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,
,
From the * Albany College of Pharmacy, the
Division of Urology, Albany Medical College,
Albany, New York; the Chang Gung Memorial
Hospital, Chia-Yi, Taiwan; and the Stratton VA
Medical Center, Albany, New York.
| Correspondence to: Dr Robert M. Levin, Albany College of Pharmacy, 106 New Scotland Ave, Albany, NY 12208 (e-mail: levinr{at}acp.edu). |
| Received for publication April 30, 2007; accepted for publication September 18, 2007. |
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Abstract |
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remained at obstruction level, whereas ROK β expression decreased
in comparison with the obstruction group. By 8 weeks of reversal, expression
of both ROK and β significantly decreased when compared with the
obstruction group. These results suggested that the poor relaxation response
at reversal of 4 weeks was associated with incomplete decreased expression of
both isoforms of ROK, whereas the incomplete recovery of the CCSM relaxation
response at reversal of 8 weeks may be associated with structural alterations
in the CC and irreversible damage from PBOO.
Key words: Erectile dysfunction, penis, reactive oxygen, Rho kinase, physiology, collagen, Western blotting
Although surgery, minimally invasive therapies, and pharmacologic therapies can improve LUTS and peak urinary flow rates, it is unclear whether LUTS treatment improves ED. Indeed, some of the therapies can cause or exacerbate ED (the incidence of ED following surgery is 10%; minimally invasive therapies, 1%–3%; and pharmacologic monotherapy or combination therapy, 3%–10%) (Miner et al, 2006; Poulakis et al, 2006). Indeed, in most of the reports about ED after LUTS treatment, it is hard to separate the ED from the therapy from that associated with the LUTS itself (Jorge G. Puente, unpublished data, 1998; Baniel et al, 2000). The impact of LUTS treatment on ED would be more obvious in a purer experimental preparation. It is well known that impairment of CC physiology and functioning may result in ED (Korenman, 1998; Lue, 2000; Sadeghipour et al, 2007). Therefore, based on previous studies, the goal of the current study was to investigate the changes in CC in rabbits after reversal of the PBOO.
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Materials and Methods |
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Experimental Animals
Twenty-four adult male New Zealand White rabbits (3–5 kg and
15–20 weeks old) (Millbrook Breeding Laboratories, Amherst, Mass) were
separated into 4 groups of 6 rabbits each. Group 1 served as sham controls.
Rabbits in groups 2 to 4 were subjected to PBOO for 4 weeks. After 4 weeks,
group 2, which served as the obstructed groups, was sacrificed. For the
reversal studies, each obstructed rabbit in groups 3 and 4 were relieved of
the obstruction and was kept for 4 weeks and 8 weeks, respectively. A second
sham operation group (to match the reversals) was not performed.
Operative Procedure for Creating and Relieving PBOO
Rabbits in groups 1 to 4 were anesthetized using 25 mg/kg ketamine/10 mg/kg
xylazine given intramuscularly. Surgical anesthesia was maintained with
pentobarbital (25 mg/kg intravenously; Abbott Laboratories, Abbott Park, Ill)
and isoflurane inhalation. Each bladder was catheterized through the urethra
with an 8 Fr Foley catheter (Mentor Urology, Santa Barbara, Calif), and the
bladder was exposed through a midline incision. The bladder neck and urethra
were cleared of fat and connective tissue. For group 1 (sham operated), the
catheter was removed and the wound closed. For groups 2 to 4, a mild
obstruction was created by placing a silicon ring (diameter x length =
6.35 mm x 2 mm) loosely around the catheterized urethra. The bladder was
returned, and the wound was closed in layers. Surgical induction of the PBOO
procedure and all materials were the same in the 3 groups. The level of
obstruction was standardized, with all obstructions performed by the same
surgical team. Sham surgery was identical in all aspects—the placement
and removal of the ring were performed. Pain medication (buprenorphine 0.1
mg/kg intramuscularly twice daily) and antibiotics (gentamicin 4 mg/kg
intramuscularly daily) were administered for the first 2 days
postoperatively.
For the reversal studies, each obstructed rabbit in groups 3 and 4 were anesthetized as above, the silicon ring removed, and the wound closed as before.
