:: Asian Journal of Andrology ::

Evaluation of corporal fibrosis in cadaveric pericardium and vein grafts for tunica albuginea substitution in rats

Somboon Leungwattanakij¹, Vaewvadee Tiewthanom², Wayne J.G. Hellstrom³

¹Department of Urology, ²Department of Nursing, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand
³Department of Urology, Tulane University Health Sciences Center, New Orleans, Louisiana, USA

Asian J Androl 2003 Dec; 5: 295-299

Keywords: Peyronie's disease; pericardial graft; vein graft

Abstract

Aim: To evaluate the degree of corporal fibrosis in rats with cadaveric pericardium or vein as grafting materials for tunica albuginea substitution. Materials and methods: Thirty male Sprague-Dawley rats (300 g ~ 325 g) were divided at random into 3 groups of 10 animals each: group 1 was the sham-operated controls and groups 2 and 3 underwent wedge excision of tunica albuginea and replacement with cadaveric pericardium and vein grafts, respectively. Four months later, rats were sacrificed and the penis removed to assess the degree of fibrosis using RT-PCR technique for TGF-b1 mRNA expression. The tissues were fixed in 10 % formalin, paraffin-embedded and stained with Massons trichrome and Verhoff's van Giesen for collagen and elastic fibers. Results: Four months after grafting, there was minimal fibrosis surrounding the patch in the vein graft rats and moderate fibrosis in the pericardial graft rats. The degree of penile fibrosis in the pericardial graft rats was significantly higher than that in the controls (P<0.01), but in the vein graft rats it was not significantly different from that of the controls (P>0.05). Conclusion: The degree of penile fibrosis of cadaveric pericardial graft was significantly higher than that of the control group, while in the vein graft group it was comparable to the latter. The authors believe that the vein graft may be a more ideal substance to be used as the tunica albuginea substitute than the pericardial graft in the surgical treatment of Peyronie's disease.

1 Introduction

Peyronie's disease is the most common localized connective tissue disorder that primarily affects the tunica albuginea of the penile corpora. The disease affects approximately 0.39 % ~ 3.2 % of the adult male population, usually occurring in patients between 40 years ~ 70 years of age [1- 4]. The disease is characterized by the formation of an inelastic scar or plaque resulting in curvature of the penis secondary to shortening of the involved region of tunica albuginea. Patients with Peyronie's disease may complain of any one or a combination of penile curvature (50 % ~ 100 %), palpable penile plaques (78 % ~ 100 %), painful erection (20 % ~ 70 %), and diminished erectile ability (40 %) [4-7].

Chemically processed and gamma-irradiated human pericardium (Tutoplast^®, Biodynamics International, Parsippany, NJ) has been used as a tunica albuginea substitute in Peyronie's disease with satisfactory results [8]. Recently, in the study of human pericardium graft in rats, the intracavernous pressure observation was promising, but penile fibrosis was distinct as compared with the control rats and rats with vein grafts [9, 10]. The present study was designed to evaluate and compare the degree of corporal fibrosis using cadaveric pericardium and vein as grafting materials for tunica albuginea substitution in rats.

2 Materials and methods

2.1 Animals and treatment

Thirty male Sprague-Dawley rats (300 g ~ 325 g) were divided at random into 3 groups of 10 animals each. A 42 mm wedge of the right side of the tunica albuginea was removed via a circumcision in all the 3 groups and in groups 2 and 3, the defect was covered with the human cadaveric pericardium (Tutoplast^® ) or the vein grafts, respectively; group 1 served as the sham-operated control. Interrupted sutures of 8-zero nylon facilitated by a 2.5 operating microscope were used for fixation of the grafts. The operation was done under intraperitoneal sodium pentobarbital at a dose of 30 mg/kg (with additional dosing to maintain a uniform level of anesthesia). Antibiotics were fed orally for 5 postoperative days. The vein graft was harvested from the right femoral vein of the same animal.

