Introduction

Peyronie's disease (PD) is a kind of disease that is characterized by fibrosis in penile tunica albuginea (PTA) and/or corpus cavernosum, involving further growth of fibrous plaques, sometimes even calcified plaques, and causing penile pain, abnormal curvature, and erectile dysfunction (ED) [1]. The patient's sexual life is severely affected, which is likely to have a serious impact on men's physical and mental health [2]. Epidemiological investigation shows that the age of PD onset is mostly in the range 45-60 years and there are some reports about its onset even in adolescents. Statistical results indicate that the incidence is 3.2-8.9% [3].

The course of PD mainly includes the acute phase and the stable phase [4]. The major symptoms in the acute phase are penile pain, tunica albuginea plaques, and mild curvature and deformation of the penis [5]. Patients are usually afraid of involving themselves in sexual intercourse because of pain; as a consequence, psychogenic ED and mental and psychological depression are likely to set in gradually [6]. The major symptoms in the stable phase include penile curvature and deformation. The degree of curvature will not change over time and the stable tunica albuginea plaques eventually become calcified plaques [7]. A few of these patients undergo organic ED with great mental and psychological pressure at the same time [8].

Currently, the etiology of PD is not very clear. Studies have shown that obesity, smoking, diabetes, hypertension, and Dupuytren's contracture may be the risk factors for causing PD [9]. In the beginning, the direct reason for PD to occur was thought to be caused because of trauma to the penis [10]. Moreover, urological specialists have always seen a couple of patients with PD after trauma to the penis every year. However, the latest investigation showed that trauma to the penis was not associated with PD [11]. Currently, repeated micro-trauma of tunica albuginea is an accepted pathogenesis of PD, which can result in long-term inflammatory reaction, then promote the formation of fibrous plaques by increasing the expression of TGF-[beta], and lead to penile curvature and malformation due to declining the compliance of the tunica albuginea [12].

The PD animal model is an important method to reveal PD mechanism and search for effective therapies. Although predecessors have sought out many approaches of animal experiments, the results of those methods are quite different and they cannot fully simulate the PD pathological process. TGF-[beta]1 is a recognized growth factor at present, which can promote fibroblast activation and accelerate its collagen secretion [13]. Currently, a local tunica albuginea injection TGF-[beta]1 model in rats has been used a lot. Single injection of TGF-[beta]1 can only make the local tunica albuginea fibrosis, but rat's penis will not be curved [14]. Besides, another researcher has shown that repeat adenovirus expressing TGF-[beta]1 injections may lead to the formation of a curvature of the rat's penis [15]. Although TGF-[beta]1 can induce the PTA fibrosis, this kind of model is unable to study the PD attack process from trauma to increases of TGF-[beta]1. The pathogenesis of PD in this model is only limited to the TGF-[beta] pathway, which further reduces the research area. Therefore, this model poses a lot of inconvenience in the PD mechanism study. PD process is a long-term inflammatory reaction, which then develops to tunica albuginea fibrosis. Exogenous chemical stimulants (chlorhexidine alcohol, chlorhexidine ethanol, ChE) may stimulate tissues producing the inflammatory response. There are some studies that showed that repeat intraperitoneal injections of chlorhexidine alcohol could build peritoneal fibrosis model in rats [16-18]. Hence, we suppose that repeat local tunica albuginea injections by ChE can build an animal model that is even more in accordance with the PD pathophysiological process.

