Wan-Li Hu¹, Li-Quan Hu¹, Jian Song², Shi-Wen Li¹, Xin-Min Zheng¹, Bei Cheng², Bing-Chun Tian¹

Keywords: cavernous nerve; transforming growth factor-b1; penis; fibrosis; collagen fiber; smooth muscle

Abstract

Aim: To investigate alterations of smooth muscle cells and collagen fibers in corpus cavernosum following cavernous neurectomy and its relation to the expression of transforming growth factor-₁ (TGF-₁). Methods: Ten adult male SD rats (neurectomy group) were subject to a bilateral cavernous nerve (CN) resection aseptically under an operating microscope, with 6 sham-operated rats as the control. Fifteen weeks after the operation, the penile specimens were collected and prepared for quantitative-analyzing of ratio of smooth muscle to collagen fibers in corpus cavernosum with confocal microscopy, and for detecting the expression of TGF-₁ by RT-PCR and western-blot. Results: Smooth muscle cells that show red color after fluorescent-labeling with tetramethylrhodamine isothiocyanate-phalloidin and collagen fibers that produce green autofluorescence after paraformaldehyde fixation were clearly identified under the confocal microscope. Quantification of fluorescent intensity showed that the ratio of smooth muscle to collagen fibers in corpus cavernosum in neurectomy group was 0.2650.125, which was significantly lower than that in sham-operated group (0.7600.196, P<0.01). RT-PCR and western-blot analyses revealed a significantly higher expression of TGF-₁ in the penile tissues of the neurectomy animals than that in sham-operated group. Conclusion: Bilateral ablation of CN can lead to fibrosis of corpus cavernosum, which may be related to an increased expression of TGF-₁ induced by hypoxia in cavernous tissue after denervation.

1 Introduction

In the present study, bilateral cavernous neurectomy rat models mimicking non-nerve-sparing prostatectomy were created and the relationship between the expression of transforming growth factor-₁ (TGF-₁) and the cavernosal fibrosis in penile tissue after denervation was investigated.

2 Materials and methods

In order to guarantee the complete ablation of the CNs, the apomorphine test was conducted 15 weeks after surgery. Apomorphine (Sigma, USA) was dissolved in 0.5 % ascorbic acid in saline, and was injected sc on the back at a dosage of 100 g/kg and the animal was observed carefully for 30 min. Penile erection would appear if the nerve was intact. After that, animals were sacrificed and the penile shafts were collected for the following tests.

The middle part of the penile shaft was immediately embedded with OCT embedding medium, quickly frozen in liquid nitrogen and sectioned (15 - 20 m) with a cryomicrotome. The frozen sections were exposed in room temperature for about 15 minutes, fixed in 4 % paraformaldehyde in 0.1 mol/L PBS (pH 7.4) for 6 - 8 hours for collagen fiber autofluorescence. After washing with PBS, sections were stained with tetramethyl-rhodamine isothiocyanate-phalloidin (1:4000 dilution in 0.1 mol/L PBS, pH 7.4, P5285, Sigma, USA) at room temperature for 30~60 min for actin filament labeling. The stained sections were scanned under the TCS-SP2 MP confocal system (Leica, Germany). Smooth muscles and collagen fibers were inspected at 543 nm and 488 nm, respectively. Quantitative analysis was made on the pictures with Scion Image 4.02 software. Ten consecutive pictures of each section were analyzed and the mean was taken as the result. Under the confocal microscope, smooth muscles combined with tetramethylrhodamine isothiocyanate-phalloidin produce red fluorescence, while collagen fibers produce spontaneous green fluorescence.

The proximal part of the penile shaft was minced into small pieces and then crushed into powder after frozen hard in liquid nitrogen. Total RNAs were isolated by the Trizol (Cat. No. 15596-026; Invitrogen, USA) method according to the specification and quantified by spectrophotometry at 260 nm. The RNA (2 g) were reverse-transcribed into cDNA in the reaction system: template RNA (the volume determined by its concentration), 10RT buffer 2 L, dNTP 2 L (10 mmol/L each), AMV reverse transcriptase 1 L (10U), RNasin 1 L (50U), Oligo (dT) primer 1 L (0.1 g/L) and ddH₂O (adjust total volume to 18 L). The RT mixture was incubated sequentially in a thermal cycler at 37 for 1 hour and 95 for 10 min. The subsequent PCR reaction was conducted in the system: cDNA template (half of the above reaction product including dNTP) 9 L, 10PCR buffer 5 L, 25 mmol/L MgCl₂ 3 L, Taq DNA polymerase 1 L (2.5 U), sense and anti-sense primer of TGF-₁ cDNA each 10 pmol/L (1 L), ddH₂O 30 L; The PCR mixture was incubated sequentially in the same thermal cycler at 94 for 5min (pre-denaturation), followed by 35 cycles' reaction (94 for 1min, 50 for 1min, 72 for 1min ) and final extension at 72 for 5 min. Take -actin as the inner control (the annealing temperature was 47 ). Equal amounts (10 L) of PCR products from each animals were analyzed on a 1.5 % agarose gel stained with ethidium bromide (EB) and photographed under UV. Primers were designed with Vector NIT 5.2 software according to the rat sequences of TGF-₁ and -actin cDNA obtained from Medline. When designing the primers, we ensured the sense and antisense primers were not located in the same exon. Each of the primers is described in Table 1.

