Effects of extra-corporeal shock waves on penile hemodynamics and histopathology in rats

Ahmet Tefekli ¹, Abdullah Armagan¹, Bulent Erol¹, Murat Celtik¹, Isi Kilicaslan ², Asiye Nurten ³, Ates Kadioglu¹

¹Department of Urology, ²Department of Pathology, Medical Faculty of Istanbul, ³Department of Neuroscience, Experimental Research Institution, University of Istanbul, Turkey

Asian J Androl 2002 Dec; 4: 249-253             

Keywords: penis; extra-corporeal shock wave; Peyronie's disease; tunica albuginea

Abstract

Aim: To study the effect of extra-corporeal shock wave (ESW) on the penile hemodynamics and histopathology in rats. Methods: Adult male Sprague-Dawley rats were divided at random into 3 groups. ESW application was performed with a Siemens Lithostar with the rats under anesthesia lying prone on the balloon probe. Rats in Group I received a total of 1000 shocks at 18 kV and immediately underwent hemodynamic evaluation performed by direct electrostimulation of the cavernous nerve and measurement of intracavernous pressure (ICP). Rats in Group II received 3 times 1000 shocks at 18 kV at weekly intervals and hemodynamic evaluation was performed 1 month after the last ESW application. Group III served as the control. Histopathological examinations of penile tissues were done on Masson's trichrome and hematoxylin and eosin stained sections. Results: Penile hemodynamic evaluation showed a trend toward a diminished mean maximal ICP, duration of erection, ICP during the plateau phase and maximal ICP/ blood pressure ratio in Group I, although there was no significant significance. The mean latency period in Groups I and II was prolonged. Petechial bleeding within tunical layers and small foci of hemorrhage within the corpora cavernosa were observed in Group I. However, histopathological examination failed to reveal any significant differences between the groups in terms of smooth muscle content, tunical thickness, organization of collagen bundles and elastic fiber-lattice framework. Conclusion: ESW has certain damaging effects on the penis.

Since its introduction in 1980, extra-corporeal shock wave (ESW) lithotripsy has quickly evolved as the treatment of choice for urinary tract stones [1]. Its stone fragmentation activity has also led investigators to extent the application in the treatment of a variety of diseases with pathological calcification, such as atherosclerotic plaques, bone corns, osteogenetic alterations and stones in pancreas and parotid [2, 3]. Recently, ESW therapy has been proposed to treat Peyronie's disease, and more or less favorable results in small groups of patients have been reported [4-7].

Peyronie's disease is a localized connective tissue disorder of penis and primarily affects the tunica albuginea of the corpus cavernosum and its adjacent erectile tissue [8]. During the initial phase of the disease, the lesions on the tunica albuginea show inflammatory infiltrate, while those of long duration exhibit hyalinization and alteration of collagen fiber arrangement, fibrosis, calcification, and sometimes ossification [9, 10]. Clinical hallmarks of Peyronie's disease include pain on erection, penile deformity and plaque formation on the penis. ESW treatment is proposed to disintegrate the Peyronie's plaques and improve the penile deformity [4-7, 10]. However, the efficacy of this treatment alternative has not been yet supported by pre-clinical and experimental studies and long-term results are awaited. Furthermore, since ESW treatment itself may cause inflammation and fibrosis, its therapeutic action in Peyronie's disease may be frequently associated with microtrauma[11]. Thus, the aim of this experimental study is to answer whether penile hemodynamics and histopathology are affected by ESW.

2 Materials and methods

2.1 Animals

A total of 20 male Sprague-Dawley rats, weighing 32020 g, were used in this study. They were divided randomly into 3 groups receiving different doses of ESW.