Materials and Methods

Study Design

Sixty-six Sprague-Dawley rats were obtained from the Animal Breeding Center at the Affiliated Drum Tower Hospital, School of Medicine, Nanjing University, Nanjing, China. The rats were divided into 7 groups, including 5 control subgroups (n = 6*5), group 2-6 (n = 6), and the TGF-[beta]1 group (n = 6). Each rat in the control group received injection of 0.9% isotonic saline (100 [micro]L) into the tunica albuginea and they were killed at the end of the last injection after day 60. Control subgroup 1 (saline 1x), injection at day 0; control subgroup 2, (saline 2x), injection at days 0 and 7; control subgroup 3, (saline 3x), injection at days 0, 7, and 14; control subgroup 4, (saline 4x), injection at days 0, 7, 14, and 21; control subgroup 5, (saline 5x), injection at days 0, 7, 14, 21, and 28. In order to create PTA fibrosis, 0.1% chlorhexidine gluconate plus 15% ethanol dissolved in saline (ChE) was premixed. Group 2: ChE (1x), a single injection of 100 [micro]L ChE and were killed at the end of day 60. Group 3: ChE (2x), twice injections of 100 pL ChE (days 0 and 7) and were killed at the end of day 67. Group 4: ChE (3x), triple injections of 100 [micro]L ChE (days 0, 7, and 14) and were killed at the end of day 74. Group 5: ChE (4x), 4 times injections of 100 [micro]L ChE (days 0, 7, 14, and 21) and were killed at the end of day 81. Group 6: ChE (5x), 5 times injections of 100 [micro]L ChE (days 0, 7, 14, 21, and 28) and were killed at the end of day 88. The rats in positive control group was injected by 100 [micro]L TGF-[beta]1 (10 ng/ mL; Sigma, USA) into PTA and killed at the end of day 60. The penile skin was incised and the tunica albuginea was exposed. And then saline, TGF-[beta]1, and ChE was injected into the tunica albuginea via a 30-gauge needle and the incision was closed. All animal experiments were approved by the Institutional Animal Care and Use Committee at the Nanjing University, Nanjing, China.

Erectile Function Assessment

The intracavernous pressure (ICP) was measured by the electric stimulation of the cavernous nerve as described previously [19]. An RM6042B/C multichannel signal-collection processing system (Chengdu Implement Company, Chengdu, China) was used to record the mean arterial pressure (MAP) and ICP. Erectile function was evaluated by the peak ICP/MAP ratio.

Penile Curvature Assessment

After the erectile function was assessed, photographs were taken with a digital camera (Powershot A590, Canon, Japan) during the artificial erection test with intracavernous injection of saline; the degree of penile curvature was then measured using a protractor by the same investigator to maintain consistency. Penile tissue was then harvested for histological analysis.

Histochemistry

All histochemical staining procedures were performed according to a previously described protocol [19]. Briefly, 4% formalin phosphate-buffered solution was used to fix the freshly dissected tissue. The specimens were stained with hematoxylin and eosin (H&E), Masson trichrome, which identifies the collagen as blue, and Hart's staining, which identifies the elastin fibers as black. For immunohistochemical staining, representative sections were incubated with 3% bovine serum albumin for 30 min at room temperature and then incubated rabbit anti-TGF-[beta]1 (1:200; Abcam Inc., Cambridge, MA, USA) antibodies at 4 [degrees] C overnight, followed by a biotinylated-conjugated secondary antibody (1:200; Boster, Wuhan, Hubei, China). Image-Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD, USA) was used in the analysis of the TGF-[beta]1 position area under the x200 power field and the concentration and length of the elastic fiber under the x400 power field. Six sections of each penile sample were analyzed and the average results were used for data analysis.

Immunofluorescence

For immunofluorescence staining, tissues and sections were prepared as previously described [19].Penile sections were incubated with mouse anti-alpha smooth muscle actin (a-SMA, 1:200; Abcam Inc., Cambridge, MA, USA) at 4 [degrees] C overnight, followed by fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Google Organisms, Wuhan, China, 1:500) for 1 h at room temperature. Then, the tissue sections were stained with Click-iT[TM] reaction cocktail (Invitrogen) and 4,6-diamidino-2-phenylindole (DAPI; Invitrogen). Fluorescent images were captured using a laser scanning confocal fluorescence microscope (Olympus, Williston, VT, USA).

Statistical Analysis

All data were presented and expressed as mean values [+ or -] SD. Statistical analysis was performed by the independent Student t test and one-way ANOVA using SPSS 16.0 and Graph Pad software. A value of p < 0.05 was considered statistically significant.

