1 INTRODUCTION

Testicular torsion (TT) is a medical term describing an important urologic condition, in which a testis rotates around its longitudinal axis and twists the spermatic cord, which results in a significant decrease in blood flow and perfusion of testicular tissue. During TT, the testicular tissue is affected by ischemia (I), heat stress, hypoxia, and oxidative and nitrosative stress.^{1, 2} The annual estimation for TT in the United States includes 13%–54% of pediatric acute scrotal upcoming cases, with an occurrence of 1 in 4000 in men over 25 years old.³

TT can be categorized into two types: intravaginal, with a higher incidence rate, which occurs within the tunica vaginalis, and extravaginal or supravaginal, which occurs outside the tunica vaginalis. The intravaginal is more common after puberty onset, while the extravaginal is more common during prenatal development up to 1 year of age when the gubernaculum can freely rotate.^4-6 Both types of TT should be considered medical emergencies. Surgical intervention (testicular detorsion [TD]) as the sole treatment option in viable cases involves counter-rotation of the twisted testis in line with possible fixation to the scrotal wall aiming to prevent recurrence.^{6, 7} TD, like other surgical interventions, in parallel with beneficial effects, induces numerous side effects. TD re-initiated reperfusion into the testis results in endothelial cells injury, microcirculation disturbances, and intense germ cells loss^{8, 9}

Prompt and accurate diagnosis of TT is of major importance since it can result in proper recognition of the best period for surgical intervention and prevention of irreversible ischemic damage to testicular tissue as well as germ cells loss.¹⁰ A study by Sheth et al. reported various symptoms for TT, such as acute unilateral pain in the scrotum, vomiting, nausea, hardened testis, absence of cremaster reflexes, and redness of the scrotal skin.¹¹ Long-term TT may lead to infertility and subfertility due to testicular atrophy,¹² loss of the testis,¹³ germ cells necrosis,¹⁴ arrested spermatogenesis,¹⁵ or diminished serum testosterone.¹⁶ Therefore, the time frame for starting appropriate treatment is of major importance. Many animal studies have shown that the contralateral testicle will not be affected during TT.¹⁷ The duration and degree of torsion have been shown as the main parameters relating to infertility and/or subfertility of torsion-induced outcomes.¹⁸

Based on the previous investigations of human cases, the optimal period for maintaining testicular viability after TT is 4–6 h.^{13, 14, 19} After this period and before 12 h, there is still a 50% chance of viability for the testis, but this chance falls to 10% after 24 h, after which there is a major risk of atrophy and loss of testis after 24 h.^{5, 13, 20} Orchiectomy is the last procedure in the case of the atrophied testis, and is necessary in 32%–42% of TT cases.^{14, 19, 21} Considering the acute nature of TT, the histopathological lesions of TT post-ischemia are mainly induced by hypoxia and progressive reactive oxygen species (ROS) generation.^{22, 23}

The current review aims to discuss experimental TT models, different time frames for TT induction, and the associated pathophysiology by emphasizing cellular and molecular events as well as different therapeutic agent applications for TT in research proposals.

2 REVIEW CRITERIA

All original research and epidemiological papers related to keywords were found and included using PubMed, Google Scholar, and Scopus search engines. Abstracts, congress papers, presentations, and full papers that were not available in English have been excluded. Regarding anti-inflammatory agents, the last 20 years’ original papers have been included. However, for antioxidant agents, original papers published during the last 5 years have been included. Moreover, regarding the model's criteria, TT-induced models with over 4 h in rats were excluded.

The following keywords were searched: “Testicular Torsion” OR “Reperfusion” OR “Histopathology” OR “Leydig Cells” OR “Sertoli Cells” OR “Spermatogenesis” OR “Germ Cells” OR “Sperm” OR “Endocrine Status” OR “Semen” OR “DNA” OR “Inflammation” OR “spermatogenesis” OR “Oxidative stress” OR “DNA fragmentation” OR “Heat stress” in combination with other search phrases relevant to the topic of testicular torsion were used.