The distal part of the penile shaft was crushed into powder as above, lysed in a lysing buffer (1mmol/L EDTA, 10 mmol/L Hepes, 50 mmol/L NaCl, 0.05 % 2-mercap-toethanol, 0.5 % Triton X-100, 0.1 mmol/L PMSF and 10 g/mL aprotinin) and centrifuged at 15 000 g for 15 min. The total protein concentration in the supernatant was measured by spectrophotometry. Equal amounts of protein were electrophoresed on 15 % SDS-Page and transferred onto a nitrocellulose membrane. The membrane was blocked in 5 % nonfat dry milk in Tris-HCl buffer saline containing Tween-20 (TBS-T) at room temperature for 1 hour and then incubated with a rabbit anti-rat TGF-₁ polyclonal antibody (1:1000, sc-146, Santa Cruz, USA) at 37 for 1 hour, followed by a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:1000 dilution in Tris-buffered saline containing Tween-20, ZDR-5401, Zhongshan Co., China) at 37 for 1 hour. The blots were visualized with diamino-benzidine and the integrated gray intensity of reactive bands was analyzed with Scion Image 4.02 software.

3 Results

Total RNA extracted from the penile tissues of rats was analyzed by RT-PCR to investigate the mRNA expression of TGF- ₁. Figure 2A shows the specifically amplified products of 526 bp (TGF-₁) and 198 bp (b-actin) fragments after gel electrophoresis. The ratio of band density of the TGF- ₁ to -actin was significantly higher (P<0.01) in the neurectomy than that in the sham-operated group (Figure 2B).

Figure 2. RT-PCR analysis for TGF-₁ mRNA expressions. A: Products of RT-PCR amplification for TGF-₁ (526 bp) and -actin (198 bp) in sham-operated (S) and neruectomy (N) groups. B: Analysis for band integrated optical density. The bars represent meansSE of the ratio of TGF-₁ mRNA to -actin mRNA. ^cP<0.01, compared with sham-operated group.

The expression of TGF-₁ protein in penile tissues was detected by Western blot and compared by band gray intensity analysis. As shown in Figure 3A, the bands representing TGF-₁ (15 kDa) were specifically reacted with the anti-TGF-₁. The expression level of TGF-₁ in the neurectomy group was nearly double of that in the sham-operated group (Figure 3B).

Figure 3. Western blot analysis demonstrating expression of TGF-₁ protein. A: Protein products of predicted size (15 kDa) were clearly seen on the membrane (M: marker S: sham-operated N: neurectomy). B: The bars represent meansSE of optical density of the bands. Expression of TGF-₁ protein was much stronger in neurectomy than that in sham-operated group. ^cP<0.01, compared with sham-operated group.

Although the high rate of ED following radical prostatectomy is well recognized, the aetiology and pathophysiology have not yet been fully explored. Recent data on the use of intracavernous injections postoperatively support the hypothesis that a prolonged interval characterized by the absence of frequent and rigid erections during the period of postoperative neurapraxia may be associated with cavernous hypoxia, fibrosis and erectile dysfunction [7]. In the present study, the facts that an increase in cavernosal collagen fibers, a decrease in smooth muscle cells and a high expression of TGF-₁ after penile denervation could indicate that long continued inability to erection after cavernous neurectomy may lead to fibrotic alterations in corpus cavernosum, which may have a close relationship to overexpression of TGF-₁.

TGF-₁ is a cytokine involved in numerous vital processes, including inflammation, stimulation of intercellular matrix formation, production of fibroblast and normal healing [8]. It has been indicated that TGF-₁ is up-regulated in ischemic conditions in different organs. In patients with supraceliac aortic clamping, the resultant splanchnic ischemia was associated with a significant elevation of TGF- [9]. Other studies have reported the relationship between hypoxia and cavernosal fibrosis. Moreland et al [10] reported that TGF-₁, which was induced by hypoxia, increased the collagen synthesis in human corpus cavernosal smooth muscle cells in culture and that prostaglandin E1 ( PGE1) suppressed the induction of collagen synthesis by TGF-₁ in human corpus cavernosum smooth muscle. Moreland et al [11] further claimed that hypoxia might induce the expression of TGF-₁ and inhibit the synthesis of PGE1 in penile tissue. Therefore, in the current study, high expression of TGF-₁ suggests that ischemia and hypoxia may have occurred in penile tissue of rats after denervation.

Recently, Leungwattanakij et al [13] reported an increase in TGF-₁, HIF-₁ and collagen III expression in rat cavernosal smooth muscle after cavernous neurotomy. They hypothesized that these alterations might be induced by loss of nocturnal and normal erections. But they did not find any morphological changes in rat penile tissue, which is not consistent with the present result. The following points may contribute to the disparity: 1) the rats were housed for 15 weeks in our experiment which is much longer than 3 months in Leungwattanakij's study; 2) the methods to study the smooth muscles and collagen fibers were different in the two studies. Perhaps, in the observation of smooth muscles and collagen fibers, using confocal microscopy to detect fluorescence is more sensitive than the Masson trichrome staining method.

It is well known that normal content of smooth muscle cells and collagen fibers in corpus cavernosum is very important for erectile function. The pathogenesis of some kinds of ED is correlated with collagen fibers and smooth muscle fibers alteration. Shen et al [14] and Shen et al [15] reported a higher collagen fiber content in the penile tissue of old man and castrated rats. El-Sakka et al [16] demonstrated an increased collagen fiber content with an overexpression of TGF-₁ in the penile tissue of a traumatic arteriogenic ED rat model. In the present study, we also found similar alterations in rats after denervation. These facts suggest that ischemia-induced fibrosis might be a common pathophysilogical process of most types of ED.

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Correspondence to: Prof. Li-Quan Hu, Research Center of Urology and Andrology, Wuhan University Zhongnan Hospital, Wuhan 430071, China.
Tel: +86-27-6781 3104, Fax: +86-27-8733 0803
E-mail: 92011552730@sina.com
Received 2003-09-01 Accepted 2004-03-16