2.2 ESW procedure

ESW application was performed with a Siemens Lithostar Plus (Siemens Medical Systems, Iselin, NJ) with rats lying prone on the balloon probe under pentobarbital sodium (i.p. 35 mg/kg) anesthesia. The focal point of ESW probe was the penis. Animals in Group I (n=6) received a total of 1000 shocks at 18 kV and immediately underwent penile hemodynamic evaluation by direct electrical stimulation of cavernous nerve. Rats in Group II (n=7) received 1000 shocks at 18 kV for 3 times at weekly intervals and their hemodynamic evaluation were performed 1 month after the last ESW treatment. Group III (n=7) served as the controls and did not receive ESW.

2.3 Penile hemodynamics

Assessment of penile hemodynamics was performed by direct electrical stimulation of the cavernous nerve and measurement of intracavernous pressure (ICP) as described elsewhere [12]. Anesthesia was maintained during the course of experimental protocol (2~3 h) by subsequent intraperitoneal injections of pentobarbital sodium (5 mg/kg~10 mg/kg) every 45~60 minutes as required. The animals were placed in supine position and the bladder and prostate were exposed through a midline abdominal incision. The major pelvic ganglia, pelvic nerves and the cavernous nerve were identified posterolateral to the prostate on both sides. The penis was denuded of skin and both corpora cavernosa were exposed. To monitor the ICP, a 23-gauge cannula filled with 250 U/mL of heparin solution and connected to a PE-50 tubing (Intramedic, Becton-Dickonson, USA), was inserted into the left corpus cavernosum. The carotid artery was also exposed and systemic arterial blood pressure (BP) was monitored via a 25-Gauga cannula. Both pressure lines were connected to pressure transducers (Grass Model, PT 300, Grass Instrument Company, USA) connected to a Grass polymetrograph (Grass model 7400, Grass Instrument Division, Astro-Med Inc., USA). The pressure transducers were calibrated in cmH₂O before each experiment.

Direct electrical stimulation of the cavernous nerve was performed with a delicate stainless steel bipolar hook electrode, 0.2 mm in diameter and the 2 poles were separated by 1 mm. Monographic rectangular pulses were delivered by a signal generator (custom-made and with a built-in constant-current amplifier; Dr. Curtis Gleason, University of California, San Francisco, USA). The stimulation frequency was 20 Hz, the pulse width 0.22 ms and the duration 1 min. The application of a 10 mA current was needed to achieve a significant and consistent erectile response.

2.4 Histopathological examination

After electrical stimulation, the animal was sacrificed and the penis immediately transferred to the Holland's solution. After fixation, tissues were processed for paraffin embedding. Five mm tissue sections were obtained, deparafinized, hydrated with distilled water and stained with Masson's trichrome and hematoxylin and eosin as previously described [13]. The thickness of the tunica albuginea, the presence of fibrosis and organization of collagen and elastic fibers were assessed.

2.5 Statistical analysis

The hemodynamic data were expressed meanSD. The Student's t-test was used to compare the mean penile hemodynamic parameters and P<0.05 was considered significant.

3 Results

3.1 Penile hemodynamics

Baseline ICP measured before electrical stimulation ranged between 8 to 20 cmH₂O and the mean values were similar among groups (P>0.05, Table 1, Figure 1). Following electrical stimulation of the cavernous nerve, an increase in ICP was noted to occur within 5 to 20 seconds and the mean latency period was slightly shorter in the control group (P>0.05, Table 1). ICP increased about 6 to 8-fold in each group during erection.

Table 1. Hemodynamic findings (mean SD). ICP: Intracorporeal pressue, BP: Blood pressure.

Figure 1. Histopathological sections of penile tissue (Masson's trichrome stain; 32) obtained; a) Immediately after one EWS application (Hemorrhage shown with blue arrow), b) 1 month after 3 ESW applications, c) Controls. Note: Penile tissue sections do not differ in terms of tunical thickness (white arrow) and none has any sign of fibrosis.

The mean maximal ICP was 108.1 26.3 cmH₂O in the control group, 110.5 40.5 cmH₂O in Group II and 94.136.7 cmH₂O in Group I (Table 1). The mean ICP during the plateau phase of electrically induced erection was slightly lower in ESW treated rats compared to the controls (P>0.05, Table 1). The duration of erection ranged between 38 to 90 (mean: 68.918.5) seconds in Group II, 32 to 90 (63.620.1) seconds in the controls, and was reduced to 43~72 (57.810.6) seconds in Group I.

