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Cavernous Neurotomy Causes Hypoxia and Fibrosis in Rat Corpus Cavernosum

From the Department of Urology, Tulane University Health Sciences Center, New Orleans, Louisiana.

Correspondence to: Wayne J.G. Hellstrom MD, Tulane University School of Medicine, Department of Urology SL-42, 1430 Tulane Avenue, New Orleans, LA 70112 (e-mail: whellst{at}tulane.edu).

Received for publication May 15, 2002; accepted for publication September 19, 2002.

	Abstract

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The etiologies of erectile dysfunction (ED) after nerve-sparing radical prostatectomy have not been clearly elucidated. The aim of this study was to evaluate the effects of cavernous nerve injury on cavernous fibrosis, and to consider measures to prevent irreversible damage to the cavernous tissues. Twenty male Sprague-Dawley rats constituted the study population. The animals were divided into 2 groups; group 1 consisted of sham-operated rats (n = 10), and group 2 consisted of rats that underwent incision of both cavernous nerves (n = 10). Three months later, all rats underwent intracavernous papaverine injection (300 and 600 mg), and intracorporal pressures were recorded. Transforming growth factor-ß₁ (TGF-ß₁) messenger RNA (mRNA) expression from rat penile tissue was measured using reverse transcriptase-polymerase chain reaction. Hypoxia-inducible factor-1 (HIF-1), TGF-ß₁, and collagen I and III protein expressions were determined by Western blot analysis and immunohistochemical staining. Erectile function as studied with intracavernosal papaverine injection and histological analysis of penile cross-sections at 3 months was similar in both groups. TGF-ß₁ mRNA expression, HIF-1, TGF-ß₁, and collagen I and III protein expressions were significantly greater in the neurotomy group. Immunohistochemical staining for TGF-ß₁, HIF-1, and collagen III were qualitatively more positive in the neurotomy group, whereas collagen I staining was similar. This study demonstrates an increase in TGF-ß₁, HIF-1, and collagen III synthesis in rat cavernosal smooth musculature after cavernous neurotomies. In theory, cavernous fibrosis may be reduced by employing various vasoactive agents or interventions that increase oxygenation to the corporal tissues during the postoperative period.

     Key words: Erectile dysfunction, radical prostatectomy, neurapraxia

Most investigators attribute ED after nerve-sparing radical prostatectomy to vascular, neurogenic, and psychogenic etiologies. Because ED is significantly more common in men who undergo non–nerve-sparing prostatectomy than in men who undergo nerve-sparing prostatectomy, a neurogenic etiology is recognized to be a main etiology of postprostatectomy ED (Gralnek et al, 2000). Moreover, in many studies, the recovery rate of erectile function due to the neurapraxia from surgery is time-related, and it may take 6 to 18 months after surgery for it to occur (Montorsi et al, 1997; Hong et al, 1999).

The aim of this study was to evaluate the isolated effects of cavernous nerve injury on cavernous oxygenation and cavernous fibrosis using a rat model. Basic knowledge about postprostatectomy ED causation may help researchers develop better techniques to prevent irreversible damage to the cavernous tissues.

	Materials and Methods

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Surgical Procedures

All animal studies received approval from the Animal Care and Use Committee of Tulane University School of Medicine. Twenty male Sprague-Dawley rats (300–325 g) constituted the study population. The animals were divided into 2 groups; group 1 consisted of sham-operated rats (n = 10), and group 2 consisted of rats that underwent incision of both cavernous nerves (n = 10). The procedures were performed under general anesthesia by intraperitoneal injection of 30 mg/kg of sodium pentobarbital. Supplemental doses of pentobarbital were administered as needed to maintain a uniform level of anesthesia.

The cavernous nerve was identified via an extraperitoneal low-transverse suprapubic incision. In the sham-operated group, the incision was closed layer by layer after bilateral identification of the cavernous nerves. In the neurotomy group, cavernous nerves were cut using an electrical cautery device. Antibiotics were fed orally to all animals for 5 days after surgery.

