The long-term protective effect of tadalal on spermatogenesis following testicular ischemia-reperfusion injury in a rat model

Testicular torsion is a urological emergency in which misdiagnosis and inappropriate treatment can lead to testicular atrophy and male infertility owing to ischemia-reperfusion injury (IRI). Although experimental studies of testicular torsion have been preceded, promising therapeutic agents based on the long-term effect for spermatogenesis have not been identied in testicular ischemia reperfusion injury (IRI) animal model. Tadalal, one of the phosphodiesterase-5 inhibitors commonly used in the treatment of erectile dysfunction, has recently reported a protective effect against IRI in several organs. In this study, we evaluated the long-term protective effect of tadalal for spermatogenesis in a rat testicular IRI model.


Background
Testicular torsion is a urological emergency in which misdiagnosis and inappropriate treatment can lead to testicular atrophy and male infertility owing to ischemia-reperfusion injury (IRI). Although experimental studies of testicular torsion have been preceded, promising therapeutic agents based on the long-term effect for spermatogenesis have not been identi ed in testicular ischemia reperfusion injury (IRI) animal model. Tadala l, one of the phosphodiesterase-5 inhibitors commonly used in the treatment of erectile dysfunction, has recently reported a protective effect against IRI in several organs. In this study, we evaluated the long-term protective effect of tadala l for spermatogenesis in a rat testicular IRI model.

Methods
Forty-eight adolescent Sprague-Dawley rats were divided into 6 groups (A-F). Sham operation was performed in group A. Group B received surgical 720-degree torsion of the left testis without any medication. Groups C, D, E, and F were operated surgical torsion with tadala l at varying doses (0.3 mg/kg, 1.0 mg/kg) and durations (single or daily administration for 4 weeks). Detorsion was performed after 3 hour of torsion in all rats except the sham group. Four weeks after operation, both testes were evaluated of spermatogenesis using Johnsen scoring. To evaluate the protective effect of tadala l against oxidative stress by IRI, malondialdehyde (MDA) and superoxide dismutase (SOD) level were analyzed via ELISA in both testes 4 hour after detorsion in the same experiments as in group A, B, and C.

Results
For the evaluation of spermatogenesis according to doses, the groups with high-dose tadala l showed a higher Johnsen scores than low-dose counterparts. The groups with daily administration for 4weeks were observed a higher Johnsen scores than those given a single administration. Furthermore, molecular markers (MDA and SOD) related with oxidative stress and histopathologic ndings showed remarkable improvement after tadala l administration.

Conclusion
Tadala l alleviated long-term deterioration of spermatogenesis and oxidative stress by restoring antioxidant status after testicular IRI rat model. Furthermore, it demonstrated a protective effect against testicular IRI in a time-and dose-dependent manner.
Background Page 4/18 Testicular torsion is a urological emergency caused by the twisting of the spermatic cord, leading to a reduction in blood ow that results in arterial obstruction, ischemia and gonadal necrosis [1,2]. Generally speaking, it is one of the common genital diseases among adolescent boys -estimated to a ict 1 in 4000 younger than 25 years of age [3], and therefore should be corrected promptly to prevent the development of ipsilateral testicular dysfunction and infertility later in life [4]. Surgical intervention by counter-rotating the testis is the standard method of treatment; however, this can give rise to inevitable ischemia-reperfusion injury (IRI) [5][6][7][8][9][10][11][12][13], which impairs spermatogenesis and triggers production of reactive oxygen species (ROS) [6,[14][15][16][17].
Salvage rates have been reported in 30-50% and impaired spermatogenesis occurs in most patients with testicular torsion despite considerable efforts [16,17]. Unfortunately, to date, there is no established standard treatment, except surgical reduction, for testicular torsion-induced injury. Theoretically, promising therapeutic candidates to prevent complications arising from testicular torsion should ameliorate ischemic injury, promote spermatogenesis, and regulate harmful immune response. Although various therapeutic agents have shown some bene ts in reducing oxidative stress as short-term results in several experimental studies, appropriate pharmacological treatments based on the bene t of long-term spermatogenesis still need to be developed.
Phosphodiesterase-5 inhibitors (PDE5is) are commonly used to treat erectile dysfunction in current urology practice [18]. The inhibition of phosphodiesterase enzymes increases cyclic guanosine monophosphate (cGMP) levels in the tissue, resulting in relaxation of smooth muscles. Loosened the smooth muscles of vessel by PDE5is increases the local perfusion and dilation of the vessels and inhibits platelet aggregation, and also some studies have suggested that PDE5is might have positive effect in the prevention of IRI [19,20]. Recently, experimental studies have suggested that PDE5i has a protective effect against IRI by regulating the antioxidant activity of several organs and disease models [6,[20][21][22]. Furthermore, several other studies have investigated the protective effect of testicular ischemiareperfusion injury in a rat testicular torsion model using different PDE5i agents, such as sildena l, tadala l, and vardena l [23][24][25][26][27][28][29]. Of PDE5is, Tadala l is frequently used as a long-term maintenance therapy with once daily administration to maintain a steady-state blood concentration compared with other PDE5is. Nevertheless, there were few studies regarding the long-term protective effect of tadala l for spermatogenesis in testicular IRI model.
In this study, we evaluated the protective effect of tadala l against testicular damage for long-term spermatogenesis in a rat testicular torsion-induced IRI model.

