Introduction

Priapism is a pathological condition of penile erection that persists beyond or is unrelated to sexual stimulation.¹ Priapism can be classified as high flow or low flow depending on the status of the penile arterial blood flow. Low flow (ischemic priapism) is more prevalent than high-flow priapism and is a true urologic emergency classically depicted by acidotic corporal sinusoids, penile venous outflow obstruction and patient pain.² If untreated, ischemic priapism can lead to corporal fibrosis and permanent erectile dysfunction.

One of the pathophysiological mechanisms of ischemia-induced fibrosis is hypoxia-induced overexpression of transforming growth factor beta 1 (TGF-₁).^{3, 4} TGF-₁ is a pleotrophic cytokine that has been shown to increase collagen synthesis in corpus cavernosum smooth muscle cells in vitro.^{5, 6} This cytokine is sufficient to initiate corpus cavernosum fibrosis and exogenous treatment with TGF- has been shown to induce 2.5–4.5-fold increase in the synthesis of type I and III collagen.⁶ Moreover, intracavernosal injection of TGF- histomorphometric analyses demonstrated dose-dependent decreases in the percentage of corporal smooth muscle with treatment.⁴ Ul-Hassan et al⁷ demonstrated increased expression of TGF-₁ as a possible mediator of fibrosis that may follow prolonged penile erection in a canine model. Thus, the authors mentioned that the use of neutralizing antibodies to TGF-₁ might be a possible tool for prevention of the fibrotic changes in priapism, which was previously successfully used in rodents to control scarring in wounds, inhibition of fibrous adhesions in abdominal surgery, reducing perineurial scarring in peripheral nerve injury and prevent subglottal stenosis.^{8, 9, 10, 11, 12} Eventually, the aim of this study is to evaluate the possible role of TGF-₁ antibodies for the prevention of fibrotic effects of priapism in a rat model. To our knowledge, this is the first study that investigates the role of TGF-₁ neutralizing antibodies in an ischemic priapism model.

Materials and methods

A total of 30 adult male Sprague–Dawley rats (250–300 gm) obtained from Institute for Experimental Medicine, Istanbul University were divided into five groups. 1—ischemic priapism with 6 h, 2—ischemic priapism with 6 h+anti-TGF-₁, 3—ischemic priapism with 24 h, 4—ischemic priapism with 24 h+anti-TGF-₁, 5—control.

Ischemic priapism was achieved with a vacuum erection device, which is a cone-tipped standard 50 cc irrigation syringe for Foley urethral catheters. The tip of the syringe was suited to the approximate diameter of the base of the flaccid penis. Before applying the vacuum device, a constriction band created from no: 5 T tube (mostly used in gall bladder surgery) was loaded around the tip. After placing the syringe to the base of the penis, the piston was gently withdrawn and suction was created which induced erection. When sufficient erection was achieved, the rubber constriction band was placed at the base of the penis by pulling from the syringe. Rubber band was left in place for 6 or 24 h according to the groups (Figure 1). During these periods, rats were observed frequently and they were excluded from the study if the band was replaced. The rats were spurted drops of urine while their penises were in full erection. During the procedure of achieving priapism, a temporarily anesthesia with ether was applied.

Figure 1.

Penile erection: visible straightening and grooming of the penis.

Full figure and legend (52K)

A total of 1 g anti-TGF-₁ (TGF-₁ (Ab-1) Cat#GF 33L, Oncogene research products) was administered intracavernosaly 1 h after the initiation of ischemic priapism in groups 2 and 4. One of the applications of this product is neutralization studies and has ability to neutralize the biological affects of TGF-₁. One half maximal neutralization occurs with 0.3–1.0 g/ml in the presence of 0.25 ng/ml of recombinant TGF-₁. This product was reconstituted in 1 ml sterile H₂O containing 100 g bovine serum albumin and 0.1% sodium azide, alloquated in smaller volumes and stored at -20°C. Upon reconstruction, this product is viable for 6 months at -20°C. After the administration of antibodies, again vacuum erection device was used for the distribution of the antibodies through the corpora as homogeneous as possible.

