± 14.9, 19.1
± 4.2, 17.5 ± 8.2, 14.2 ± 4.4, and 10.0 ± 3.8,
respectively, vs 50.2 ± 2.8), but TEST animals maintained or had an
increase in ICP in comparison with WT mice (48.0 ± 1.4, 52.3 ±
1.3, 60.8 ± 7.6, 80.5 ± 2.1, and 81.5 ± 1.2,
respectively). Mean systemic arterial pressure remained approximately 80 mm Hg
irrespective of treatment. CAST mouse penis specimens revealed decreased
-actin and CD-31 immunoreactivity only at the 4-week interval, compared
with WT and TEST specimens. Doppler ultrasound flow rates (centimeter per
second), taken before, during, and immediately after cavernous nerve
stimulation, were WT 45.4 ± 7.3, 30.6 ± 5.2, 55.3 ± 8.2
vs CAST (2 weeks) 22.2 ± 2.5, 25.0 ± 1.5, 23.1 ± 2.0 vs
TEST (2 weeks) 30.5 ± 6.5, 25.7 ± 2.0, 45.2 ± 4.5. This
prominently showed that intrapenile flow was not reduced normally during
erectile stimulation in CAST mice. This is the first described mouse model of
castration-induced veno-occlusive erectile dysfunction. Erectile response
abnormalities as measured by ICP and Doppler ultrasound studies in CAST mice
may be attributed to hypogonadal effects on erectile tissue function.
Morphologic changes in the cavernosal tissue of CAST mice coincide with these
abnormalities to some extent. This study defines an androgen-dependent
mechanism of veno-occlusive erectile function in the mouse. The castrated
mouse model can be applied in future studies of veno-occlusive erectile
dysfunction.
Key words: Penile erection, castration, penis, androgen, testosterone, animal model
The mammalian erectile response is dependent on the dilation of arterioles carrying blood into the corpora cavernosa. Veno-occlusion is equally important in providing a pressure dependent reduction of blood draining from the corpora cavernosa. It is the combination of increased arterial blood inflow and decreased venous blood outflow that leads to a rise in intracavernosal pressure and subsequent penile erection. A low outflow resistance whether because of a primary trabecular smooth muscle dysfunction or true lack of venous occlusion can lead to venoocclusive erectile dysfunction.
Androgens play an important role in the development of secondary sexual characteristics and have been shown to influence penile morphology (Mills et al, 1996; Baskins et al, 1997; Shabsigh, 1997; Shabsigh et al, 1998, Traish et al, 1999). Previous animal data particularly in the rat reveal that androgens support erectile function through a direct effect on erectile tissue. Castration results in an impaired erectile response to central and peripheral stimulation as well as a measured decrease in nitric oxide synthase tissue concentrations in the rat penis (Burnett et al, 1995; Chamness et al, 1995; Lugg et al, 1996; Vanhatalo et al, 1996; Gonzales-Cadavid et al, 2000). In addition, the induction of apoptosis in rat penile tissue following castration has been demonstrated (Shabsigh et al, 1998; Zhang et al, 1999). Interestingly in the rabbit, castration also significantly reduces trabecular smooth muscle content of the penis, but neural nitric oxide synthase protein expression and total activity were not altered significantly by castration (Traish et al, 1999). Exogenous testosterone replacement has been shown to reverse these abnormalities. However, a generalization regarding androgen control in mammalian erectile physiology remains uncertain. In humans, data regarding castration and erectile function are even less clear than in animal studies.
In an effort to advance the study of erection physiology with the help of animal models, a mouse model was selected. This model affords a unique approach to the field of sexual dysfunction research when compared with other animal models. Not only is this species useful for erection experiments and affordable, but mice also allow molecular biological approaches through the use of transgenic technology (Burnett et al, 1996; Huang, 1999; Hedlund et al, 2000; Sezen and Burnett, 2000). We have explored the role of androgens in the physiology of erectile function in mice and described a mouse model for veno-occlusive erectile dysfunction.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Surgical Procedures
Mice were anesthetized with 40 mg/kg sodium pentobarbital intraperitoneally
(Nembutal, Abbot Laboratories; Chicago, IL). Surgical castration was performed
via a midline scrotal incision allowing bilateral access to the hemiscrotal
contents. After exposing each testicle, a 3-0 Vicryl suture was used to ligate
the spermatic cord and then remove the testicle. In the TEST group, a 5.0
mg/kg testosterone pellet (60-day release) was placed into the empty right
hemiscrotum and fastened within the tissue using a 3-0 Vicryl suture. The dose
of the implanted testosterone pellet attains physiologic levels of
testosterone (1.3-4.57 ng/mL) (Crispens,
1976; Foster et al,
1982). The skin was then closed with 3-0 Vicryl suture.