The BP measured during electrical stimulation ranged between 73 to 140 cmH₂O in all rats and the mean values were not significantly different between groups. The mean maximal ICP/BP ratio was also reduced in Group I (Table 1).

3.2 Naked eye observation

Multiple sites of petechial hemorrhage were observed within the tunical layers of penis of all the rats in Group I immediately after ESW application (Figure 1). Further-more, rectal bleeding was observed in 2 rats of this group.

3.3 Histopathological data

Histopathological examination of corpora cavernosa and the surrounding tunica did not reveal any significant differences between groups in terms of tunical thickness, organization of collagen bundles and elastic fiber-lattice framework (Figure 1). Small foci of hemorrhage with red blood cell extravasation were observed in the corpora cavernosa in the majority of rats in Group I.

4 Discussion

In Peyronie's disease, the normal arrangement of collagen bundles and elastic fibers is lost [8, 9, 14]. Repetetive microtrauma to erect or flaccid penis, mechanical stress, autoimmunity, inheritance, chromosomal abnormalities, sexually transmitted diseases and various medications have all been blamed to be responsible of this connective tissue disorder of penis [10]. Histopathological studies have shown that the elastic fiber content of the tunica albuginea in Peyronie's disease is reduced and often fragmented, leading to decreased compliance[8, 9, 14]. Collagen fibers of the tunica are densely packed and nodule-like clumps of excessive collagen deposits circumscribed by a ring of elastic fibers are observed [9, 10, 14]. This packed configuration constrains the fibers and limits their movement, thus creating a focal area of veno-occlusive dysfunction, as well as fibrous plaque formation and thickening of the tunica albuginea [10, 14]. Recent studies, both in rats and humans, have clearly demonstrated that TGF-b plays a major role in the pathogenesis of fibrosis and Peyronie's disease [15].

In general, medical treatment modalities, such as intralesional verapamil, oral colchicine, can be initiated in the acute phase of Peyronie's disease, while surgical treatment is deserved for those with stable penile deformity and disabling sexual intercourse [10]. Recently, ESW has been introduced for the treatment of Peyronie's disease and an improvement in pain (41.6 % to 74.9 %), penile deviation (31 % to 72.5 %) and sexual intercourse (37 % to 74.9 %) [4-7]. The mechanical plaque disintegrating activity of ESW is proposed to be the major mechanism of the observed effects. An increase in the microcirculation around the plaque with consecutive resorption of fibrotic tissue and calcification has been suggested as another action mechanism of this treatment. In addition, increased vascularization around the plaque may lead to inflammatory reaction, resulting in lysis of the plaque and removal by phagocytosis. Quick stabilization of plaque metabolism and a change in the milieu of the free radicals or direct interference in the pain receptor are assumed to relieve the pain.

However, in a recent case-controlled clinical trail on ESW treatment of Peyronie's disease, Hauck et al [7] could not demonstrate any significant effect on the plaque size, calcification, pain, quality of life and general subjective improvement; they suggested that the decrease in curvature following ESW treatment could be the natural course of the disease. In our study the hemodynamic changes observed during electrical stimulation of cavernous nerve immediately after ESW might be explained by the hypothesis that shock waves may temporary diminish the neural transduction, the neurotransmitter transport, the corporeal smooth muscle relaxation or the intracellular communication in the erection pathway. However, these postulates remain to be proved by further studies.