After 8 weeks, all rats underwent electrical stimulation of the cavernous nerve. They were anesthetized with pentobarbital sodium (50 mg/kg. i.p.). During the preparation of rats for cavernosal nerve stimulation, the penis was incised and the prepuce was degloved to fully expose both corpora cavernosa. A 26-gauge needle was inserted just proximal to the angulation of the penis, which was previously found as the best site for accurate and repeatable insertion of the recording needle.¹³ The technique for cavernosal stimulation was performed with the technique described in the literature by different authors.^{14, 15} Stimulation was performed with an electric generator (created by Dr Curtis Gleason, University of California, San Francisco) with a frequency of 20 Hz and 0.2 ms pulse duration for 1 min. During the procedure intracavernous and systemic blood pressures were recorded with polymetograph (Grass Model 7400, Grass Instrument Division, West Warnick, RI, USA).

Basal ICP (intracavernosal pressure), latent period for erection, duration of erection, duration of plateau, maximum ICP, mean ICP during plateau phase, max ICP/BP were the parameters evaluated during the electrical stimulation of cavernosal nerve in all groups. After obtaining the measurements with electrical stimulation, penile tissue was collected just prior to the killing of animals. Each rat penis was fixed in neutral-buffered formalin and processed for paraffin embedding. The sections of cavernosal tissue were stained with Masson's trichrome stain, which determines the relative proportion of collagen to stromal smooth muscle.¹⁶ Also, degree of fibrosis in different groups of priapism was evaluated with a computer-based programme (Analysis^®, Soft Imaging System, GMBH, Münster, Germany), which calculates the area of sinusoidal spaces in digital images of the tissue sections. For this purpose, a light microscope equipped with a digital CCD camera (Nikon CoolPix 5000) was used. The area of sinusoidal spaces was determined for five different fields (magnification 150) in tissue sections arbitrarily.

All data were presented as means.e. Differences between treatment groups were evaluated by ANOVA, followed by the Tukey's multiple comparison test for post hoc comparison of means against the control. Values are considered as significant at P<0.05.

Results

Pressure measurement studies

The hemodynamic parameters of the rats obtained with the cavernosal nerve stimulation worsened with the duration of priapism. The mean plateau intracavernosal pressures for group 1 and 3 were 33.310.2 and 29.710.6 cmH₂O, respectively, while it was 91.617.51 cmH₂O for the control group. Ratio of maximum intracavernosal pressure to systemic blood pressure was 0.220.05 for group 1 and 0.220.007 for group 3 and it was 0.640.17 for the control group.

Best results were found in groups 2 and 4 for the hemodynamic parameters of cavernosal nerve stimulation. Notably, hemodynamic values of group 2 were more closed to the control group. Briefly, the mean value for the plateau ICP in groups 1 and 2 were 33.310.6 and 62.526.9 cmH₂O, respectively, which was 29.710.6 and 38.714.9 cmH₂O for groups 3 and 4. Thus, as the most important parameter of the hemodynamic evaluation, ratio of ICP over BP (ICP/BP) was significantly better in groups 2 and 4 (0.480.26 vs 0.220.05) compared with groups 1 and 3 (0.330.05 vs 0.220.07) which means, anti-TGF-1-treated rats showed significantly higher ICP pressures to cavernous nerve stimulation and had higher ICP/BP ratios when compared to nontreated rats. This state was more significant when the antibody was administered within 6 h, since the value of max ICP/BP for group 2 and 4 was 0.480.26 and 0.330.05, respectively, while it was 0.640.17 for the control group. Hemodynamic parameters of the rats were summarized in Tables 1 and 2.

Table 1 - Hemodynamic parameters of cavernosal nerve stimulation according to anti-TGF-1-treated and nontreated rats.

Full table

Table 2 - Comparison of the groups for the hemodynamic parameters with each other.

Full table

Histopathologic changes

Histopathologic examination of the corpora under light microscopy demonstrated the obliteration of the sinusoidal spaces and replacement of smooth muscle cells with collagen in group 1. In group 2, fibrotic changes were strikingly less severe and sinusoidal spaces were open (Figures 2 and 3). In both groups 3 and 4, severe fibrosis was identified similar to group 1 but a few sinusoidal spaces were observed in group 4. On the other hand, the total area of sinusoidal spaces determined with the computer program revealed a significant difference between group 2 and the other groups. The total area of sinusoidal spaces in group 2 were closed to the control group (3161627 vs 32291227 m²). Briefly, the mean area of the sinusoidal spaces in groups 1 and 2 were 350153 and 3161627 m² (P<0.01), respectively, which was 338255 and 1332379 m² (P<0.05) for groups 3 and 4.

