In this study, we investigated the protective effect of tadalafil in a rat testicular torsion-induced IRI model. We observed that tadalafil attenuated oxidative stress caused by testicular torsion and had a protective effect against testicular IRI for long-term spermatogenesis in a time- and dose-dependent manner. To our knowledge, this is the first study on the protective effects of PDE5i dose and duration on testicular IRI. As a clinical implication, our data indicate that administering tadalafil to patients with testicular torsion may be beneficial against IRI in the early- as well as long-term duration.
Twisting of the spermatic cord, called testicular torsion, is not rare emergent urological condition occurring in adolescent males. Testicular torsion requires prompt diagnosis and surgical detorsion known as a the current standard management for preventing histological damage and reduction of male reproductive ability [2]. Several studies have identified possible mechanisms for the progression of this emergent disease responsible for testicular atrophy and altered spermatogenesis [5–12]. The main pathophysiological consequence of testicular torsion is ischemia and reperfusion, which are generated by the twisting of the spermatic cord followed by detorsion [13]. Reperfusion involves the generation of toxic ROS with the return of blood flow after a period of ischemia. Subfertility after unilateral testicular torsion exists in nearly 40% of cases, and semen analysis may result in abnormal results in more than half of the patients with testicular torsion during long-term follow-up [4, 33]. Unfortunately, there is no established standard treatment for testicular torsion-induced injury without surgical detorsion to date. Unfortunately, to date, there is no established standard treatment for testicular torsion-induced IRI without surgical reduction. Therefore, there is a search for new promising therapeutic approaches to attenuate related pathological signals. Although several therapeutic agents have shown some benefit in reducing testicular IRI, appropriate pharmacological treatments for clinical practice still need to be developed.
Several studies have attempted to suppress testicular damage via medications and surgical maneuvers, such as postconditioning in an animal model with torsion-induced IRI [24–29, 34–44]. However, previous studies have usually focused on the change of molecular markers and histopathological change in early phase, mostly within hour, after ischemia-reperfusion injury. Most studies that have evaluated PDE5i as an attenuating medication have performed similar experimental methods with the torsion lasting 2–4 hour, followed by a detorsion period within 4 hour, following which the rats were sacrificed and biochemical and histopathological analysis after bilateral orchiectomy was performed. The biochemical markers of oxidative stress, such as MDA, SOD, and glutathione peroxidase, were measured owing to their effects in the early phase (several hours). Although the early phase after detorsion is an appropriate time to assess changes in substances related to oxidative stress, it is a questionable period for the assessment of spermatogenesis. Generally, testicular torsion occurs during puberty; however, it is meaningful to measure spermatogenesis in terms of fertility in adulthood. In addition, testicular torsion results in testicular epithelial tissue degeneration via apoptosis secondary to testicular ischemia and reperfusion [36]. This composite pathological cascade is responsible for testicular atrophy and impaired spermatogenesis observed at later stages. Therefore, it is desirable to operate testicular IRI in the adolescence period and subsequently assess the histopathological changes regarding spermatogenesis in adult period in animal study. Previously, we conducted a study to identify the ischemic time of when a long-term serious histopathological change occurs after testicular IRI in adolescent rats. We confirmed via histopathological analysis 4 weeks after testicular IRI that a serious deterioration in spermatogenic activity was induced after 3 hour of ischemia rather than after shorter ischemic times. Therefore, we selected 3 hour of ischemia as the duration of torsion for confirming the long-term spermatogenic results of the current study [31].