3 EXPERIMENTAL SURGERY METHODS

Torsion ischemia induction techniques were applied to simulate ischemic damages in testicular tissues. Testicular artery ligation and clamp, aiming to stricture/obstruction in testicular blood flow and ischemic damages first introduced by Oettle and Harrisson in 1952²⁴ is still effectively applied in different studies.^25-27 Jhunjhunwala et al. used a different approach in a way to a vertical incision on the scrotum after opening subcutaneous, tunica dartos and tunica vaginalis was applied, and one of the testes was exposed and exteriorized carefully for torsion in a targeted degree (720° is the most used one).²⁸ In the mentioned method, the testis should be fixed by suturing tunica albuginea to the scrotal cavity via non-absorbable suture materials like silk or polyglactin. The final step will be suturing the applied incision with silk, vicryl, or catgut.^{14, 29-34}

The differences in surgical technique between each animal model are due to a variety of species. For instance, in dogs and rabbits, tunica vaginalis usually does not get incised and dissected^{35, 36}; in the mice model, the surgical incision should be done in the lower ventral midline and gubernaculum in line with dissection of the testicular vascular membrane before TT induction.¹⁵ The size of suture materials usually depends on animal size, but in the experimental TT surgery procedure, 3–0 and 4–0 materials are the most commonly utilized suture materials.^{29, 33, 36, 37}

4 THE TIME FRAME USED IN RATS AND MICE TO INDUCE A SUFFICIENT AND STANDARD MODEL

The main goal of using TT-induced animal models is to mimic the condition that has been illustrated in patients. Numerous studies during the last decades have been conducted to demonstrate the best time for inducing TT in rats and mice. Although time is an important factor in TT induction, TT degree and ischemia (I)-induced damages are also playing key roles in the induction of TT condition.^{14, 38, 39} Prolonged TT induction leads to testicular infarction and necrosis, making a kind of limitation for a proper evaluation of TT condition. For instance, it has been revealed that atrophied testis )half of the normal testicular mass) represents a complete spermatogenesis arrest and aspermatogenic condition.¹⁷ Thus, it seems necessary to utilize a model reflecting key pathological aspects and testicular dysfunction, while testicular tissue remains viable to investigate.

Accordingly, as mentioned above a minimum of 1 h 720^o TT is needed to induce ischemia in rats¹⁷ and pigs.⁴⁰ Induced TT in this manner is sufficient to induce blood flow obstruction,^{17, 41} spermatogenesis dysfunction,¹⁴ germ cell arrest,⁴² and apoptosis.³⁹ However, in order to induce the same condition in mice, at least 2 h of 720^o torsion is required.⁴³ Moreover, Shamsi et al. illustrated that more than 4 h of 720^o TT induction in rats causes testicular necrosis, which could be the source of irreversible damage to the tissue.^{14, 42} Talebi and Farahpour demonstrated that 4 h TT induced germ cells, and cell cycle machinery arrest, which was reversible, at least 8 h post TD.⁴⁴ Therefore, a time frame between 1 and 4 h to induce TT condition in rats is acceptable.

5 ISCHEMIA-INDUCED OXIDATIVE STRESS IMPACT ON TESTICULAR TISSUE

Early conducted studies illustrated that TT negatively impacts spermatogenesis. The histopathological changes demonstrated loss of germ cells in TT-induced conditions independent of Leydig and Sertoli cells disruption.^{17, 45} Based on previous reports, it has been suggested that TT-induced germ cells loss is associated with intrinsic/extrinsic induction of apoptosis.^{17, 45} Furthermore, Bozlu and colleagues suggested that germ cells loss in TT-induced condition is linked to testicular lipid peroxidation products, nitric oxide (NO) content, and myeloperoxidase (MPO) level.^{46, 47} Different investigations revealed that TT leads to decreased seminiferous tubule diameter, germinal epithelial cell thickness, and spermatozoal maturation.^{45, 48} Regarding sperm quality, it has been shown that TT can diminish sperm concentration in parallel with enhanced sperm abnormalities but without changes in sperm motility.⁴⁹

During the last decades, various studies including those from our group revealed that oxidative stress plays a pivotal role in TT-induced damages.^{14, 39, 42} The ischemia/reperfusion (I/R)-induced condition results in testicular lipid peroxidation increment, reduced total antioxidant capacity (TAC), superoxide dismutase (SOD), catalase, and glutathione (GSH) peroxidase (GPx) enzyme activity in testicular tissue. Thus, induced oxidative stress does not only happen due to reduced oxygen supply but also can be more severe when the testicular antioxidant defense mechanism suppresses.^{14, 39}