Methods
This study was approved by the institutional animal care and use committee of College of Medicine in Yeungnam University (YUMC-AEC2018-029). All surgeries were performed under anesthesia, and all possible efforts were made to minimize suffering.
In total, 72 male healthy 6-to 8-week-old Sprague-Dawley rats (200-250 g) correlating with adolescent period in humans were purchased from KOATECH Co. (Pyeongtaek-si, Gyeonggi-do, Republic of Korea). According to 3R (reduction, replacement, re nement) of Institutional Animal Care and Use Committee (IACUC), we chose the minimal number of animals used. A 1-week acclimatization period was allowed before any surgical procedure. All rats had free access to food and water under a 12-hour light/dark cycle.
All rats were anesthetized via intramuscular injection of Rumpun and Zoletil once with the addition of one-third of the initial dose if anesthesia was not attained. We euthanized rats using CO 2 gas or cervical dislocation. In a previous study, we identi ed the aggravated testicular damage in ischemic time dependent manner and the serious deterioration of spermatogenic activity developed 3 hour after ischemia in a rat testicular torsion model [31]. Therefore, we selected 3 hour of ischemia as the torsion duration in this study. Rats received surgical torsion for 3 hour and subsequent detorsion of left testis.
After left scrotal incision, unilateral testicular torsion was made by a 720-degree clockwise rotation of the left testis followed by the xation to the scrotal wall with 4 − 0 silk suture (Fig. 1). After maintaining the torsion state for 3 hour, the testis was counter-rotated to its natural position and reinserted in the scrotum.
To evaluate the long-term effects of tadala l on spermatogenesis, 48 healthy rats were randomly allocated into six groups (A-F) as follows: Group A sham operation (surgical incision without testicular torsion/detorsion)