Figure 2.

Obliteration of sinusoidal spaces and replacement of smooth muscle cells with collagen (group 1— 150).

Full figure and legend (117K)

Figure 3.

Sinusoids are open, less fibrotic changes than 6 h priapism (group 2— 150).

Full figure and legend (130K)

Discussion

The AFUD Sexual Function Health Council and AUA consensus panel on Priapism recently stated that despite the recent explosion of laboratory and clinical research data concerning erectile physiology, there has been limited understanding of the pathophysiology of the priapism.^{1, 17} However, the pathophysiology of the priapism can be simplistically viewed as dysfunctional hemodynamic process of the penis, whereby the genital organ excessively endures blood engorgement and the major point of this phenomenon is the anoxia of penile tissue.¹⁸ Briefly, in anoxic state, smooth muscle of corpus cavernosum has minimal basal tension and has no spontaneous contractile activity.¹⁹ Under anoxic conditions, -adrenergic agonists produce poorly sustained phasic contractile responses. From the therapeutic view, this explains the responsiveness of corpus cavernosum to phenylephrine in priapism.²⁰ On the other hand, hypoxia causes inhibition of oxidative phosphorilation with loss of high-energy phosphates, necessary for the homeostasis of calcium and the contractile mechanism of the smooth muscle.²¹ Thus, increased lipid peroxidation products (reactive oxygen species) and fibrogenic cytokines like TGF-1 contributes to the triggering of the biochemical cascade that leads to cellular injury in ischemic state.^{3, 22} Nevertheless, when effective therapy for ischemic priapism is performed for increasing corporal partial pressure of oxygen, reperfusion causes erectile tissue injury owing to the presence of reactive oxygen species. Munarriz et al²³ in a rabbit model showed a significant increase in myeloperoxidase activity and lipid peroxidation after corporal reperfusion and demonstrated adhesion of the PMN leukocytes to the corporal endothelium and their infiltration to the underlying tissues. Consequently, all these factors contribute to penile fibrosis, which is more severe in priapism induced by sickle cell disease, due to micro-occlusion of the venous outflow by sickled erythrocytes. For this reason, it was suggested to perform early implantation of penile prosthesis to reduce the technical difficulties and complications associated with penile prosthetic insertion in priapism.²⁴

TGF- plays an important role in tissue repair by transiently attracting fibroblasts to the wound site and by stimulating synthesis of matrix molecules such as fibronectin and collagen, ultimately leading to the incorporation of these compounds into the cellular matrix.^{25, 26, 27} Previously, Ul-Hassan et al ⁷ demonstrated that TGF-1 might be the mediator of fibrosis in priapism. In a canine model with papaverine-induced ischemic priapism, they detected the expression of TGF-1 mRNA in 66.7% of the experimental group compared with 16.7% of the control group, indicating that the agents that neutralize TGF-1 should be the target of therapeutic interventions to overcome the induction of fibrosis. Recently, Leungwattanaku et al²⁸ demonstrated an increase in TGF-1, hypoxia-inducible factor-1 (HIF-1) and collagen I and III synthesis in cavernous neurotomized rats, which is a mild form of hypoxia compared with priapism. Although the cavernous neurectomy did not cause significant morphological and functional changes in the corpus cavernosum due to the short period of nerve injury, the results of hypoxia in this model in molecular level is strikingly similar to priapism.²⁸ Thus, we have previously demonstrated that decorin (a TGF- neutralizing substance) treatment has an antifibrotic effect in TGF--induced rat model in Peyronie's disease.²⁹

An animal model for priapism can be created in two ways. With a pharmacological agent like papaverine and PgE1 or with a collar or constrictor band placed in the root of the penis. For most of the investigators, it is financially prohibitive to obtain larger animals like monkeys, dogs or cats. Small laboratory animals like rats have the advantage of being obtained more easily and being less expensive. However, up to date, there is no well-established priapism model in rats. The reason is impossibility of maintaining an erection for a long period of time in a rat model. Chen et al¹³ demonstrated that the maximal intracavernous pressure was induced with 0.4 mg of papaverine and the longest effect was with 0.8 mg of papaverine in which the intracavernosaly pressure returned to baseline over the next 40–60 min. For inducing a priapism with duration of 6 h, one should inject papaverine 6–9 times. Every time, a vasoactive agent has to be injected with at least 0.05–0.1 ml of saline to corpora and this may lead to acute fluctuations in intracavernosal pressure. Also, all these procedures should be performed under general anesthesia. If one would like to investigate the priapism after 24, 48 or 72 h, the procedure becomes messier. On the other hand, application of constrictor band is easy and may be performed under temporary anesthesia. With an appropriate band, rats can spurt drops of urine while preserving full erection, which prevents discomfort due to urinary retention. In this model, the constrictor band is tight enough for compression of the subtunical venules, but not much for the corruption of the urethra or skin necrosis. In our study, we encountered no skin necrosis. One other point to be underlined is the source of blood coming to the corpora, which is mainly venous with lower partial oxygen pressure that mimics priapism better.