It should be noted that TT-induced oxidative stress has the potential to induce cell cycle arrest in germ cells.⁴⁴ Talebi and Farahpour conducted a study illustrating an increase in p21 protein expression in a TT model experiment. The p21 increment inhibits CyclinD1/cyclin-dependent kinase 4 (Cdk4) interaction, resulting in cell cycle machinery disruption in the G1/S stage of mitotic division.^{44, 50} It has been shown that at least 8 h of testicular reperfusion (TR) is required to repair the I/R-induced DNA damage through PCNA and re-initiate the cyclin D1 and CDK-4 expression in germ cells.⁴⁴ Moreover, I/R-induced oxidative stress after 1 h of TT triggered the intrinsic apoptosis pathway through p53 activation in oxidative stress-induced DNA damage.³⁹ In accordance with these findings, Shamsi-Gamchi et al., in a recently conducted study, reported a time-dependently increment of p53, p21, and Bax proteins in TT-induced condition. Indeed, increased oxidative stress in TT-induced condition led to a reduction in mitochondria membrane integrity, Bax protein increment, and Bcl-2 protein decrement. Moreover, p21 inhibited the cell cycle machinery of germ cells during mitotic division and led them to apoptosis. Indeed, p21 in association with p53 triggered p21/p53-dependent apoptosis in testicular germ cells.⁴²

Not only oxidative stress plays the most important role in I/R-induced pathological impacts but also ATP (adenosine triphosphate) supply decrement, diminished glycogen level, and increased intracellular level of Ca²⁺ may play key roles in TT-induced permanent injury.⁵¹ It has been reported that TT-induced oxidative stress in rats leads to heat shock protein-70 (HSP-70) decrement in a time-dependent manner. However, this decrement occurs until 4 h post-TT induction. TD could not initiate HSP-70 expression after 4 h of TT, illustrating irreversible aponecrosis occurrence 4 h after TT induction in rats. Similarly, diminished expression of caspase-3 mRNA and protein levels in testicular tissue revealed the necrosis initiation in TT following intensive I/R-induced tissue damage¹⁴

6 ANTIOXIDANT TREATMENT IN TT AND TD-INDUCED CONDITIONS

Considering numerous studies used antioxidant therapy, here in this study, only the last 5 years’ conducted studies were used. Antioxidants in the current studies have been divided into only antioxidant agents (Table 1) and agents with antioxidant activity (Table 2). Antioxidant treatment in TT-induced condition has been used as protective (a single dose before TT/TD), co-treatment (a single dose before TT/TD and continued after reperfusion), and treatment (only used after TD and during the reperfusion period) protocols.

In the case of protective agents, antioxidants have been administrated PO (per oral) before TT induction and before TT termination (commonly i.p or IV). For instance, taxifolin (50 mg/kg) PO administration 1 h before TT induction in rats reduced malondialdehyde (MDA) levels and increased GSH and SOD levels in TT-treated animals, compared to non-treated control.⁵² Oral administration of an antioxidant agent such as taxifolin or others as mentioned in Tables 1 and 2 before TT protects the testis from I/R-induced damages. However, the protocol cannot be applicable in the clinical situation since the practitioner starts the treatment procedure after the occurrence of TT.

Accordingly, most of the protective protocol studies tried to use a single dose of antioxidants before TD. Indeed, the main aim of this method is to protect the testis from TD-induced severe oxidative stress condition. For instance, using myricetin (1 mg/kg; i.p) 30 min before TD, reduced (p = 0.004) testicular MDA level and increased (p = 0.003) Johnsen score in I/R-treated rats in comparison to control counterparts. However, evaluating testicular SOD and catalase enzyme levels showed no significant difference between treated and non-treated groups.⁵³ Moreover, berberine (200 mg/kg i.p. [intraperitoneally]) 30 min before TD was able to diminish (p < 0.05) testicular MDA, total oxidant status (TOS), and oxidative stress index (OSI) levels.⁵⁴

Conversely, chrysin i.p. administration (100 mg/kg) 30 min before TD not only reduced (p< 0.05) testicular MDA, TOS, and OSI levels but also increased SOD enzyme activity in the testis.⁵⁵ These results are in agreement with different studies (Tables 1 and 2) suggesting that administration of antioxidant agents before TD surgery can protect the testis from I/R-induced injuries in a dependent and /or independent of antioxidant enzyme activity manner. Moreover, this treatment method could stimulate cell survival gene expression such as proliferating cell nuclear antigen (PCNA), Bcl-2, cAMP-responsive element modulator-τ.^56-58 The pro-apoptotic and apoptotic-related gene expression is reduced in the protective protocol as presented in Tables 1 and 2.