2.2 Penectomy

Four months later rats were anesthetized and placed on a thermo-regulated surgical table. The trachea was cannulated (PE 240 polyethylene tubing) to maintain a patent airway to the room air. The skin overlying the penis was incised and only the graft replacement part of all rat penises were cut and saved.

2.3 Masson's trichrome and Hart's stains

One-half of the rat penis specimen underwent histological examination using Masson's trichrome and Hart's elastic staining. Penis was fixed in neutral buffered formalin and processed for paraffin embedding. Sections of 5 mm were applied to charged slides, deparafinized and hydrated with distilled water. The sections were then stained with Masson's trichrome stain which determines the relative proportion of collagen to stromal smooth muscle as previously described [11, 12].

2.4 RNA isolation and RT-PCR

The degree of penile fibrosis was assessed using RT-PCR technique for TGF-1 mRNA expression. Total RNAs were isolated using approximately one-half of the penile tissue by the TriZol method. RNA concentrations were quantified by spectrophotometer at 260 nm. The total RNAs (2 mg) was reverse-transcribed using RT-PCR (GeneAmp PCR system, Model 2400 Perkin-Elmer, USA). The RT reaction was performed in a reaction mixture (reaction volume 20 mL) containing Moloney murine leukemia virus reverse transcriptase, 1mmol/L each of the dNTPs (dATP, dCTP, dGTP and dGTP), 20 U RNase inhibitor, oligo (dT) primer, 10?standard buffer and MgCl₂. The RT mix was incubated in a thermal cycler at 42 for 15 minutes, 99 for 5 minutes and 5 for 5 minutes sequentially. Subsequent polymerase chain reactions were carried out in the presence of 20 pM sense and antisense primer, 2.5 mL 25 mmol/L MgCl2, 0.4 mL Taq DNA polymerase, 1 mL of each dNTP, 2.5 mL 10buffer and 3 mL of template cDNA in a total volume of 25 mL. The primer sequences used for PCR amplification are shown in Table 1. Oligonucleotide primers for rat b-actin were used as a positive control in each sample. PCR was performed initially after the denaturation step at 95 (2min) 30 cycles for b-actin and 37 cycles for TGF-1. Each PCR cycle for b-actin consisted of denaturation at 94 (45 sec), annealing at 61 (30 sec) and extension at 72?C (30 sec). Each PCR cycle for TGF-1 consisted of denaturation at 94 (5 sec), annealing at 55 (5 sec) and extension at 72 (10 sec). The PCR products were analyzed on a 2.0 % agarose gel stained with ethidium bromide and finally visualized under UV light. The band density was measured by a densitometer (Biorad, USA) and computerized by Multi-Analyst software.

2.5 Statistics

The band densities of RT-PCR results were expressed as optical density (OD.)/mm2. All data were analyzed using an ANOVA test. P<0.05 was considered statistical significant.

3 Results

3.1 Histological observation

All animals survived the procedure without any adverse complications.

Four months after pericardial graft replacement, the penile tissue revealed moderate degrees of fibrosis surrounding the patch (Figure 1A and B) as compared to the controls (Figure 2A and B). The elastic fibers of the tunica albuginea around the graft material were oriented in only a circular direction (Figure 1A and B). The tunica albuginea of the control group demonstrated elastic fibers, which were oriented in two directions similar to the human tunica albuginea (Figure 2A and B).

Figure 1. Four months after pericardial grafting, elastic fibers of graft (PCD) were less than the control group and oriented only in circular direction with moderate degree of fibrosis (F) under graft. (A) Trichome stain, 40; (B) Hart stain, 40; TA = tunica albuginea; C = corpus cavernosum.

Figure 2. Tunica albuginea of control rat 4 months after sham operation showing many collagen bundles oriented in two directions: inner circular (*) and outer longitudinal (#) with abundance of elastic fibers similar to composition of human tunica tissue. (A) Trichome stain, 100; (B) Hart stain, 40.

At 4 months, the vein graft group revealed minimal degree of fibrosis surrounding the patch (Figure 3A, B). Mild foreign body reaction around the nylon suture material was present.