RNA Isolation and Reverse Transcriptase-Polymerase Chain Reaction

Three months after the sham and cavernous nerve injury operations, total RNAs were isolated using approximately of each rat penis by the TriZol method. RNA concentrations were quantified by spectrophotometry at 260 nm. Total RNAs (2 µg) were reverse-transcribed using reverse transcriptase–polymerase chain reaction (RTPCR) (GeneAmp PCR system, model 2400; Perkin-Elmer Corp, Norwalk, Conn). The RT reaction was performed in a reaction mixture (20 µL reaction volume) containing Moloney murine leukemia virus RT, 1 mM of each deoxynucleotide triphosphate (dNTP) (dATP, dCTP, dGTP, and dGTP), 20 units of RNase inhibitor, oligo(dT) primer, 10x standard buffer, and MgCl₂. The RT mix was incubated sequentially in a thermal cycler at 42°C for 15 minutes, at 99°C for 5 minutes, and at 5°C for 5 minutes. Subsequent PCRs were carried out in the presence of 20 pM sense and antisense primer, 2.5 µL of 25 mM MgCl₂, 0.4 µL of Taq DNA polymerase, 1 µL of each dNTP, 2.5 µL of 10x buffer, and 3 µL of template complementary DNA in a total volume of 25 µL. The primer sequences used for PCR amplification are shown in Table 1. Oligonucleotide primers for rat ß-actin were used as a positive control in each sample. PCR was initially performed after the 2-minute denaturation step at 95°C (30 cycles for ß-actin and 37 cycles for TGF-ß₁). Each PCR cycle for ß-actin consisted of denaturation at 94°C for 45 seconds, annealing at 61°C for 30 seconds, and extension at 72°C for 30 seconds. Each PCR cycle for TGF-ß₁ consisted of denaturation at 94°C for 5 seconds, annealing at 55°C for 5 seconds, and extension at 72°C for 10 seconds. The PCR products were analyzed on a 2% agarose gel stained with ethidium bromide and finally visualized under UV light. The band density was measured with a densitometer (Bio-Rad, Hercules, Calif) and computerized with Multi-Analyst software (version 1.1).

Protein Extraction and Western Blot Analysis

One-fourth of each penile tissue was homogenized separately in a buffer containing 0.32 M sucrose, 10 mM Tris-HCl pH 7.4, 2 mM ethylenediamene tetraacetic acid (EDTA), 10 µL/mL of Protease-Arrest (Protease Inhibitor, Genotech, St Louis, Mo). The homogenate was centrifuged at 900 x g for 10 minutes, and the resulting supernatant was centrifuged at 100000 x g for 1 hour. The final pellet was resuspended in a buffer containing 50 mM Tris-HCl, 10 mM EDTA, 100 mM NaCl, and 8 mM MgCl₂ pH 7.4. The protein concentration was measured using a colorimetric assay for protein quantitation (Labsystem, Multiskan, MS) and then calculated with Genesis software version 3.04 (Life Sciences, United Kingdom). After denaturation of extracted membrane protein at 95°C for 5 minutes, membrane proteins (70 µg of protein/lane) were applied onto a 10% discontinuous sodium dodecyl sulfate–polyacryamide gel electrophoresis. The resolved membrane proteins were transferred onto a 0.2-µm nitrocellulose sheet by semidry electroblotting for 1.5 hours, and then soaked in 5% nonfat dry milk in Trisbuffered saline containing Tween-20 (TBS-T; 10 mM/L Tris-HCl pH 7.2, 250 mM/L NaCl, and 0.05% Tween-20) at 4°C for 1 hour. The membrane was incubated with polyclonal rabbit anti-rat TGF-ß₁, mouse anti-rat hypoxia inducible factor-1 (HIF-1, Novas), and collagen I and collagen III monoclonal primary antibodies (1:500, 1:1000, 1:1000, and 1:2000 dilutions in TBS-T, respectively) at room temperature for 1 hour, subsequently incubated with biotinylated secondary antibody (1:5000 dilution in TBS-T) at room temperature for 1 hour, and reacted with peroxidase-conjugated streptavidin at room temperature for 1 hour. Specific bands were visualized with chemiluminescence (ECL plus Western Blotting Kit; Amersham-Pharmacia Biotech, Piscataway, NJ). Each band density was measured with a densitometer (Bio-Rad) and computerized with Multi-Analyst software (version 1.1).

Immunohistochemical Stains

The remaining of the rat penile specimens were removed and washed in an ice-cold 0.9% normal saline solution to remove any blood. The extracted tissues were fixed by immersion in 4% paraformaldehyde for 24 hours and cryoprotected in 20% sucrose phosphate buffer for 24 hours. Ten-micrometer sections were cut on a cryomicrotome. Sections were thaw-mounted on the probe on pluscharged slides (Fisher Scientific, Pittsburgh, Pa) at room temperature, placed in the cryostat, and then stored at -70°C until used for immunohistochemical staining.