Isolation of CC and Physiology
Each penis was surgically removed en bloc. The CC was then procured via
sharp dissection from the surrounding tunica and either immediately suspended
in an organ bath or frozen in liquid nitrogen and stored at –80°C.
In addition, a 2- to 3-mm piece of intact penis was prepared in paraffin
blocks for histologic study.
The CC strip was suspended under 2 g of resting tension and allowed to equilibrate for 1 hour in organ chambers containing 30 mL of Tyrode buffer (24.9 mM NaCl, 2.5 mM KCl, 23.8 mM NaHCO3, 0.5 mM MgCl2·6H2O, 0.4 mM NaH2PO4·H2O, 1.8 mM CaCl2, and 5.5 mM dextrose) at 37°C gassed with 95% oxygen and 5% carbon dioxide. During this time, the Tyrode buffer was replaced every 15 minutes with fresh solution. One end of each strip was connected to a force displacement transducer, and changes in muscle tension were measured and recorded with a polygraph (model 7D; Grass Technologies, East Warwick, RI). After a 1-hour incubation, each tissue was contracted with 100 µM phenylephrine. When contraction reached a plateau, the strip was subjected to relaxation in response to EFS using sequential frequencies of 2, 8, and 32 Hz (80 V, 1-millisecond duration, 15-second train, and 5-minute train interval). The strips were allowed to return to baseline precontractile tension between tests at each frequency. Following EFS, the relaxation responses to ATP, acetylcholine, and sodium nitroprusside (SNP) of phenylephrine-preincubated CC strips were investigated. Each strip was washed 3 times at 15-minute intervals with fresh oxygenated Tyrode solution between pharmacologic agents. ATP (2 mM) relaxes CCSM through increases in cAMP levels; acetylcholine (500 µM) is used to assess endothelium-dependent relaxation; and SNP (100 µM) is a nitric oxide (NO) donor and activates cGMP synthesis.
Histology and Trabecular SM to Collagen Fibers Ratio
Paraffin blocks were prepared from sections of the mid region of the penis
containing CCSM. Each fixed section of CC was embedded on edge to provide a
cross-sectional view of CC after microtome sectioning.
Five-micrometer–thick sections were cut from each block and mounted on
positively charged slides, deparaffinized in xylene, and stained with a Masson
trichrome kit (Richard Allan Scientific, Kalamazoo, Mich). The ratio of
collagen to SM within the CC was measured with image analysis on the
trichrome-stained slides. The blue-stained collagen and red-counterstained SM
were highlighted for each image using Image-Pro Plus software (Media
Cybernetics Inc, Bethesda, Md) by manually selecting the pixel values of each
using the color cube–based tool in the count/size application.
Photomicrographs at 200x magnification were taken of 4 areas from each
tissue and analyzed for the ratio of SM to collagen in well-defined areas of
the tissue. It should be noted, however, that this method does not allow the
quantitation of the amount of collagen per unit bladder
mass.
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Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis and Western Blotting
Frozen CC tissues (100 mg) from each rabbit were ground to a fine powder in
a mortar cooled in liquid nitrogen and homogenized in a buffer containing 20%
glycerol, 50 mM Tris·HCl (pH 6.8), 0.5% (vol/vol) Tween-20, and
protease inhibitors (0.5 mM phenylmethylsulphonyl fluoride, 2 µM pepstatin,
2 µM antipain, and 0.1 mg/mL trypsin inhibitor). After addition of sodium
dodecyl sulfate (SDS; 1% final concentration), the sample was boiled for 4
minutes and centrifuged at 13 300xg for 15 minutes. The protein
concentration in the supernatant was measured using the a bicinchoninic acid
protein assay kit (Pierce Biotechnology, Rockford, Ill). Equal amounts (20
µg) of total protein from CC obtained from the 4 different groups of
rabbits were loaded on 8% SDS polyacrylamide gels and transferred to
Immobilon-P membranes (Millipore Corp, Billerica, Mass) with Towbin buffer (25
mM Tris, 192 mM glycine, and 20% [vol/vol] methanol). The membranes were
blocked with 5% nonfat milk in 0.05% Tween-20 in phosphate-buffered saline for
1 hour at 37°C and then incubated with a primary antibody (1:1000
dilution; anti–Rho kinase (ROK) clone 21; Transduction
Laboratories, Lexington, Ky) or a 1:250 dilution of an anti-ROKβ antibody
(clone C-19; Transduction Laboratories) in a shaking incubator. After
treatment with the primary antibody, the membranes were washed with 20 mM
Tris, 500 mM NaCl, and 0.05% Tween-20 and incubated with a secondary antibody
(goat anti-mouse immunoglobulin G at 1:5000). Substrates were visualized by
using enhanced chemiluminescence (Amersham Pharmacia Biotech, Buckinghamshire,
United Kingdom) and exposing the membranes to autoradiographic films (Kodak
X-OMAT; Sigma-Aldrich, St Louis, Mo). Films were scanned and analyzed with a
Kodak Image Station 440CF and Kodak ID image analysis software (Scientific
Image System, Rochester, NY). Equivalent sample loading was confirmed by
stripping membranes with 0.2 M NaOH and washing with 20 mM Tris, 500 mM NaCl,
and 0.05% Tween-20. Then the membranes were stained in Bradford reagent (0.01%
Coomassie blue G-250, 9.5% ethanol, 8.5% H3PO4) for 10
minutes and destained in 50% methanol and 1.0% acetic acid until background
was reduced and total proteins were clearly visible.