ESW has been accepted as an effective noninvasive treatment of urinary tract stones. However, the popularity of ESW is based on the false perception that this technology is entirely safe and free of adverse effects [1]. It has been shown that ESW induces acute and chronic changes in the kidney and the acute ESW-induced changes in renal morphology and function resemble those after blunt trauma [1]. Animal studies demonstrated that blood vessels were particularly susceptible to shock-wave injury, resulting in focal and diffuse hemorrhage [16]. Direct delivery of shock waves to skeletal muscles can induce irreversible changes that lead to degeneration of myofibers [17]. In addition to vascular damage, shock waves also injures the parenchymal cells of the targeted organ [16]. Nerve function may also be diminished by shock waves, possibly due to cavitation activity of the wave [18]. The penis, as an organ composed of blood vessels, smooth muscle and nerves is theoretically vulnerable to these bioeffects of ESW. Vascular damage induced by ESW includes disruption of the vessel wall, particularly the endothelium, with extravasation of blood cells into the surrounding tissue space and clot formation at the site of the rupture [19]. Electronmicroscopy shows distinct sites of endothelium rupture passing completely through the cell and its underlying basement membrane [20]. Permanent morphological changes in the kidney after a long ESW treatment consist of interstitial fibrosis, focal calcification, nephron loss, dilated veins, hyalinization to acellular scars running from the cortex to the medulla, glomerular hyalinization and sclerosis, interstitial inflammation and edema, large distensive hematomas, rupture and blockage of veins and arteries, and breakage of glomerular and peritubular capillaries [16]. As Peyronie's disease is a result of excessive fibrosis of the tunica albuginea in response to repetitive microtrauma, ESW treatment seems to aggravate, but not ameliorate the pathogenesis of the disease. Furthermore, it can be speculated that the improvement reported in penile curvature with ESW is not a consequence of plaque disintegration or scar tissue dissolution at the concave side of the curvature, but rather a result of new fibrotic reaction and scar formation at the opposite, convex side of the curvature.

The acoustic properties are also fundamental electromagnetic ESW action [11]. If the acoustic impedance is mismatched between two neighboring materials, wave reflection and transmission will occur, and fragmentation takes place. However, if the acoustic impedance is matched at the boundary of two dissimilar materials, the shock waves travel through the boundary without a significant loss of energy. In the treatment of Peyronie's disease with ESW, the acoustic impedance of scar tissue without calcification can be considered to match the surrounding tissue and thus the shock waves can be regarded to exit through the penis without any significant energy loss. Furthermore, if the calcified plaque tissue is regarded to be the site of shock wave reflection and transmission, than only those Peyronie's cases with calcified plaques would have been benefited from ESW treatment. However, recent studies with ESW failed to show any difference in the treatment outcome in cases with or without calcified plaques [4, 5, 7]. Only Husain et al mentioned that they achieved a higher success rate in cases in their chronic phase [6]. It must also be underlined that the whole tunica is effected in Peyronie's disease, and that a local treatment will not heal the entire pathology [8, 9]. In the rat model of Peyronie's disease, plaque and calcification in the tunica albuginea, observed after TGF-b injection, may result in a different acoustic impedence. Thus, further studies in the rat model of Peyronie's disease are needed to clarify the effects of ESW.

An other debate concerning the basic principals in the fragmentation and disintegration actions of ESW is its cavitation activity, which is defined as the formation of bubbles in a liquid [11]. Since Peyronie's plaques are not surrounded with a liquid media, the cavitation activity, which is a major action in fragmentation, can not take place in the penis. In addition, it is also obscure how the disintegrated plaque is cleared or how the dissolved fibrotic tissue is digested in the penis following ESW.

In our study, histopathological examination revealed only small foci of hemorrhage in the corpora cavernosa of ESW treated rats. This traumatic action of ESW was also evident by the small petechial bleeding within the tunical layers. However, we failed to demonstrate any significant chronic changes, such as fibrosis in the multiple session ESW treated rats. Further studies are needed to clarify the exact action of ESW treatment.

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Correspondence to: Ahmet Tefekli, M.D., Department of Urology, Medical Faculty of Istanbul, University of Istanbul, 34390-Capa, Istanbul, Turkey.

Tel: +90-212-663 34 57 Fax: + 90-212-532 1059

E-mail: atefekli@anet.net.tr

Received 2002-07-18    Accepted 2002-11-22