Tadalafil is one of the mostly used PDE5i to treat erectile dysfunction and maintain a steady-state blood concentration that repairs damaged endothelium and improves erectile function, while sildenafil, vardenafil, and udenafil are more rapidly absorbed. Therefore, tadalafil is frequently used once daily as a long-term maintenance therapy [18, 19]. Inhibition of PDE5 enzyme may cause increased cGMP levels, ultimately resulting in smooth muscle relaxation [18, 19]. There have been several reports regarding the protective effects of PDE5i therapy against IRI in several organs, including kidney, heart, intestine, and lung tissues, in experimental models via anti-inflammation, anti-ROS, and anti-apoptosis activities [19, 20]. Furthermore, several studies have reported that various types of PDE5is may have a protective effect on testicular torsion-induced IRI animal models [23–29, 42]. However, there have been contradictory results regarding the effect of PDE5is on testicular torsion animal models. In agreement with our results, Ozmerdiven et al [25], Ameli et al [26], Yildirim et al [27], and Wu et al [42] have reported the beneficial effects of tadalafil administration in testicular IRI rat model. They reported increased antioxidant enzyme levels, decreased lipid peroxidation levels, germ cell apoptosis, and attenuated histopathological results in the early phase. Similar results can be also found in other studies based on sildenafil and vardenafil [23, 24, 29, 45]. On the other hand, some studies have shown controversial results regarding the effects of PDE5is after testicular IRI. Utsun et al reported that sildenafil and vardenafil caused exaggerated testicular apoptosis and increased nitric oxide synthase levels after 1 hour of ischemia and 2 hour of reperfusion injury [39]. Additionally, Istanbulluoglu et al suggested that vardenafil worsened histopathological changes related to oxidative stress and had no protective effect on testicular IRI in a pig model [28]. Regarding on the conflicting outcomes of PDE5i in testicular IRI, we assumed that the differences were caused because of varying torsion and reperfusion times, doses of PDE5is, and animal species used in the studies. In addition, it is well documented that changes in the testes depend on torsion duration; however, the exact time presenting serious testicular deterioration is unclear in previous studies. In the current study, we identified 3 hour as the exact torsion time related to serious damage via our previous study and confirmed the protective effect of tadalafil based on an established torsion-induced IRI rat model.
Oxidative stress is caused by an imbalance in the production of oxidants and the scavenger, a product of the defense system. Although ROS is maintained at physiologically low levels by the endogenous antioxidant defense system in tissue under normal conditions, overgeneration of oxidative stress, in which ROS generation exceeds the defense mechanisms capacity to control it, such as in the course of testicular ischemia and reperfusion, contributes to reversible or irreversible cell injury [46, 47]. Several studies have established that testicular torsion-induced IRI increases oxidative stress and decreases the antioxidant scavenger [23–29, 42]. MDA is a stable end product of lipid peroxidation generated by ROS and is usually used as an indirect indicator of oxidative damage [48, 49]. In addition, SOD is a key component in cell growth, differentiation, and protection and acts as one of the enzymatic antioxidant defense systems against ROS and oxidative damage [47]. The results of our study showed that tadalafil can protect spermatogenic activity in the late phase as well as attenuate oxidative stress markers in the early phase. This may be due to the antioxidant property and ability of tadalafil in testicular torsion-induced IRI model [19]. These results agree with those of previous studies using tadalafil [25–27, 42]. In addition, our study demonstrated that tadalafil can alleviate histopathological damage after testicular torsion-induced IRI in a time- and dose-dependent manner in the late phase. Although further studies are needed regarding the exact mechanism, our study supports the notion that tadalafil, which is suitable for dose escalation and long-term use, has encouraging preventive effects with a positive correlation with dose and exposure duration against testicular injury. We assumed that this phenomenon is due to the persistent and cumulative antioxidant property of tadalafil as an excellent scavenger against free radical generation.
The data on damage to the contralateral testis after ischemia–reperfusion injury are conflicting. Some studies have reported contralateral testis impairment after ipsilateral testicular ischemia–reperfusion injury in experimental models [50, 51]. Although the exact mechanism of contralateral damage is not fully understood, decreased testicular blood flow by reflex sympathetic response, ROS overproduction, and autoimmunization against the spermatogonia are hypothesized to explain this phenomenon [52]. However, there were several studies that have reported no effect of testicular torsion on contralateral testis [53, 54]. Recently, Ozgur et al [24] and Unsal et al [38] observed no pathological changes in the contralateral testis after ipsilateral testicular IRI, similar to the findings of our study. This difference is thought to be a result of diverse animal models and evaluation methods for contralateral damage to the testis and should be clarified via additional studies using standardized animal models in the future.
Our study has several limitations that need to be considered. Tadalafil has a relatively long duration of action, with a higher half-life than other PDE5is. In addition, the recommended initial dose of tadalafil is 2.5–5 mg for maintenance therapy in humans and can be increased up to 20 mg. In our study using doses of 0.3 mg/kg and 1 mg/kg, the higher dose of tadalafil showed greater efficacy; however, this dose is not within normal clinical use limits for humans. Therefore, further studies are warranted to elucidate the optimal dose and duration of tadalafil administration to maximize efficacy without possible adverse effects. Furthermore, there is concern regarding the long-term use of PDE5is because some reports have shown that chronic administration of tadalafil to rats may result in degenerative changes in the testis and a decline in semen parameters [55]. Therefore, additional studies regarding the long-term adverse effects of tadalafil itself on the normal or pathological testes will be required. Finally, further studies are essential to clarify the mechanism of chronic use of tadalafil and to establish a protocol applicable to humans for future clinical application.