Results

Repeat Penile Albuginea Injuries of ChE Will Lead to

Penile Curvature

We have found that there were no significant changes in the shape of the rat's penis including the length and curvature at 60 days in all control subgroups (Fig. 1; online suppl. Tables 1, 2; for all online suppl. material, see www.karger.com/doi/10.1159/000475601). Moreover, the shape of the rat's penis did not significantly change 60 days after single injury of ChE via artificial erectile experiment induced by injecting saline into the corpus cavemosum compared with the control group (Fig. 1; online suppl. Tables 1, 2). However, it was found that the rat's penis was obviously curved after repeated injuries of ChE. Along with increased frequencies of injuries, the angle of curvature was enlarged and there was an increase in the incidence of curvature and deformation as shown in Figure 1. Above all, almost all rats eventually appear curvature and malformation of penis in 5x ChE group and the curved angles in this group is among 40[degrees] to 50[degrees]. In addition, we have found that the angle of penile curvature in rats of the ChE 5h group is increased compared to a single tunica albuginea injection of TGF-[beta]1.

Repeat PTA Injuries Will Lead to the Formation of Penile Fibrous Plaques

Rat's penile tissue structure is found almost fully recovered after 60 days in control groups and single damage of ChE group via H&E staining. Interestingly, the tunica albuginea and corpus cavemosum appear as an irreversible tissue structure disorder after repeat injuries of ChE (Fig. 2). By further assessing the impacts of damages on the rat's penile tissue structure, we have discovered that the rat's normal penile histological structure is replaced by massive collagen fibers after repeat injuries of ChE via Masson staining, thereby forming penile fibrous plaques (Fig. 3; online suppl. Table 3).

Impacts of Repeat Penile Albuginea Injuries on a Rat's Erectile Function

It is found that there were no significant differences of ICP/MAP value in control groups, the ChE 1c group, and the TGF-[beta]1 group by electrical stimulation of erectile nerves. ICP/MAP value is also not changed obviously after twice, 3 and 4 injuries of ChE. Otherwise, ICP/MAP value is slightly reduced only at the ChE 5x group compared to the control group (Fig. 4).

TGF-[beta]1 and [alpha]SMA Expression Are Increased in the Rat's Penis Tissue after Repeat PTA Injuries

To investigate the forming mechanism of fibrous plaques induced by repeat injuries, we have tested the TGF-[beta]1 (main function is to activate fibroblasts) and [alpha]SMA (an activated fibroblast markers) expression in the rat's penis tissue. There were no significant differences of [alpha]SMA and TGF-[beta]1 expression in all control subgroups (online suppl. Table 4). However, when compared to control groups, we have found that [alpha]SMA and TGF-[beta]1 expression among the tunica albuginea and corpus cavemosum significantly increase in the repeated injury of ChE groups (Fig. 5, 6; online suppl. Table 4).

The Length of Elastic Fibers Is Shortened after Repeat PTA Injuries

Elastic fibers breaking will lead to a decline of tissue compliance. Compared with the single ChE injury group, the maximum length of elastic fibers among the tunica albuginea and corpus cavemosum was significantly shorter in the ChE 5x group. It will be highlight that the expression of elastic fibers is compensatorily increased and the distribution of elastic fibers is more chaotic in ChE 5x group than in the single ChE injury group either in the tunica albuginea or corpus cavernosum (Fig. 7).