In this study, we demonstrated that the decrease in erectile capacity is proportional with the duration of priapism. Plateau ICP of the group 1 and 3 were 33.3510.69 and 29.710.61 cmH₂O, respectively. The small difference between the two groups indicates the critical importance of the first 6 h of priapism, because the future erectile capacity is to be mainly determined in this critical phase of the state. In this phase, with an adequate treatment, future erectile capacity may be preserved.

On the other hand, we obtained a statistically significant difference between the rats with 6 h of priapism treated with anti TGF-1 and the other groups in terms of plateau ICP and maximum ICP/systemic blood pressure. That means, the application of anti-TGF-1 in the early phases of priapism may protect the future erectile capacity, especially if the duration of priapism is short (<6 h). From the clinical window, we similarly know that most of the patients have no sequel in terms of erectile capacity when they are treated within 6 h of priapism. However, in our study, there was a notable difference between control group and group 2 in terms of plateau ICP and maximum ICP/systemic blood pressure. Briefly, rats in group 2 have nearly two-thirds of the erectile capacity of the control group. This may show that this model may be heavier than seen in routinely clinical basis. Although, the duration of ischemia is the most important factor for estimating the erectile capacity in the future, we detected no difference in terms of maximum ICP/BP between groups 1 and 3 after 8 weeks; this again shows the importance of early intervention within 6 h since any therapeutic intervention may not be helpful for the future erectile capacity after 6 h.

Spycher and Hauri³⁰ demonstrated that when the episode of priapism lasted more than 24 h, severe cellular damage and widespread necrosis occur. After 48 h, trabecular inflammation becomes conspicuous and most of the smooth muscle is either transformed to fibroblast-like cells or undergoes necrosis. Corpora then replace with collagen. In this study, histopathologic examination of the group 1 revealed obliteration of the sinusoidal spaces and the replacement with collagen. Contrary, TGF-1-treated rats (group 2) had less fibrotic changes and sinusoidal spaces were open which demonstrates the positive effects of the treatment on histopathology. On the other hand, histopathologic findings of groups 3 and 4 were similar to group 1 which again point out the critical importance of the first 6 h of priapism for the future erectile capacity. In this study, observations of an experienced and referenced pathologist (VU) were also confirmed by calculating the area of sinusoidal spaces with a computer program by using the digital images of the sections of the cavernous tissue.

The AFUD Sexual Function Health Council stated that treatments initiated beyond 72 h may have benefits in relieving the unwanted erection and associated pain, but have little documented benefit in terms of potency preservation.¹ This opinion refers us to perform further studies to show the effects of treatment on potency preservation with a rat model with the duration of priapism beyond 24 h. In this study, we applied the antibodies in the very early phase of the priapism, because our major aim was to investigate if any response to the anti-TGF-1 treatment exists, and we found out that treatment with anti-TGF-1 may be a possible treatment for the prevention of fibrotic effects of priapism. With further refinement, application of the antibodies in 2, 4, 12, 16, 36, 48 and 72 h should be performed to investigate the therapeutic response of the antibodies in the moderate and late phases of priapism.

Conclusion

TGF-1 is one of the mediators that induce fibrosis in ischemic priapism. In this study, TGF-1 neutralizing antibodies decreased the fibrotic effects of TGF-1 especially in cases with short duration of priapism. Furthermore, in the management of ischemic priapism, administration of TGF-1 antibodies intracavernosaly in the early phases of priapism may be a possible tool for limiting the level of cavernosal fibrosis. This study also demonstrated that the erectile capacity of the penis is correlated with the duration of priapism and early intervention is mandatory to preserve the future erectile capacity.

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