Statistical Analyses
Data are expressed as means ± SEM. Analysis was performed using
1-way or 2-way analysis of variance followed by Bonferroni's multiple range
tests. P < .05 was considered significant.
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Results |
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Expression of ROK at the Protein Level
There was a mild increase in the expression of ROK in the cavernosal
tissue of the bladder obstruction group that is not significantly different
than that of the control. At 4 weeks after reversal, there was a significant
increase in the expression of ROK, which fell below control levels by 8
weeks after reversal (Figure 6A and
B). The expression of ROKβ significantly increased following
obstruction and then decreased significantly after 4 and 8 weeks of reversal
(Figure 7A and B). Equivalent
sample loading was confirmed when the membranes were shown to contain clearly
visible total proteins (Figure
8).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Our previous study (unpublished data) confirmed that PBOO results in a significant decrease in the relaxant response to EFS (as well as to ATP, acetylcholine, and SNP). That study also demonstrated that reversal of PBOO resulted in a time-dependent increase in the responses to EFS, acetylcholine, and SNP. EFS was used to assess nerve-mediated relaxation of CCSM, which is composed of cholinergic, adrenergic, and nonadrenergic noncholinergic nerves components. A previous study verified the decreased innervation of the CCSM from rabbits with PBOO by immunohistochemical staining (Chang et al, 2005). The gradual recovery of the relaxation response could be a reflection of progressive reinnervation in CCSM, like reinnervation in detrusor, after reversal of PBOO. Some hypotheses have been developed for pathophysiology leading to decreased innervation in the CCSM of rabbits with PBOO. The study showed that there was constant compression of the nerves by urethral ligation and of vessels in the base of the bladder, which led to denervation and ischemia of the CCSM (Chang et al, 2002). Consequently, we investigated whether relief of PBOO would terminate these injuries and provide recovery for the CCSM. The results of the shamoperated control group ruled out that denervation or ischemia is caused by surgery per se. In addition, the operative site and position of ligature were distant from the penis, which meant that structural changes are unlikely to be caused by direct trauma from operations.
In addition, we hypothesized that the decreased innervation of CC is caused by production of free radicals secondary to PBOO. Nitrotyrosine, which has been demonstrated to be a marker of free radical damage due to reactive nitrogen species, has been shown to be significantly elevated with PBOO (Levin et al, 2005; Conners et al, 2006; Mannikarottu et al, 2006). Also, nitrotyrosine has been associated with neuronal ischemic injury and numerous neurodegenerative diseases (Smith et al, 1997). Therefore, like penis injury from cigarette smoke mediated by large numbers of free radicals, penis injury from either denervation or ischemia could be mediated by free radicals generated after PBOO (Ota et al, 1997; Gocmez et al, 2005). Because decreased expression of nitrotyrosine and reinnervation of detrusor have been found to occur after reversal of PBOO, we theorized that this could also occur in the penis and contribute to restoration of the relaxation response of CCSM (unpublished data). The greater magnitude of recovery in the relaxation response of CCSM at reversal of 8 weeks, when compared with reversal at 4 weeks, could also imply a time-dependent improvement for ischemia after reperfusion. Therefore, we hypothesized that there could be increased removal of free radicals and increased reinnervation for reversal at 8 weeks than at 4 weeks. However, further study is needed for this hypothesis.