Discussion

PD is a kind of disease featured with PTA and/or corpus cavernosum fibrosis. The main causes of tissue fibrosis are repeat injuries and long-term inflammatory re sponse, such as liver fibrosis [20]. The most accepted mechanism of PD is repeatedly micro-injury of PTA during sexual intercourse. Building a reliable PD animal model is extremely important for intensively studying PD mechanism and searching therapies for PD. By comparing tunica albuginea single chemical minute damage and multiple chemical minute damages, this trial has found that after repeat injuries of ChE (1) local collagen deposition happened in the PTA, as well as fibrous plaques were formed; (2) penis was curved and deformed; (3) erectile function was not changed significantly and it slightly decreased only at the 5 times injury; and (4) the TGF-[beta]1 and [alpha]SMA levels were increased in the PTA and corpus cavernosum and the elastic fibers are broken and disordered, though the concentration of elastic fibers is compensatory increased. Therefore, we have found that repeated occurrence of micro-injury by repeatedly injecting ChE in the tunica albuginea can successfully build a PD animal model.

This trial has built a PD animal model for the first time via repeatedly administering local injections of ChE in the tunica albuginea, which preferably simulates the onset procedure, continuously repeat micro injuries, of PD. Rat has undergone apparent penile curvature; fibrous plaques were formed in the local tunica albuginea; there was slightly decreased erectile function after repeat injuries. Predecessors have completed many researches in PD animal models. Lue TF set up the first PD animal model via injection of Cytomodulin (a type of molecule with TGF-[beta]-like actions) in the PTA. Histochemistry showed that the tunica albuginea was thickened, the collagen was proliferated, and the elastic fibers were broken [21]. Nehra et al. [22] and Bivalacqua et al. [14] injected TGF-[beta]1 at local penile albuginea of rabbits and rats, respectively, and both of them discovered the above-mentioned phenomenon. In order to maintain a constant and stable TGF-[beta]1 expression, Ryu et al. [23] built the PD animal model via 3T3 cell transfecting TGF-[beta]1 and single tunica albuginea injection and they found out apparent fibrous plaques in the albuginea, decline of ICP/ MAP value, and mild ED in the molding group. An animal model built by Piao et al. [15] via adenovirus transfecting TGF-[beta]1 and repeat tunica albuginea injections not only showed the histological changes mentioned above, but also discovered that the mild penile curvature occurred only in the high-dose group and it disappeared in a short time. The current mainstream animal model is PTA injection of TGF-[beta]1; however, this type of model is unable to study the process of PD attack from micro-trauma to TGF-[beta]1 rise and it also cannot reproduce the penile curvature sufficiently, which will further greatly reduce the focus of the study. Somers and Dawson [24] revealed that the fibrin levels in the PD plaques were significantly higher than those of normal tunica albuginea tissue for the first time from cells and animal levels. Davila et al. [25] built another PD animal model in 2003 via the single tunica albuginea injection of fibrin. Fibrous plaques remarkably turned up in the PTA 3 weeks after molding, as well as collagen proliferation. And fibrin can constantly stimulate the production of TGF-[beta]1. Unfortunately, this model is also unable to bring about penile curvature and ED. Moreover, the early-stage preparations of fibrin are rather tedious. Although the anticoagulation is added in, fibrin is still very easy to be freezing. Hence, using a small needle to inject fibrin is extremely difficult [26]. Some researchers induced rats spontaneously forming PD using the gene mutation technology. Lucattelli et al. [27] observed that fibrillin-mutated C57B1/6J rats turned up PD-like fibrosis phenomenon in the penis 12 months after birth. However, because this model leads to extensive fibrosis disease in the whole body and also because molding time is too long, its extensive use is limited enormously. In addition, people try to set up PD animal models by inflicting trauma to tunica albuginea. El-Sakka et al. [28] in the 1990s, built a PD model by trying to cut off partially the rat's penile albuginea. This method only led to the formation of the acute inflammatory response and the tissue structure returned to normal after the acute phase. Acikgoz et al. [29] and Zargooshi et al. [30] also discovered that single trauma to penile albuginea would not lead to PD via prospective studies one after another. However, a latest meta-analysis has shown that penile fracture, a kind of penile trauma, could result in ED, penile plaques and curvature, though the incidence of these complications was only approximately 13% [31]. Ferrettil built a PD animal model in 2014 via allogenic albuginea transplantation [32]. This model not only had apparent penile induration and plaques; these plaques also turned up calcification or ossification. Rat's penis was significantly curved in the allogenic albuginea transplantation group compared to the autologous transplantation group and the angle of curvature was over 25[degrees]. Early erectile function did not change significantly, but the erectile function was declined after 12 weeks. However, it still has some limitation. The demands for tunica albuginea incision and transplantation operation are really high. The size and thickness of dissected tunica albuginea are hard to control.