Figure 3. Four months after vein grafting, elastic fibers of graft (V) were less than control group; mild degree of fibrosis (F) under graft. (A) Trichome stain, 40; (B) Hart stain, 40; TA = tunica albuginea; C = corpus cavernosum; S = suture material.

3.2 RT-PCR analysis

The results of PCR amplification for TGF-1 are shown in Figure 4. For the positive controls, b-actin was included in this study. Amplification products at the predicted sizes were clearly detected (TGF-1: 278 bp and b-actin: 218 bp) (Figure 4). The band density of TGF-1/-actin ratio was significantly higher in the pericardial graft group than that in the controls (P<0.05), but not significantly different from that of the vein graft group (P= 0.162) (Figure 5). However, the band density of TGF-1/-actin ratio in the vein graft group was similar to the control (P=0.153).

Figure 4. PCR amplification for TGF-b1 (278 bp) and b-actin (218 bp) in 3 groups. Densitometer reading of -actin was similar in all groups.

Figure 5. Optical density ratio (OD) of TGF-1/-actin was significantly higher in pericardial graft group comparing with control group (^cP<0.01). OD between pericardial and vein graft groups (P=0.162) and between control and vein graft groups (P=0.153) were similar.

4 Discussion

Surgical therapy is indicated in Peyronie's disease patients who fail to improve with medical or intralesional therapies and who have significant curvature and/or loss of penile rigidity that impairs sexual function. The surgical procedures can be divided into three categories: (1) plication or wedge excision to correct the curvature [5,13,14]; (2) plaque excision with the use of an autograft (e.g., dermis, dura mater, tunica vaginalis, vein, temporalis fascia, penile crura, etc.) [15-19] or an inert substance (e.g., Dacron mesh and Gore-tex) [20-22] and (3) prosthesis placement with or without excision or incision of the plaque and grafting.

Several investigators have proposed the use of autologous and non-autologous graft materials for the surgical treatment of Peyronie's disease. However, there has been only one graft material, vein graft in the rat model, that has any scientific data to validate its use in human cases. We demonstrated that vein graft was an advisable substance to be used as a tunica albuginea substitute in the surgical treatment of Peyronie's disease, because of the minimal fibrosis around the graft [10]. However, the disadvantages of vein graft and other autologous grafts include the necessity for a second incision for pre-grafting preparation, postoperative contracture and scarring and the meticulous preparation necessary to remove fat and other connective tissues. Moreover, the size of vein graft is insufficient for graft replacement in some cases [23]. Therefore, many investigators have been looking for allografts or xenografts, which have low tissue reactions as autografts.

In this study, we investigated the mRNA expression of TGF-1 as a parameter for comparing the degree of penile fibrosis. The penile section was obtained from the mid penile shaft where the patch graft had been placed. Four months after grafting, the band density of TGF-1/-actin ratio was significantly higher in the pericardial graft group than in the controls, while that of the vein graft group was similar to the controls, both being consistent with the histology study.

In conclusion, the degree of penile fibrosis of cadaveric pericardial graft was significantly higher than that of the control group, while in the vein graft group it was comparable to the latter. It may be reasonable to attribute this fibrosis to differences in antigenicity between the human pericardial graft and the rat tunica albuginea.