PBOO inhibits both NO-dependent (acetylcholine-stimulated) and NO-independent (ATP- and SNP-stimulated) relaxation of CCSM, which are also endothelium-dependent and endothelium-independent vasodilators, respectively. SNP-activated cGMP synthesis directly induced relaxation of CCSM (Sadeghipour et al, 2007). Adenosine has potent relaxant activity on the CC, acting through a mechanism different than the NO pathway (Mantelli et al, 1995). The poor relaxation response at reversal at 4 weeks could be due to ischemiareperfusion injury to endothelium and the NO/cGMP pathway; whereas partial recovery of the relaxation response after 8 weeks of recovery implies that some irreversible damage to either endothelium, ATP, or the NO/cGMP pathway occurred.
One of our striking findings was that PBOO significantly increases the distribution of collagen within the CC, which remained unchanged after reversal. Similar to castration and aging, the decreased ratio of trabecular SM to collagen would be expected to contribute to some degree of irreversible loss in the relaxation response of the CCSM after reversal of PBOO (Bakircioglu et al, 2001; Traish et al, 2003). The significantly decreased proportion of SM would result in a decrease in the net contraction of the strip to phenylephrine, even if the SM components responded with increased tension. We theorized that the decreased proportion of SM accounted for the decreased response to phenylephrine in both obstructed and reversal groups. In addition, the decrease in phenylephrine-induced tone in the obstruction group might also arise from ischemia because hypoxia, acidosis, or glucopenia alone or in combination showed a sustained reduction in the tone (Muneer et al, 2005). Moreover, CCSM did not regain the control level of phenylephrine-induced tone after the reversal of 8 weeks. Conversely, following a relatively shorter period of hypoxia, the reversibility of phenylephrine-induced tone of CCSM has been reported in some studies (Kim, 1996; Muneer et al, 2005). Hence, we speculated that ischemia from 4 weeks of PBOO results in irreversible CCSM dysfunction.
In addition to mediating phosphorylation, ROK isoforms are involved in the
noradrenergic contractile pathway in the CCSM of the penis
(Rees et al, 2001). An
increase in ROK isoforms expression/activity would be expected to make it more
difficult for the CCSM to relax (Chang et
al, 2005). On the other hand, inhibition of ROK was beneficial for
erectile function in old rats (Rajasekaran et al, 2006). A clinical study by
Omer et al (2006), which
demonstrated that maximum relaxation responses of CCSM following ROK inhibitor
(Y-27632) administration were significantly higher in ED patients in a
BOO-positive group compared with those in a BOO-negative group, also supports
the proposal. Therefore, in comparison to reversal at 4 weeks, the decreased
expression of both isoforms at 8 weeks could account for the higher relaxation
responses of CCSM. These findings imply that the higher relaxation response of
CCSM obtained from the reversal of PBOO is associated with decreased activity
in both ROK isoforms. However, the exact functional differences between
ROK and ROKβ are not currently known, and further study is
indicated.
In general, the lack of physiologic in vivo experiments to support our in vitro findings make it difficult to extrapolate from this study about the relationship between cessation of LUTS and ED in men. However, the rabbit model with reversal of PBOO could still be a reliable tool in investigating the issue. According to a report by Elliott et al (2004), obstructive instead of irritative LUTS is correlated with and is predictive of ED. Additionally, reversal of PBOO in a rabbit model can be easily performed without complications of treatments for benign prostatic hyperplasia. Our laboratory and others have demonstrated that bladder contractile function can be restored after reversal of PBOO (Levin et al, 1985; Malmgren et al, 1990; Lin et al, 1998). Hence, further in vivo study of the penis would clarify the status of erectile function after restoration of bladder function. To conclude, our present study indicated that the magnitude of the restoration of the relaxation responses of CCSM is proportional to the time course of recovery of bladder function. Also, after relief of PBOO, a certain degree of irreversible loss in both CCSM relaxation and contraction responses is associated with a permanent increase in collagen content and distribution observed in the CC. Moreover, the expression of ROK isoforms is consistent with the alteration in relaxation of CCSM after reversal of PBOO.
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Footnotes |
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