This trial has found that TGF-[beta]1 expression is increased in local penis after repeat injuries and the increased TGF-[beta] may promote the activation of fibroblasts. Increases of [alpha]SMA are the same as our testing results, which mean a large amount of fibroblasts are activated. These activated fibroblasts secrete massive extracellular matrix and the main component is collagen.

Many studies suggest that the tissue will be restored to normal after a single or a mild injury, while repeat or severe injuries may lead to fibrosis [33, 34]. Platelets adhere to the damaged location and this is promoted by the clotting mechanism after a single or mild injury so as to activate platelets releasing various chemotactic factors (such as interleukin [IL]-1 and IL-6, etc.), thereby making inflammatory cells gather to the injured place [35, 36]. Subsequently, these inflammatory cells will release cytokines (such as TGF-[beta]1 and TNF, etc.) to activate fibroblasts and turn them into myofibroblasts. These myofibroblasts not only have features of fibroblasts but also play roles of partial smooth muscle cells. On the one hand, myofibroblasts can secrete collagen fibers and other ECM to repair injured parts; on the other hand, myofibroblasts can shrink wound and promote tissue repair [37]. Once external stimulus is removed, the inflammatory response will gradually fade away. ECM will be degraded under actions of matrix metalloproteinases (MMPs) [38]. Myofibroblasts turn up anoikis due to lack of ECM and regain the normal histological structure [39]. When having repeat or severe injuries, the inflammatory response remains for a longer duration and does not vanish, or the inflammatory response has not finished, but the external stimulation comes back again. Therefore, inflammatory cells are continuously activated. Those inflammatory cells release a large number of cytokines (such as TGF-[beta]1) and continue to activate fibroblasts, thereby increasing its transformed ability into myofibroblasts and greatly enhancing the function of secretion and contraction. When stopping external stimulation, MMPs may be unable to fully degrade massive ECM [40]. The integrin of ECM will activate TGF-[beta]1 and then activate fibroblasts again via the Smad-dependent approach and the Smadnon-dependent approach. Activated fibroblasts further secrete plenty of ECM resulting in fibrosis in the end [41]. The apoptosis of myofibroblasts is reduced. A lot of collagen deposition is turned up and the tissue structure changes resulting in hardly ever irreversible fibrosis. Currently, a majority of scholars believe that the main cause of PD is repeated micro-injuries during sexual intercourse. PD's pathophysiological manifestation shows repeated micro-injuries leading to local separation and evulsion of 2 layers of the PTA structure, which is closely connected. The body starts the process of inflammatory response and a large number of inflammatory cells enter into the injured area, thereby inducing pain in the PD acute phase; releasing multiple cytokines (such as TGF-[beta]1) by inflammatory cells; promoting tissue repair; inducing fibroblasts into myofibroblasts; and releasing abundant fibrin and collagen [42, 43]. Excessive fibrin and collagen are deposited locally, while most elastic fibers are destroyed or even broken. Morbid repair leads to penile induration and the final formation of plaques. Because the elasticity of tunica albuginea in the plaque area is far poorer than the normal PTA, the penis will be curved toward the lesion side clinically. The penile erection is a process of arterial hyperemia. Erectile veins of corpus cavernosum will be pressed by tunica albuginea and then closed. The elasticity of tunica albuginea under PD is not as good as the normal tunica albuginea, thereby leading to venous valvular inadequacy and ED [44].