In the case of co-treatment protocol, the antioxidant agents have been used starting before TD until the end of trial.^{59, 60} The main aim of this approach is not only to inhibit reperfusion-induced injury but also trying to evaluate antioxidant enzyme activity during the first days post TT. It has been demonstrated that selenium (0.5 mg/kg; i.p) for 7 continuous days post operation was able to diminish MDA and increase TAC, SOD, and GSH activity in TT/TD co-treated rats, compared to TT/TD non-treated controls.⁶⁰ Similarly, using baicalin (25, 50, 100 mg/kg; i.p) before and 12 h after TD reduced MDA and NO levels and increased SOD and GPx levels. Moreover, baicalin co-treatment increased Bcl-2 and reduced Bax, caspase-3, caspase-9, and Fas ligand (Table 1) expression in the co-treatment protocol.⁵⁹

Similar to other treatment protocols using antioxidants post-TT operation reduced oxidative stress metabolites such as MDA,^61-63 NO,⁶⁴ MPO, and xanthine.⁶⁴ Antioxidant treatment increased SOD,^61-64 GSH,⁶² and GPx⁶³ enzymes level. However, antioxidants increased testosterone levels by protecting Leydig cells⁶³ and stimulating testosterone synthesis by increasing serum cholesterol, and LDH (lactate dehydrogenase).⁶⁴ Omega-3 fatty acid treatment increased lactate transporters number⁶⁴ and stimulated mitochondria-dependent anti-apoptotic gene expression such as Bcl-2 in testis.^{56, 63} In contrast, pro-apoptotic and inflammatory gene expressions such as phosphorylated mammalian target of rapamycin and caspase-3.⁶⁵ Bax,⁶⁵ tumor necrosis factor alpha (TNF-α), and interleukin (IL)-1β⁶⁴ reduced in omega-3 treated animals testis, compared to TT/TD-induced animals.

Comparing all the three protocols, it is blatant that protective antioxidants administration reduces testis damages facing I/R. However, it is not clinically relevant to use different antioxidants before TT, while in real life it is not sure when and how the TT will happen. On the other hand, using antioxidants, as a co-treatment or treatment, showed the potency to reduce the TT and TD-induced damage to the testis. A recently published article by Tangül et al. compared the protective and co-treatment protocols in relation to N-acetylcysteine in the TT model. The results revealed that the protective effects of both methods are still significant; however, co-treatment administration reduced testis MDA level more pronounced in comparison to the single-dose (protective) method.⁶⁶ Studies by our group showed that TD-induced injuries start soon after the operation.^{14, 39, 42} Thus, the protective protocol, which is to start using antioxidant treatment before TD and continue the treatment for a couple of days after TD, might be the most effective protocol to protect the testis. Despite various studies focusing on the protective effects of different antioxidant agents in I/R condition, it is still unclear which antioxidant administration protocol can protect testis more efficiently, and finding the best antioxidant with a sufficient protective mechanism against TT warrants further studies with different antioxidants and the TT model.

7 CALCIUM CHANNEL BLOCKERS

Antioxidant administration has been used as a routine treatment option to protect or treat testis in the I/R condition. However, considering the intracellular Ca²⁺ key role in testicular germ cell apoptosis, inhibiting intracellular Ca²⁺ accumulation in testicular I/R condition is another treatment option.⁵¹ Accordingly, nifedipine administration (100 mg/kg) as a Ca²⁺/Na⁺ channel blocker, 30 min before TD showed the ability to reduce apoptotic germ cells and apoptotic tubules number in treated animals testis, compared to non-treated animals. Moreover, SOD, GPx, Johnsen score, and mean seminiferous tubular diameter level showed an increment in the nifedipine administrated group.³⁰