For detection of HIF-1, TGF-ß₁, and collagens I and III, endogenous peroxidase was blocked with 0.3% H₂O₂ in methanol for 30 minutes. The sections were washed in TBS-T and then blocked for 1 hour with normal horse serum in TBS-T and incubated overnight at 4°C with rabbit anti-rat TGF-ß₁ polyclonal primary antibody, mouse anti-rat collagen I, collagen III, and HIF-1 monoclonal primary antibodies (TGF-ß₁, 1:250; collagen I, 1:500; collagen III, 1:1000; and HIF-1, 1:500 dilution in TBS-T; in the control study only TBS-T was used). After washing in TBS-T the sections were incubated with biotinylated anti-rabbit immunoglobulin G (1: 200 dilution in TBS-T; DAKO, Carpinteria, Calif) for 1 hour at room temperature. After rinsing, the sections were further incubated for 1 hour in streptavidin (1:200 dilution in TBS-T; DAKO) at room temperature and visualized by employing diaminobenzidine. Tissue sections were lightly stained with hematoxylin.

The slides were blindly evaluated by 4 independent observers and were scored on a basis of 1 to 5 points depending on the degree of positive staining. Score differences of more than 2 points were considered significant. These techniques have been reported previously (Bivalacqua et al, 2001).

Measurement of Erectile Responses

Three months after the sham and cavernous nerve injury operations, all rats were anesthetized and placed on a thermo-regulated surgical table. The trachea was cannulated (PE-240 polyethylene tubing) to maintain a patent airway, and the animals breathed room air enriched with 95% O₂/5% CO₂. The left carotid artery was cannulated (PE-50 tubing) and systemic arterial pressure was measured continuously with a Viggo-Spectramed (Oxnard, Calif) transducer connected to a computerized system for data acquisition (Windaq DI 400, DATAQ, Data Systems International, St Paul, MN). The right jugular vein was cannulated (PE-50 tubing) in order to administer fluids and supplemental anesthesia.

The skin overlaying the penis was incised, and the right crus was exposed by removing part of the overlaying ischiocavernous muscle. A 25-gauge needle filled with 250 U/mL of heparin and connected to PE-50 tubing was inserted into the right crura. Systemic arterial and intracavernosal blood pressures were measured with a Statham P23 pressure transducer connected to a computerized system for data acquisition. Each rat was injected intracavernously with 300 µg of papaverine to achieve a significant and consistent erectile response. The effect of papaverine on cavernosal pressure was measured until cavernosal pressure returned to a stable baseline. Subsequently, a second intracavernous papaverine injection of 600 µg was made 10–15 minutes after the baseline pressure had been achieved. These procedures have been previously described (Bivalacqua et al, 2000).

Statistics

The hemodynamic data of intracavernous pressures are expressed as means ± SE. The band density of RT-PCR and Western blot results are expressed as optical density (OD)/mm². All data were analyzed using a paired t-test. A P value of < .05 was used as the criteria for statistical significance.

	Results

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RT-PCR Analysis

To investigate the mRNA expression of TGF-ß₁ in rat penes, total RNAs of both groups were analyzed via RT-PCR. The results of PCR amplification for TGF-ß₁ are shown in Figure 1. A positive control for ß-actin was included in this study. Amplication products at the predicted sizes were clearly detected (TGF-ß₁, 278 bp and ß-actin, 218 bp; Figure 1A). The band density of the TGF-ß₁:ß-actin ratio was significantly greater in the neurotomy group (Figure 1B).

View larger version (48K): [in this window] [in a new window]   Figure 1. (A) The results of PCR amplification for TGF-ß1 (278 bp) and ß-actin (218 bp) in sham-operated and neurotomy groups. (B) Densitometer readings for ß-actin were similar in both groups (P = .418). Densitometer measurement of TGF-ß1 was significantly higher in the neurotomy group (P = .014). The optical density ratio of TGF-ß1:ß-actin was significantly greater in the neurotomy group. *P < .05; significantly different than sham-operated control animals.