References

[1] Carson CC. Francois Gigot de la Peyronie (1678-1747). Invest Urol 1981; 19: 62-3.
[2] Levine LA. Peyronie's disease: a difficult sexual dysfunction problem. West J Med 1998; 169: 168-9.
[3] Gelbard MK, Dorey F, James K. The natural history of Peyronie's disease. J Urol 1990; 144: 1376-9.
[4] Schwarzer U, Klotz T, Braun M, Reifenrath B, Engelmann U. Prevalence of Peyronie's disease: results of an 8,000 men survey. J Urol 2000 (Suppl); 63: s742A.
[5] Nooter RI, Bosch JL, Schroder FH. Peyronie's disease and congenital penile curvature: long-term results of operative treatment with the plication procedure. Br J Urol 1994; 74: 497-500.
[6] Carson CC. Peyronie's disease: medical and surgical management. In: Hellstrom WJG, editor. The Handbook of Sexual Dysfunction. Lawrence, Kansas: Allen Press; 1999. p 93-8.
[7] Erdogru T, Savas M, Yilmaz N, Faruk Usta M, Koksal T, Ates M, et al. Evaluation of penile hemodynamic status and adjustment of treatment alternatives in Peyronie's disease. Asian J Androl 2002; 4: 187-90.
[8] Leungwattanakij S, Bivalacqua TJ, Reddy S, Hellstrom WJ. Long term follow-up on use of pericardial graft in the surgical management of Peyronie's disease. Int J Impot Res 2001; 13: 183-6.
[9] Leungwattanakij S, Bivalacqua TJ, Caulfield JJ, Hellstrom WJ. Evaluation of cadaveric pericardium in the rat for the surgical treatment of Peyronie's disease. Urology 2000; 56: 1075-80.
[10] Leungwattanakij S, Bivalacqua TJ, Yang DY, Hyun JS, Hellstrom WJG. Comparison of cadaveric pericardial, dermal, vein and synthetic grafts for tunica albuginea substitution using a rat model. BJU Int 2003; 92: 119-24.
[11] Hunter GC, Henderson AM, Westerband A, Kobayashi H, Suzuki F, Yan ZQ, et al. The contribution of inducible nitric oxide and cytomegalovirus to the stability of complex carotid plaque. J Vasc Surg 1999; 30: 36-49.
[12] El-Sakka AI, Hassan MU, Nunes L, Bhatnagar RS, Yen TS, Lue TF. Histological and ultrastructural alterations in an animal model of Peyronie's disease. Br J Urol 1998; 81: 445-52.
[13] Lamberger RJ, Bishop MC, Bates CP. Nesbit's operation for Peyronie's disease. Br J Urol 1984; 56: 721-3.
[14] Gholami SS, Goharderakhshan RZ, Lue TF. Correction of penile curvature using minimal tension paired plications and papaverine: a review of 84 patients. J Urol 2000 (Suppl); 163:s993A.
[15] Hicks CC, OBrien DP, Boswick J, Walton KN. Experience with the Horton-Devine dermal graft in the treatment of Peyronie's disease. J Urol 1978; 119: 504-6.
[16] Fallon B. Cadaveric dura mater graft for correction of penile curvature in Peyronie's disease. Urology 1990; 35: 127-9.
[17] Das S. Peyronie's disease: excision and auotgrafting with tunica vaginalis. J Urol 1980; 124: 818-9.
[18] Brock G, Nunes L, von Heyden B, Martinez-Pineiro L, Hsu GL, Lue TF. Can a venous patch graft be a substituted for the tunica albuginea of the penis? J Urol 1993; 150: 1306-9.
[19] Teloken C, Ros CD, Fornari A, Soares FC, Alegre CSP. Penile staightening in Peyronie's disease with crural graft of the corpus cavernosum. J Urol 2000 (Suppl); 163: s991A.
[20] Lowe DH, Ho PC, Parsons CL, Schmidt JD. Surgical treatment of Peyronie's disease with dacron graft. Urology 1982; 19: 609-10.
[21] Bazeed MA, Thuroff JW, Schmidt RA, Tanagho EA. New surgical procedure for management of Peyronie's disease. Urology 1983; 21: 501-4.
[22] Ganabathi K, Dmochowski R, Zimmern PE, Leach GE. Peyronie's disease: surgical treatment based on penile rigidity. J Urol 1995; 153: 662-6.
[23] Backhaus BO, Muller SC, Albers P. Corporoplasty for advanced Peyronie's disease using venous and/or dermis patch grafting: new surgical technique and long-term patient satisfaction. J Urol 2003; 169: 981-4.

Correspondence to: Somboon Leungwattanakij, M.D. Associate Professor of Urology, Department of Surgery, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand.
Tel: +66-2-201 1315, Fax: +66-2-201 1316
E-mail: ptuaja@hotmail.com
Received 2002-12-02 Accepted 2003-09-16