Similarly, using amlodipine (AML), another Ca²⁺/Na⁺ channel blocker, 30 min before TD and 90 min after TD (5 and 10 mg/kg) reduced MDA level, TNF-α, and TGF-β1 mRNA levels and increased SOD, GSH levels in I/R animals. Using 5 mg/kg AML before TD reduced TNF-α and TGF-β1 mRNA levels, compared to TT-induced condition; however, using the same dose of AML as a treatment agent could not diminish TNF-α and TGF-β1 mRNA levels, compared to TT-induced condition. Minding this impact, it can be suggested that using Ca²⁺ blockers as protective agents and administrating them before TD surgery may reduce I/R-induced inflammation more effectively, compared to using them in the treatment protocol.⁶⁷

8 TT-INDUCED INFLAMMATION IMPACT ON TESTICULAR TISSUE AND ANTI-INFLAMMATORY THERAPY

It has been previously demonstrated that injuries due to TT and TD are able to up-regulate pro-inflammatory cytokines expressions such as TNF-α,⁶⁸ IL-1β,⁶⁹ and IL-6⁷⁰ in testis. It has been reported that TNF-α and IL-1β provoke monocyte chemoattractant protein-1 expression in TT-induced testis.⁷¹ Moreover, mitogen-activated protein kinase (MAPK) family proteins activation, such as extracellular regulated kinase 1/2 and c-Jun-N terminal kinase⁷² have been illustrated as a key player in TT.

The MAPK pathway's vital role in the phosphorylation of effector proteins and transcription proteins, such as nuclear factor-kappaB (NF-κB) in testis, has been revealed in various studies.^{73, 74} Indeed, active NF-κB leads to pro-inflammatory cytokine IL-1β and IL-18 expression in testis,⁷⁵ demonstrating TT promotes various cytokines expression through different pathways such as MAPK.^{76, 77} NF-κB activation in the case of I/R condition, in TT-induced testis, has been shown previously.⁷⁸ In addition, TNF-α increment has been observed in TT-induced rats.⁷⁹

Considering these impacts, different anti-inflammatory agents have been used in protective, co-treatment, and treatment manners as summarized in Table 2. For instance, using single-dose erythropoietin (3000 IU/kg, i.p) as a protective anti-inflammatory agent has diminished IL-1, IL-6, and TNF-α levels in TT-induced rats testis, leading to lower apoptotic germ cells number.⁷⁰ In the case of co-treatment, Zhao et al. used sulfasalazine before TT induction and 3 days after TD. The results revealed diminished NF-κB expression and apoptotic germ cells number in sulfasalazine co-treated I/R-induced rats, compared to I/R-sole-induced rats⁷⁴ Similarly, dexamethasone (10 mg/kg) single-dose treatment in I/R-induced rats has reduced germ cells apoptosis ratio and vascular neutrophils adhesion, compared to sole I/R rats.⁸⁰

Using anti-inflammatory therapy could inhibit pro-inflammatory cytokines production in TT-induced testicular tissue. However, anti-inflammatory agents such as pirfenidone protected testicular tissue by inhibiting ROS accumulation in TT-induced testis. The results have shown an increment of antioxidant enzymes such as SOD and GPx, and diminished lipid peroxidation level, edema, and hemorrhage in pirfenidone-treated animals.⁸¹ Moreover, it has been revealed that anti-inflammatory agents usage as protective,⁸² co-treatment,⁸³ or treatment⁸¹ can reduce adverse histopathological effects of I/R in testis.

9 VASODILATOR AGENTS

As blood drainage bypass is the main issue in the TT condition, various studies tried to use numerous vasodilator agents. For instance, milrinone, a non-glycosidic phosphodiesterase (PDE) 3 inhibitor, as a vasodilator, acts through the induction of heart muscle.⁸⁴ Milrinone (0.5 mg/kg, immediately after TT) administration, reduced inflammation (p = 0.001), haemorrhage (p < 0.001), edema (p = 0.001), congestion (p < 0.001), and increased Johnsen score (p < 0.001) in treated animals’ testis, compared to non-treated rats. Moreover, milrinone administration increased SOD, GPx, total antioxidant status (p < 0.001) and decreased protein carbonyl, MDA, TNF-α, IL-1β, and TOS, compared to the non-treated group.⁸⁵