Cavernous nerve stimulation for the induction of penile erection was performed as previously described (Burnett et al, 1996). Neurostimulated erections were evaluated by intracavernosal pressure (ICP) monitoring (Sezen and Burnett, 2000). ICP monitoring was performed on animals (CAST and TEST) at 24-hour, 72-hour, 1-week, 2-week, and 4-week time points. A control group of WT mice also underwent ICP measurements for comparison. At the end of the experiment, the aorta was cannulated with PE-60 tubing and connected to a pressure transducer to evaluate mean arterial pressure.
Immunohistochemistry
Whole-penis specimens (n = 4) were removed from WT, CAST (1-week and
4-week), and TEST (1-week and 4-week) mice. Specimens were divided into
proximal and distal segments and quickly frozen. Transverse sections (6 µm
thick) were cut on the cryostat and mounted onto gelatin/chrome alum-coated
slides. Slides were then fixed in cold acetone (-30°C) for 2 minutes.
Immunohistochemistry Protocol for CD31— After fixation, slides were rinsed 3 times in 0.1M phosphate buffered saline (PBS), pH 7.4, for 2 minutes. Endogenous peroxidase activity was blocked by incubating the slides in 0.3% H2O2 solution in PBS for 10 minutes, followed by rinsing slides 3 times in PBS for 2 minutes. Purified rat antimouse CD31 or platelet endothelial cell adhesion molecule-1 monoclonal antibody (BD PharMingen, San Diego, CA, #550274) at 1:80 dilution with PBS was applied directly to the slide and then incubated for 1 hour at room temperature. After rinsing in PBS, biotinylated secondary antibody at 1:50 dilution with PBS was applied directly to the slide for 30 minutes. Staining was visualized with an avidin-biotin-peroxidase system (Vector Laboratories, Burlingame, CA) with diaminobenzidine as the chromagen. Finally slides were rinsed with water and then dehydrated with alcohol and xylene. In addition, specimens were processed in the absence of the primary antibody.
Immunohistochemistry Protocol for -actin—
Slides were blocked with 2% bovine serum albumin for 30 minutes at room
temperature. Slides were then incubated with monoclonal anti-A-smooth muscle
actin antibody at a 1:30 dilution with PBS for 1 hour (Sigma Chemical Co., St
Louis, MO, #A5691). Slides were rinsed with PBS and subsequently incubated
with substrate NBT/BCIP (Sigma, #B5655) for 3 minutes. Finally the sections
were rinsed with water, dehydrated, and mounted. Specimens were also processed
in the absence of primary antibody.
Histomorphometry—
For each mouse group, 8 representative sections consisting of a
cross-section of penile tissue were studied. Each cross-section of corpus
cavernosum was analyzed at a 10x power under the microscope. Using the
Image Pro-plus software (Mediacybernetics; Carlsbad, CA), the percent of
positive staining representing immunoreactivity of the corpus cavernosum for
CD31 or -actin was recorded.
Doppler Flow Studies
The Agilent Sonos 5500 Doppler ultrasound with continuous wave measurement
(Agilent Technologies, Palo Alto, CA) was employed to measure penile blood
flow in the mouse. Waveforms were measured before stimulation (pre), during
stimulation (intra), and after stimulation (post) of the cavernous nerve.
Using a small vascular probe, measurements were obtained at the lateral
aspects at the base of the penis. The penis had previously been denuded of
skin during cavernosal nerve stimulation and ICP measurements, with the
ischiocavernous muscles dissected off bilaterally to expose the penile crus.
Continuous-wave Doppler images recorded penile blood flow as video images and
audible signals. The video images were later converted to digital images using
Videum image capture software (Winnov, Sunnyvale, CA) and analyzed using Adobe
Photoshop (Adobe; San Jose, CA) and Matlab software (Mathworks, Natick, MA).
Flow rates (centimeters per second) were measured and compared among WT, CAST
(2-week), and TEST (2-week) mice (n = 5 per group).
Statistical Analysis
Data were analyzed using the Student's t test or two-way analysis
of variance with Sigmastat 2.0 software (SPSS, Chicago, IL). Results are
expressed as mean values ± SEM. A P value of less than .05 was
considered significant.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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ICP measurements (mm Hg) were compared between WT and CAST mice at 24-hour,
72-hour, 1-week, 2-week, and 4-week time points (50.2 ± 2.8 vs 41.9
± 14.6, 19.1 ± 4.2, 17.5 ± 8.2, 14.2 ± 4.4, and
10.0 ± 3.8). In the CAST animal group, a statistically significant
difference in maximal ICP was established at 72 hours and all subsequent time
points (P 
.05). In TEST animals, ICP measurements were
maintained in comparison with WT results. In addition, at the 2-week time
point a significant increase in ICP was noted (50.2 ± 2.8 vs 48.0
± 1.4, 52.3 ± 1.3, 60.8 ± 7.6, 80.5 ± 2.1, and
81.5 ± 1.2; P .01 at 2 and 4 weeks). These data suggest
that exogenous testosterone sustains erectile function but may also enhance
erectile function in mice possibly causing a super-physiologic response to
stimulation. Likewise, the sudden removal of testosterone induces erectile
dysfunction after 72 hours.