Western Blot Analysis

To investigate the protein expressions of TGF-ß₁, HIF-1, and collagens I and III, protein expression from rat penile tissues from both groups were analyzed by Western blot analysis, and these data are summarized in Figure 2. Protein products of predicted sizes were clearly detected (TGF-ß₁, 15 kd; collagen I, 300 kd; collagen III, 330 kd; and HIF-1, 120 kd; Figure 2A). Protein expressions of TGF-ß₁, HIF-1, and collagens I and III were all significantly greater in the neurotomy group as determined by densitometry (Figure 2B).

View larger version (42K): [in this window] [in a new window]   Figure 2. (A) Western blot analysis demonstrating expression of TGF-ß1, collagen I, collagen III, and HIF-1 in sham-operated and neurotomy corpus cavernosum. (B) Bar graphs demonstrate that the optical density/mm2 of TGF-ß1, collagen I, collagen III, and HIF-1 were all significantly greater in the neurotomy group as determined by densitometry. *P < .05; significantly different than sham-operated control animals.

Immunohistochemical Stains

Immunohistochemical localization of TGF-ß₁, HIF-1, and collagens I and III was determined in both the sham-operated and cavernous neurotomy penes, and these data are summarized in Figure 3. When 3 independent observers scored the histological sections, TGF-ß₁, HIF-1, and collagen III were noted to be more intense in the cavernosal sinusoids and smooth musculature of the neurotomy group, with the staining for collagen I in both groups not subjectively different (Figure 3 and Table 2).

View larger version (90K): [in this window] [in a new window]   Figure 3. Representative immunohistochemical staining of TGF-ß1, collagen I, collagen III, and HIF-1 in sham-operated and neurotomy rat penes. There was more staining for TGF-ß1, collagen III, and HIF-1 in corpus cavernosum in the neurotomy group as observed by 4 independent observers. There was no difference in collagen I staining between both groups.

Erectile Responses to Intracavernous Papaverine Injection

The effects of sham and neurotomy operations on erectile responses induced by intracavernosal injection of papaverine were studied in sham-operated and neurotomy rats, and these data are summarized in Figure 4. At 3 months after the surgery, baseline intracavernosal pressures (ICPs) of the sham-operated (15.3 ± 1.6 mm Hg; SE) and the neurotomy animals (14.6 ± 1.8 mm Hg) were similar (P = .75). The maximal ICPs of the sham-operated (45.9 ± 6.9 mm Hg) and neurotomy animals (47.8 ± 6.3 mm Hg) after intracavernosal injection of papaverine at 300 µg were similar (P = .83). The pressure differences between the maximum ICP after intracavernosal injection of papaverine at 300 µg and baseline ICP of sham-operated (30.6 ± 6.4 mm Hg) and neurotomy rats (33.3 ± 6.2 mm Hg) were also similar (P = .77; Figure 4).

View larger version (13K): [in this window] [in a new window]   Figure 4. Bar graph showing the increase in ICP in response to direct intracavernosal injection of papaverine, a nonspecific phosphodiesterase inhibitor, in sham-operated and neurotomy rats. In vivo erection experiments were conducted 3 months after neurotomy surgeries. n, number of experiments.

Intracavernosal injection of papaverine using 600 µg produced a maximum increase in ICP that was similar (P = .70) in sham-operated (62.7 ± 9.4 mm Hg) and neurotomy animals (58.3 ± 7.9 mm Hg; Figure 4). The pressure difference between the maximum ICP after intracavernosal injection of papaverine at 600 µg and baseline ICP of sham-operated (44.8 ± 8.4 mm Hg) and neurotomy rats (42.7 ± 7.3 mm Hg) were also similar (P = .85) (Table 2).

	Discussion

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The cause of nerve-sparing ED after radical prostatectomy is still an active area of investigation. In 30 patients, Montorsi et al (1997) reported 67% recovery of spontaneous erections that were sufficient for satisfactory sexual intercourse after nerve-sparing radical retropubic prostatectomy by using early intracavernous alprostadil injections 3 times a week for 3 months, compared with only 20% recovery in the nerve-sparing surgery group without postoperative alprostadil injections. Those authors hypothesized that programmed vasoactive injections improve cavernous oxygenation, thereby limiting the development of hypoxia-induced tissue damage and subsequent fibrosis during the period of postoperative neurapraxia.