Group B 3 hour torsion/detorsion
Group C 3 hour torsion/detorsion with low-dose tadala l single intraperitoneal injection (single intraperitoneal injection of 0.3 mg/kg tadala l dissolved in 0.9% NaCl 1 hour before detorsion) Group D 3 hour torsion/detorsion with low-dose tadala l once daily administration for 4weeks (single intraperitoneal injection 1 hour before detorsion + daily medication orally of 0.3 mg/kg tadala l dissolved in 0.9% NaCl) Group E 3 hour torsion/detorsion with high-dose tadala l single intraperitoneal injection (single intraperitoneal injection of 1.0 mg/kg tadala l dissolved in 0.9% NaCl 1 hour before detorsion) Group F 3 hour torsion/detorsion with high-dose tadala l once daily administration for 4weeks (single intraperitoneal injection 1 hour before detorsion + daily medication orally of 1.0 mg/kg tadala l dissolved in 0.9% NaCl).
In all rats, bilateral orchiectomies and testicular tissue sampling were performed 4 weeks after 3 hour torsion-detorsion procedures to evaluate spermatogenesis.
And we also evenly randomized 24 healthy rats into three groups (G-I) to evaluate the protective effect of tadala l against oxidative stress.
Group G sham operation (surgical incision without testicular torsion/detorsion) Group H 3 hour torsion/detorsion Group I 3 hour torsion/detorsion with low-dose tadala l single intraperitoneal injection (single intraperitoneal injection of 0.3 mg/kg tadala l dissolved in 0.9% NaCl 1 hour before detorsion) In all rats of three group (G-I), bilateral orchiectomies and testicular tissue samplings for histopathological/molecular analysis were performed 4 hour after 3 hour torsion-detorsion procedures.
Excised testes were para n embedded and stained with hematoxylin and eosin. Histopathological analysis was evaluated independently by a single pathologist blinded to the rat experimental group using a light microscope. The Johnsen scoring system was used to evaluate long-term spermatogenesis between groups A through F [32]. The value of Johnsen score in each testis was the mean point value from at least 10 seminiferous tubules. The severity of germ cell injury was quali ed via Johnsen score from 10 to 1 points, as shown in Table 1. To analyze the protective effect of tadala l against oxidative stress between group G-I, malondialdehyde (MDA) and superoxide dismutase (SOD) level were analyzed via ELISA in both testes 4 hour after detorsion. Molecular analysis, such as MDA and SOD, was assessed in a left to right ratio.
We used the lipid peroxidation assay kit (BioVision, K739-100, USA) for MDA assessment in accordance with the appropriate procedure according to the manufacturer's instructions. In brief, testicular tissues (10 mg) were homogenized in MDA Lysis Buffer (with 3 µl BHT (100x), then centrifuged (13,000 x g, 10 min) to obtain supernatant. Each sample was added 600 µl of TBA reagent. The mixtures were incubated at 95 °C for 60 minutes. After cooling with ice, 300 µl of n-butanol was added and centrifuged (3 min at 16,000xg). N-butanol was removed and the MDA-TBA adduct was placed into a 96-well plate and absorbance was measured at 532 nm. MDA content was calculated with MDA standards.
SOD activity was analyzed by a commercially available kit from Cayman Chemicals, USA (Cat no 706002). Brie y, approximately 100 mg of testicular tissue was homogenized in 20 mM HEPES buffer containing 1 mM EGTA, 210 mM mannitol and 70 mM sucrose. After homogenization, homogenates were centrifuged at 1500 x g for 5 min at 4 °C. The supernatant was then obtained. The supernatant was diluted with sample buffer (10 x diluted) 50 times to produce absorbances within the linear range of the standard curve. Analysis was carried out according to the manufacturer's instructions, read at 450 nm.
Molecular analysis, such as MDA and SOD, was assessed in a left to right ratio.
The one-way analysis of variance (ANOVA) and Tukey's post-hoc test after one-way ANOVA were used for statistical analyses. All values were expressed as the mean ± SD. Results are representative of at least three experiments. All data were reported as the sample mean ± SD, and p values less than 0.05 were considered signi cant. Figure 2 shows the representative gross ndings of the extracted left testes and the mean value of maximal longitudinal length of the left testicle compared with that of the right between groups A through F. Group D, E, and F showed signi cantly higher longitudinal length ratio of left to right testis than group B (group A, 1.013 ± 0.043; group B, 0.313 ± 0.063; group C, 0.372 ± 0.050; group D, 0.385 ± 0.040; group E, 0.472 ± 0.094; group F, 0.510 ± 0.010). In addition, tadala l alleviated testicular atrophy after testicular IRI depending on dose and duration. Figure 3 showed the histopathological ndings and Johnsen score of groups A-F. Although the testes in the sham group (group A) showed normal morphology, those in group B showed serious testicular damage, including severe edema, apoptotic bodies, venular ectasia, and lobular coagulative necrosis. However, Groups C-F (with tadala l administration) showed improvement in histopathological ndings compared with group B. The histopathological ndings among Groups C-F also showed improvement depending on the dose and duration of tadala l. The Johnsen scores of group A (sham group), B (control group), C (low-dose tadala l/single injection), D (low-dose tadala l/daily administration), E (high-dose tadala l/single injection), and F (high-dose tadala l/daily administration) were observed mean 10 ± 0, 2 Histological evaluation of testicular tissues revealed normal tubular structures with the interstitium in group G (sham operation). In group H (control group), serious degeneration, including disorganization of tubular epithelium interstitial edema, hemorrhage, and less distinct seminiferous tubule borders, was found when compared with group G. In group I, tadala l administration mitigated the deterioration of tubular histology compared with group H. In addition, molecular markers related to oxidative stress (MDA and SOD) showed a signi cant difference between groups G-I. MDA levels was signi cantly decreased (group G, 93.5 ± 23.7, group H, 177.3 ± 34.5, group I, 107.3 ± 38.4; p < 0.001 between groups G and H and p = 0.002 between groups H and I) and SOD levels was signi cantly increased in groups G and I compared with group H (group G, 102.3 ± 7.5, group H, 79.2 ± 10.0, group I, 115.5 ± 9.5; p < 0.001 between groups G and H and p < 0.001 between groups H and I; Fig. 4). Molecular markers related to oxidative stress and histopathological ndings showed remarkable improvement after tadala l administration.