Immunohistochemistry
Immunoreactivity for CD-31 and -actin was confirmed in penile
specimens and also validated with negative controls. At the 1-week time point,
immunohistochemical staining for CD-31 and -actin revealed little
histomorphometric difference among WT, TEST, and CAST mice (CD31: WT 47.9
± 0.9, TEST 48.0 ± 0.5, CAST 47.7 ± 0.5; -actin:
WT 23.4 ± 0.6, TEST 23.5 ± 0.4, CAST 22.7 ± 0.8). At the
4-week time point, however, the CAST animals demonstrated reduced CD-31 and
-actin immunoreactivity when compared with WT and TEST animals (CD-31:
WT 47.9 ± 0.9, TEST 48.1 ± 0.44, CAST 45.3 ± 0.5;
-actin: WT 23.4 ± 0.6, TEST 23.4 ± 0.4, CAST 21.5
± 0.8). These differences were statistically significant for both CD-31
and -actin (P .05)
(Figure 2).
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Doppler Flow Studies
Penile tumescence relies on venous occlusion. Venous occlusion occurs with
the passive compression of emissary veins between the tunica albuginea and
corpus cavernosum. If the veno-occlusive mechanism fails, this results in
erectile dysfunction. Doppler ultrasound flow rates (centimeters per second)
performed in CAST mice suggested a mechanism of veno-occlusive erectile
dysfunction by the persistent intrapenile flow signal observed during the
induction of penile erection. Flow rates taken pre-, intra-, and immediately
postcavernous nerve stimulation were WT 45.4 ± 7.3, 30.6 ± 5.2,
55.3 ± 8.2 vs CAST (2 weeks) 22.2 ± 2.5, 25.0 ± 1.5, 23.1
± 2.0 vs TEST (2 weeks) 30.5 ± 6.5, 25.7 ± 2.0, 45.2
± 4.5. A statistically significant difference (P .01) was
observed when comparing WT vs CAST pre and post cavernosal nerve stimulation.
WT and TEST mice displayed higher flow rates than that of the CAST mice both
pre and post stimulation, indicating an impaired vascular function in the CAST
penis. During stimulation (intra), flow rates were reduced in WT and TEST mice
in contrast to CAST mice, which remained unchanged, indicating an impaired
veno-occlusive mechanism. Exogenous testosterone given to TEST mice maintained
flow rates in the penis similar to WT mice. Pilot studies performed at various
time points from surgery revealed that the 2-week CAST and TEST animals had
the most consistent detectable flow rates. At 4 weeks the CAST animals did not
demonstrate detectable Doppler penile blood flow rates, and earlier time
points revealed a greater variability in results.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Also, the addition of parenteral testosterone would have provided TEST mice with normal physiologic levels of testosterone, but this may not have been equivalent to a normal diurnal physiologic release of testosterone, which is seen in WT mice. Thus, a high normal steady state of testosterone may have been achieved and may have contributed to the observed improvement in erections in TEST mice over the 4-week study period.
Doppler ultrasound to assess penile blood flow in rats has been employed by other researchers (Collin et al, 1996; Mills et al, 1998). The use of the Doppler ultrasound to assess penile blood flow in mice is a novel technique and complemented our observed ICP measurements. It also provided unique information with regard to vascular properties in the mouse penis after castration.
Immunohistochemistry showed at 4 weeks that CAST animals did have a reduction in corpus cavernosal smooth muscle and endothelial cell content. This may be due to the fact that significant structural changes may take longer to be observed than functional erectile loss after castration in mice. TEST animals preserved penile morphology when compared with WT animals. Our data also revealed little difference in the structural content of corpus cavernosal smooth muscle and endothelial cells of WT and CAST animals at the 1-week time point.
The mouse model for studying vascular phenomena in the penis offers several advantages to other animal models. Economic feasibility and practicability are two major reasons for use. The application of transgenic technology and availability of molecular biological approaches to investigate genetic determinants of erectile function are also unique to mouse models.
The basis for the variability in the regulatory roles of androgens involved in penile erection physiology between different species remains unclear. In the future, animal models of penile erection may remain useful, but judgment remains necessary before assigning clinical relevance to human erectile dysfunction. The castrated mouse model can be considered for future studies of veno-occlusive erectile dysfunction.
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Footnotes |
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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