Trapidil (5-methyl-7-diethylamino-triazolopyrimidine), an anti-anginal drug, is a growth factor inhibitor derived from PDE and platelet.⁸⁶ Using Trapidil in TT/TR-induced rats reduced ROS formation⁸⁶ and histopathological damages.⁸⁷ Sildenafil is another vasodilator drug, which selectively inhibits (guanosine 3',5'-cyclic monophosphate (cGMP)-specific PDE-5 and is commonly used for erectile dysfunction.⁸⁸ Sildenafil administration 1 h before TD resulted in a reduced MDA level. However, the antioxidant enzyme profile remained unchanged. Thus, the authors suggested that sildenafil reduces MDA levels through nicotinamide adenine dinucleotide phosphate oxidase inhibition.⁸⁹

Carvedilol (CVO) is a selective blocker of α1-receptors and a non-selective blocker of β1 and β2-adrenoceptors. CVO has been used in various I/R conditions such as ovary I/R^{90, 91} and renal I/R⁹² conditions. Using a single dose of CVO (30 mg/kg, 30 min before TD) reduced MDA and increased SOD and GPx in the testis and serum of I/R-treated rats, compared to non-treated ones. Moreover, CVO administration increased Johnsen's score non-significantly in the I/R-treated group.⁹³

10 CONCLUSION AND FUTURE PERSPECTIVE

Testicular torsion is a medical emergency, and the only treatment available is surgical testicular detorsion. Rapid and accurate diagnosis is fundamental because after 6 h, the damage may be irreversible. Testicular torsion causes germ cell necrosis, arrested spermatogenesis, and diminished testosterone levels, with consequent infertility. Among different involved pathophysiological impacts, testicular torsion/testicular detorsion-induced ischemia seems to play the key role by leading the tissue toward other series of events in the testis. However, during the last decades, no classification was reported for testicular torsion-induced condition due to various variables such as time and degree, as well as the many underlying events, such as ischemia, hypoxia, heat stress, and oxidative stress. In our opinion, a classification might be considered as severe (reactive oxygen species generators such as ischemia, hypoxia, heat, oxidative stress as severe), moderate (inflammation), and mild (hormonal imbalance) involved pathophysiologies.

Numerous studies have used adjuvant antioxidants, calcium channel blockers, anti-inflammatory agents, or vasodilating agents in order to decrease these effects as discussed. Similar to pathophysiology, using of different agents in numerous doses, different time frames of administration, and numerous protocols result in different variables’ involvement at the same time, inhibiting the possibility of well-organized classification. Based on the severity of ischemia condition leading to oxidative stress (in the short turn) and inflammation (in the long run), using the ameliorating antioxidants and anti-inflammatories in different comparable conditions can improve our knowledge, creating a foundation to use these agents in clinical trials. Although experimental data have demonstrated a benefit from these therapies, associated with testicular detorsion, the critical need for well-designed human clinical studies should be considered.

To the best of our knowledge, no previously conducted study examined therapeutical agents’ beneficial effects post clinical ischemia/reperfusion condition in humans. Different agents targeting different pathophysiological conditions were used to ameliorate the ischemia/reperfusion-induced condition in animal models; however, none of the administrated agents were tested in human cases. Although considering testicular detorsion surgery is still the golden method to reverse the testicular torsion condition and the surgical approach is undeniable, the evaluated agents (Tables 1–4) with beneficial effects, need to be investigated furthermore in clinical conditions. In order to achieve a clinical condition examination, there are still some question marks that remained to be answered in furthermore studies. The main issues including, comparing the administration of agents before and/or after testicular detorsion, using antioxidants, anti-inflammatories, and so forth, separately or in a combination form, needs to be uncovered. Considering that most of these agents in a safe dose have no adverse impact on testicular tissue or other organs, performing organized clinical studies simultaneously with animal model studies might be possible to examine the beneficial impacts. Thus, furthermore clinical studies and case reports are required to approve the animal models proposed agents’ beneficial impacts.

AUTHOR CONTRIBUTIONS

Aram Minas conception and design of the study. Aram Minas, Sina Mahmoudabadi, Naeimeh Shamsi Gamchi, and Arash Alizadeh collected the data and wrote the article. Arash Alizadeh, Mariana Pereira Antoniassi, and Ricardo Pimenta Bertolla interpreted the data, discussed the results, and revised the article.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.