Discussion
In this study, we investigated the protective effect of tadala l in a rat testicular torsion-induced IRI model. We observed that tadala l 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 rst study on the protective effects of PDE5i dose and duration on testicular IRI. As a clinical implication, our data indicate that administering tadala l to patients with testicular torsion may be bene cial 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 identi ed possible mechanisms for the progression of this emergent disease responsible for testicular atrophy and altered spermatogenesis [5][6][7][8][9][10][11][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 ow 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 bene t 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][25][26][27][28][29][34][35][36][37][38][39][40][41][42][43][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 sacri ced 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 con rmed 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 con rming the long-term spermatogenic results of the current study [31].
Tadala l 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 sildena l, vardena l, and udena l are more rapidly absorbed. Therefore, tadala l 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-in ammation, 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][24][25][26][27][28][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 bene cial effects of tadala l 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 sildena l and vardena l [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 sildena l and vardena l 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 vardena l worsened histopathological changes related to oxidative stress and had no protective effect on testicular IRI in a pig model [28]. Regarding on the con icting 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 identi ed 3 hour as the exact torsion time related to serious damage via our previous study and con rmed the protective effect of tadala l based on an established torsioninduced 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][24][25][26][27][28][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 tadala l 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 tadala l in testicular torsioninduced IRI model [19]. These results agree with those of previous studies using tadala l [25][26][27]42]. In addition, our study demonstrated that tadala l 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 tadala l, 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 tadala l as an excellent scavenger against free radical generation.
The data on damage to the contralateral testis after ischemia-reperfusion injury are con icting. 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 ow by re ex 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 ndings 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 clari ed via additional studies using standardized animal models in the future.
Our study has several limitations that need to be considered. Tadala l has a relatively long duration of action, with a higher half-life than other PDE5is. In addition, the recommended initial dose of tadala l 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 tadala l showed greater e cacy; 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 tadala l administration to maximize e cacy without possible adverse effects. Furthermore, there is concern regarding the long-term use of PDE5is because some reports have shown that chronic administration of tadala l 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 tadala l itself on the normal or pathological testes will be required. Finally, further studies are essential to clarify the mechanism of chronic use of tadala l and to establish a protocol applicable to humans for future clinical application.

Conclusions
To our knowledge, this is the rst study of the effects of PDE5is concerning dose and duration on testicular IRI. This study demonstrates that tadala l can alleviate histopathological changes and oxidative stress by restoring antioxidant status in testicular IRI in a rat testicular torsion model. In addition, it has a protective effect against testicular IRI in a time-and dose-dependent manner for longterm spermatogenesis. As a clinical implication, our data indicate that administering PDE5is to patients with testicular torsion may be bene cial for early-and long-term follow-up, providing a promising approach that may be modi ed and used in the near future. Availability of data and materials

Abbreviations
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
TGK: Conception and design JNL: Conception and design All authors have read and approved the manuscript.

Figure 1
Experiment of testicular torsion. After left scrotal incision, unilateral testicular torsion was made in a 720°c lockwise rotation on the left testis followed by the xation to the scrotal wall with 4-0 silk suture.

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