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Sources of Funding: none

Introduction

Testicular torsion makes testis ischemic. Surgery is immediately needed to reestablish blood flow. Even if the surgery succeed, testicular atrophy is often appear and lead to spermatic dysfunction. However, the etiology is still controversial in terms of pathophysiological changes. Recently it was reported that loss of the formation in any tissue after ischemia-reperfusion (IR) was involved in necroptosis, which is one of programmed cell death series. Necrostatin-1 (Nec-1) blocks both necroptosis and indoleamine 2,3-dioxygenase (IDO). In our previous research, we elucidated that IDO inhibitor decrease inflammation in testis and epididymis. First, we investigated pathophysiological change of testicular IR researching histological and biochemical phase in this study. Subsequently to the analysis, we tried to inhibit Receptor-interacting protein kinase 1 (RIPK1) and clarify a function of necroptosis in testicular IR.

Methods

Twelve weeks old ICR male mice were used in this study. Their unilateral testicular artery was clamped under general anesthesia. Declamping three hours later, their testicular blood flow were resumed. After the procedure, bilateral testes were removed in time dependent manner (at day 1, 3, 5 and 7). Histological and biochemical change were evaluated by immunostaining and ELISA methods. Spermatic analysis from the epididymal cauda were evaluated by computer aided sperm analysis (CASA). In the following research, Nec-1 4 µg/g was administrated (iv) after declamping testicular blood flow. After the treatment, bilateral testes were removed in time dependent manner. Then, histological, biochemical and semen analysis were evaluated. Sham surgery was performed as control. Their experiments were duplicated at least.

Results

Regarding histological change, invasion of lymphocyte-predominant inflammatory cells accumulated at day 3, 5 and destruction of seminiferous structure were observed at day 5, 7. Necroptosis cell using RIPK staining was abundantly expressed. In semen analysis, significant decreased spermatic concentration was observed at day 5 and 7 compared to control (p<0.05). Significant decreased spermatic motility was observed at day 1, 3, 5 and 7 compared to control (p<0.05). Interestingly, in contralateral (unaffected side) testes, significant decreased spermatic motility was observed at day 5 and 7 compared to control (p<0.05). Some candidates were picked up as molecular marker. Significant increased E-selectin expression, which is a marker of leukocyte-endothelial cell adhesion molecule, was observed at day 1 compared to control (p<0.05). Significant increased IL-6 expression, which is a marker of inflammation, was observed at day 3 compared to control (p<0.05). Significant increased 8-OHdG expression, which is a biomarker of oxidative stress, was observed at day 7 compared to control (p<0.05). Interestingly, significant increased the highest expression of E-selectin, IL-6 and 8-OHdG were observed in contralateral testes. Same results were introduced in immunohistochemical staining. After treatment of Nec-1, testicular structure were maintained and little invasion of inflammatory cells were observed. Significant decreased germ cell death (necroptosis) in seminiferous tubules were statistically observed. Significant decreased E-selectin, IL-6 and 8-OHdG were also observed. Importantly, Spermatic motility also maintained in both treated testis and contralateral testes.

Conclusions

Oxidative stress via inflammation and necroptosis should induce spermatogenetic dysfunction in IR testis. First, prominent inflammation was occurred in testicular ischemia-reperfusion model and not only expression of cytokines were increased, but also in contralateral testes. Subsequently, oxidative stress highly expressed and cell death appeared. Therefore, their change of bilateral testis would be one of pathophysiology in patients with ischemic testis. To inhibit RIPK1 would be contribute to protection of testicular tissue and spermatogenesis in IR model.

Funding

none