The relationship of oxygen tension and cavernosal fibrosis has been clearly documented in several studies (Moreland et al, 1995; Azadzoi et al, 1996; Moreland, 1998). TGF-ß₁ increases collagen synthesis in human corpus cavernosal smooth muscle cells in culture, and is induced by hypoxia (Moreland et al, 1995). The production of prostaglandin E₁, (PGE₁), which suppresses the induction of collagen synthesis by TGF-ß₁ in human corpus cavernosum smooth muscle, is also oxygen-dependent (Daley et al, 1996). Moreland (1998) observed that hypoxia can induce TGF-ß₁ expression and inhibit PGE synthesis (Moreland, 1998). The PO₂ level in his study was based on measurements of intracavernous PO₂ during penile erection and flaccidity. During flaccidity, the penile blood PO₂ was 25 to 40 mm Hg, whereas during erection it measured 90–100 mm Hg (Kim et al, 1993).

It is customary for normal men to have 3 to 5 nocturnal erection episodes, each lasting from 30 to 45 minutes (1.5– 3 hours total) per night (Fischer et al, 1965). There is an increase in blood PO₂ in the corpus cavernosum during these erectile events, which allows for the synthesis of substances such as prostanoids and nitric oxide, the production of which is favored by a higher oxygen tension. In a cell culture study, one application of PGE₁ was sufficient to significantly suppress TGF-ß₁–induced collagen synthesis (Moreland et al, 1995).

To study the effects of nerve injury alone, neurotomy was performed by electrical cauterization of the cavernous nerves in order to assure that all rat penes had been denervated for an identical period of time. HIF-1 is a transcription factor that is expressed when mammalian cells are subjected to hypoxia. HIF-1 activates the transcription of genes encoding proteins that are important for maintaining oxygen hemostasis (Chiappe-Gutierrez et al, 1998). The results of this study demonstrate that protein expression and immunohistochemical staining of HIF-1 were significantly higher in the neurotomy group, confirming the theory that hypoxia of rat penes after iatrogenic nerve injury was induced by the loss of nocturnal erections. Unfortunately, in order to quantitate the absolute diminishment in nocturnal erections, a rigiscan-like device has not been developed for rats.

Protein and mRNA expression of TGF-ß₁, a cytokine recognized to induce fibrosis (Diegelmann, 1997), was significantly increased in the neurotomy group. These results are consistent with human corpus cavernosal cell culture studies that demonstrate that hypoxia increases the production of TGF-ß₁ (Moreland, 1998). Increased immunohistochemical staining for TGF-ß₁ and HIF-1 were recorded in the neurotomy group, which is consistent with both protein and mRNA expression.

Protein expression of collagens I and III was significantly higher in the neurotomy group, which is consistent with an increased expression of TGF-ß₁. Immunohistochemical staining for collagen III was more positive; however, collagen I could not be easily discriminated, most likely because of the large amount of collagen I fibers normally found in the rat corpus cavernosum. Histological results with Massons trichome stain demonstrated similar findings. This study showed that cavernous nerve neurectomy did not cause significant morphological or functional changes in the penile erectile tissue of rats (Martinez-Pineiro et al, 1995). Because of the relatively short period of nerve injury, we believe that the changes caused by the hypoxic conditions may be detected initially only at the molecular level. Longterm studies will likely reveal progressive cavernous fibrosis and erectile dysfunction.

	Conclusion

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The results of this study demonstrate an increase in TGF-ß₁, HIF-1, and collagen synthesis in cavernous neurotomized rats, which can be hypothesized to be induced by loss of nocturnal and normal erections. These findings may explain in part why recovery and response rates to therapies for ED in patients who have undergone nerve-sparing postradical prostatectomy are lower than they are in other kinds of ED. In theory, fibrosis may be down-regulated by the application of vasoactive modalities such as intracavernous PGE₁, which improves cavernosal blood flows and increases oxygenation to the corpus cavernosal smooth muscle cells (Daley et al, 1996).

Such interventions for prevention of cavernous fibrosis needs to be initiated early. Unfortunately, the precise time and frequency of these preventive treatments remain speculative. Future therapies may focus on noninvasive measures, such as topical PGE₁ application, to increase patient compliance. Topical PGE₁ is recognized to have a lower response rate in inducing penile rigidity (McVary et al, 1999). However, hypoxic prevention measures do not necessarily require a rigid erection to increase oxygenation and benefit the cavernous smooth muscle cells under such postoperative circumstances.

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