Conclusion

This trial has built a PD animal model for the first time via repeatedly administering local injections of chlorhexidine alcohol in the tunica albuginea and continuously repeating small injuries, which preferably simulates the onset procedure of PD. Rats have suffered from apparent phallocampsis; fibrous plaques in the local tunica albuginea and/or corpus cavernosum; broken elastic fibers; slightly decreased erectile function; and increased TGF-[beta]1 and [alpha]SMA expression after repeat injuries. This model has provided a new impetus to further study the PD mechanism and investigate effective treatment methods.

Acknowledgments

The study was supported by the National Natural Science Foundation of China (No. 81170563 and 81270694).

Disclosure Statement

There are no conflicts of interest for all authors.

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Hesong Jiang Qingqiang Gao Xiaoyan Che Leilei Zhu Zheng Zhang Yun Chen Yutian Dai

Department of Andrology, Drum Tower Hospital, Affiliated to School of Medicine, Nanjing University, Nanjing, China

DOI: 10.1159/000475601

Received: December 16, 2016

Accepted after revision: April 8, 2017

Published online: November 18, 2017

Yutian Dai

Department of Andrology, Drum Tower Hospital

Affiliated to School of Medicine, Nanjing University

#321 Zhong Shan Road, Nanjing 210008 (China)

E-Mail 13913957628@163.com

Caption: Fig. 1. Photographs of rat penis after the artificial erectile experiment. a Normal control (saline 1x); b normal control (saline 5x); c single injection of ChE at day 60. d Two injections of ChE at day 67. e Three injections of ChE at day 74. f Four injections of ChE at day 81. g Five injections of ChE at day 88. h Single injection of TGF-[beta]1 at day 60. The illustration of rat penis was on right and the degree of penile curvature is shown as [alpha].

Caption: Fig. 2. Hematoxylin-Eosin staining of rat penis. a Normal control (saline 1x); b normal control (saline 5x); c single injection of ChE at day 60. d Two injections of ChE at day 67. e Three injections of ChE at day 74. f Four injections of ChE at day 81. g Five injections of ChE at day 88. h Single injection of TGF-[beta]1 at day 60. x100 magnification; the disordered structure is indicated by black arrows.

Caption: Fig. 3. Masson Trichrome staining of rat penis. a Normal control (saline 1x); b normal control (saline 3x); c normal control (saline 5x); d single injection of ChE at day 60. e Three injections of ChE at day 74. f Five injections of ChE at day 88. x20 magnification.

Caption: Fig. 4. Evaluation of erectile function. a Normal control (saline 1x); b normal control (saline 5x); c single injection of ChE at day 60. d Repeated (5) injections of ChE at day 88. e Single injection of TGF-[beta]1 at day 60. f The results of erectile function are expressed as the ICP/MAP ratio. * p < 0.05 compared with the normal control (saline 5x). ICP, intracavernous pressure; MAP, mean arterial pressure.

Caption: Fig. 5. Immunohistochemical staining for TGF-[beta]1 in the rat penis. a Normal control (saline 1x); b normal control (saline 5x); c single injection of ChE at day 60. d Three injections of ChE at day 74. e Five injections of ChE at day 88. f Single injection of TGF-[beta]1 at day 60. x200 magnification. g The statistical charts represent the expression of TGF-[beta]1. * p < 0.05 compared with the normal control (saline 5x); # p < 0.05 compared with the single injection group.

Caption: Fig. 6. Representative images of immunofluorescent staining for [alpha]SMA in the rat penis. Red fluorescence indicates [alpha]SMA and blue fluorescence represents the nucleus. x200 magnification. TA, tunica albuginea; CC, corpus cavernosum.

Caption: Fig. 7. Analysis of elastic fibers. a Hart's staining of the tunica albuginea and the corpus cavernosum. x400 magnification. Elastic fibers are designated by black arrows. b Statistical chart represents the concentration of elastic fibers. * p < 0.05 compared with the single damage group. c Statistical chart represents the maximum length of the elastic fibers. * p < 0.05 compared with the single damage group. TA, tunica albuginea; CC, corpus cavernosum.