Determination of M1/M2 macrophage polarization in the ipsilateral and contralateral rat testicular tissue after unilateral torsion/detortion

Spermatic cord torsion induced by testicular torsion or the rotation of the structures belonging to the cordon on their axis is a serious urological problem and causes male subfertility by causing testicular damage. It is known that unilateral testicular torsion causes damage to the contralateral testis. The mechanism and pathogenesis of contralateral testicular injury after testicular torsion has not been fully elucidated. According to the M1/M2 macrophage polarization, which is a new concept recently introduced in relation to macrophages; M1 macrophages cause tissue damage by producing superoxide anions, oxygen radicals, and nitrogen radicals, while M2 macrophages play a role in fibrosis by increasing cell proliferation and tissue repair. Most of the testicular macrophages are M2 macrophages. On the other hand, testicular interstitial areas contain various immune system cells such as mast cells, dendritic cells, and lymphocytes as well as macrophages. In the present study, it was aimed to determine whether unilateral testicular torsion caused an increase in the number of M1 macrophages and a change in macrophage polarization, which is one of the causes of tissue damage in the interstitial area. In the study, 8-12 weeks old 63 male Spraque-Dawley rats were used. Rats were randomly divided into 9 groups: Control, Sham 4 hours, Sham 24 hours, Sham 7 days, Sham 14 days, Torsion 4 hours, Torsion 24 hours, Torsion 7 days, Torsion 14 days. The rats in the control group kept without any treatment during the experimental period. The surgical procedure was performed on all rats in the torsion and sham groups. In animals belonging to the sham groups, the abdomen was surgically opened, kept for 4 hours without any operation, and was closed at the end of the period. In all torsion groups, the abdomen was surgically opened, the left testis was removed and rotated 720 degrees counterclockwise and created torsion. After 4 hours, detorsion was performed and the testes were taken back into the abdomen and closed. Then, the rats were decapitated after 4 hours, 24 hours, 7 days or 14 days. Spermiogram analysis to determine the effects of torsion on sperm motility, morphology, number, and acrosome integrity; ELISA for determination of hormone profiles; hematoxylin-eosin, PAS-hematoxylin and Mallory-Azan stainings for tissue damage detection; Toluidine blue staining for visualization of mast cells; TUNEL method for the determination of apoptotic cells, and immunohistochemical staining of CD 68 and CD 163 for the determination of M1/M2 macrophage polarity. In light microscopic evaluations, no histopathological changes were observed in the sham groups, but it was determined that testicular torsion caused edema on both sides, especially on the ipsilateral side, vascular congestion, disruption and degeneration in the seminiferous tubule epithelial organization, immature germ cells pouring into the lumen, metaphase arrest in some spermatocytes, detachment of the tubule basement membrane and necrotic tubules. It was observed that the severity and prevalence of these findings continued to increase from the 4th hour to the 14th day in the torsion groups. Cosentino scores of all groups were also consistent with histopathological findings. On the other hand, Johnsen scores, which are a marker of spermatogenesis, were also low in torsion groups with increased seminiferous tubular degeneration. Consistent with increased tubule damage in ipsilateral testes, it was determined that seminiferous tubule diameters and germinal epithelial thickness decreased significantly, especially in the Torsion 14 days group. When Toluidine blue staining for the detection of mast cells was evaluated, no change was observed in the sham and torsion groups in the contralateral testes compared to the control. In the contralateral testicles, mast cell increase was observed only in the Torsion 14 days group. When the mast cells of the ipsilateral testes were analyzed, there was no difference between the control and sham groups; there was a significant increase in mast cell numbers in all torsion groups. When the TUNEL method performed for counting cells with apoptosis was evaluated, there was no significant difference between the groups in the contralateral testes. In the ipsilateral testes, it was determined that the number of apoptotic cells increased in Torsion 4 hours, Torsion 24 hours, and Torsion 7 days groups. In the torsion 14 days group, it was found that the apoptotic index decreased in accordance with the increasing necrotic tubules. When the sperm motility values were analyzed in the experimental groups, there was no difference between the control group and the Sham groups. However, it was observed that motility was significantly decreased in Torsion groups compared to the control group. When the torsion groups were evaluated among themselves, there was a remarkable decrease in the sperm motility in Torsion 7 days and Torsion 14 days groups. There was no significant difference between the groups in terms of sperm density and abnormal head, abnormal tail, and total abnormal sperm ratio. The effects of testicular torsion on male hormone levels were determined by ELISA analysis. As a result of the analysis, the testosterone levels showed a significant decrease in the Sham and Torsion groups compared to the control group. However, Torsion groups were similar in terms of Testosterone levels. While FSH levels did not show a significant difference between the control group and Sham groups; It was determined that FSH levels decreased in Torsion 24 hours, Torsion 7 days, and Torsion 14 days groups compared to the control group. On the other hand, changes in M1/M2 macrophage polarization were detected after torsion-detorsion. In our torsion-detorsion groups, an increase in M1 macrophage was observed in the contralateral testicular tissue. Although the reasons for the changes in the contralateral testes after torsion-detorsion are still not fully elucidated, the findings obtained from the present study show that the mechanisms related to immune cells involving macrophages and mast cells may play an important role in this process.