Fig 1.
Rats in every group (except for the sham) were subjected to 3 hours of testicular torsion, followed by 3 hours of detorsion. Four cycles of intermittent ischemia/reperfusion stimuli were performed in the liver and limb ahead of testicular torsion for the induction of liver or limb ischemic preconditioning. BL: baseline; sham: sham-operated; TI/R: testicular ischemia/reperfusion; RIPC (liver): remote liver ischemic preconditioning; RIPC (limb): remote limb ischemic preconditioning. Arrow indicates the time point when U0126 was administrated.
Fig 2.
Remote ischemic preconditioning reduces ipsilateral testicular damage after testicular torsion/detorsion injury.
A. Typical images of ipsilateral testes after TI/R injury. sham: sham-operated; TI/R: testicular ischemia/reperfusion; RIPC (liver): remote liver ischemic preconditioning; RIPC (limb): remote limb ischemic preconditioning. B. TW/BW ratio of the ipsilateral testicular tissues postI/R injury. TW: testicular weight; BW: body weight. ***P<0.001, versus sham-operated rats. ##P<0.01, versus TI/R. n = 7 per group (by one-way ANOVA). C. Changes in ipsilateral testicular MDA levels postdetorsion. MDA: malondialdehyde. ***P<0.001, versus sham-operated rats. ###P<0.001, versus TI/R. n = 6 per group (by one-way ANOVA). D. Changes in ipsilateral testicular SOD levels postdetorsion. SOD: superoxide dismutase. ***P<0.001, versus sham-operated rats. ##P<0.01, versus TI/R. n = 6 per group (by one-way ANOVA). E. Changes in ipsilateral testicular GSH levels postdetorsion. GSH: glutathione. **P<0.01, ***P<0.001, versus sham-operated rats. #P<0.05, versus TI/R. n = 6 per group (by one-way ANOVA).
Fig 3.
The effect of remote ischemic preconditioning on ipsilateral testicular morphology postI/R.
A. Typical H&E-stained ipsilateral testicular sections after TI/R injury. sham: sham-operated; TI/R: testicular ischemia/reperfusion; RIPC (liver): remote liver ischemic preconditioning; RIPC (limb): remote limb ischemic preconditioning. Scale bars, 50 μm. n = 5 rats per group. B. Johnsen score in the ipsilateral testicular tissues postI/R. ***P<0.001, versus sham; ###P<0.001, versus TI/R. n = 5 rats per group (by one-way ANOVA). C. Cosentino score in the ipsilateral testicular tissues postI/R. ***P<0.001, versus sham; ###P<0.001, versus TI/R. n = 5 rats per group (by one-way ANOVA).
Fig 4.
Remote ischemic preconditioning ameliorates ipsilateral testicular apoptosis and the inflammatory response postI/R.
A. TUNEL-stained ipsilateral testicular sections. Green: TUNEL-positive nuclei; Blue: DAPI-stained total nuclei. sham: sham-operated; TI/R: testicular ischemia/reperfusion; RIPC (liver): remote liver ischemic preconditioning; RIPC (limb): remote limb ischemic preconditioning. Scale bars, 20 μm. n = 5 rats per group. B. Apoptotic index (TUNEL-positive neclei*100%/total nuclei). n = 5 per group. ***P<0.001, versus sham; ###P<0.001, versus TI/R (by one-way ANOVA). C. The immunostaining for Bcl-2 and Bax in ipsilateral testicular sections after detorsion. Scale bars, 50 μm. D. The level of immunoreactivity of Bcl-2. n = 5 per group. ***P<0.001, versus sham; ###P<0.001, versus TI/R (by one-way ANOVA). E. The level of immunoreactivity of Bax. n = 5 per group. ***P<0.001, versus sham; ###P<0.001, versus TI/R (by one-way ANOVA). F. Bcl-2/Bax ratio. n = 5 per group. ***P<0.001, versus sham; ###P<0.001, versus TI/R (by one-way ANOVA). G. The immunostaining for IL-6 and TNF-α in ipsilateral testicular sections after detorsion. Scale bars, 50 μm. H. Testicular immunoreactivity levels of IL-6 protein. n = 5 per group. ***P<0.001, versus sham; ###P<0.001, versus TI/R (by one-way ANOVA). I. Testicular immunoreactivity levels of TNF-α protein. n = 5 per group. ***P<0.001, versus sham; ##P<0.01, versus TI/R (by one-way ANOVA).
Fig 5.
Remote ischemic preconditioning reduces contralateral testicular damage after testicular torsion/detorsion injury.
A. Typical images of contralateral testes after TI/R injury. sham: sham-operated; TI/R: testicular ischemia/reperfusion; RIPC (liver): remote liver ischemic preconditioning; RIPC (limb): remote limb ischemic preconditioning. B. TW/BW ratio of the contralateral testicular tissues postI/R injury. TW: testicular weight; BW: body weight. **P<0.01, versus sham-operated rats. ##P<0.01, versus TI/R. n = 7 per group (by one-way ANOVA). C. Changes in contralateral testicular MDA levels postdetorsion. MDA: malondialdehyde. ***P<0.001, versus sham-operated rats. ###P<0.001, versus TI/R n = 6 per group (by one-way ANOVA). D. Changes in contralateral testicular SOD levels postdetorsion. SOD: superoxide dismutase. **P<0.01, ***P<0.001, versus sham-operated rats. #P<0.05, versus TI/R. n = 6 per group (by one-way ANOVA). E. Changes in contralateral testicular GSH levels postdetorsion. GSH: glutathione. **P<0.01, ***P<0.001, versus sham-operated rats. #P<0.05, versus TI/R. n = 6 per group (by one-way ANOVA).
Fig 6.
The effect of remote ischemic preconditioning on contralateral testicular morphology postI/R.
A. Typical H&E-stained contralateral testicular sections after TI/R injury. sham: sham-operated; TI/R: testicular ischemia/reperfusion; RIPC (liver): remote liver ischemic preconditioning; RIPC (limb): remote limb ischemic preconditioning. Scale bars, 50 μm. n = 5 rats per group. B. Johnsen score in the contralateral testicular tissues postI/R. ***P<0.001, versus sham; ###P<0.001, versus TI/R. n = 5 rats per group (by one-way ANOVA).
Fig 7.
Remote ischemic preconditioning ameliorates contralateral testicular apoptosis and the inflammatory response postI/R.
A. TUNEL-stained contralateral testicular sections. Green: TUNEL-positive nuclei; Blue: DAPI-stained total nuclei. sham: sham-operated; TI/R: testicular ischemia/reperfusion; RIPC (liver): remote liver ischemic preconditioning; RIPC (limb): remote limb ischemic preconditioning. Scale bars, 20 μm. n = 5 rats per group. B. Apoptotic index (TUNEL-positive neclei*100%/total nuclei). n = 5 per group. ***P<0.001, versus sham; ###P<0.001, versus TI/R (by one-way ANOVA). C. The immunostaining for Bcl-2 and Bax in contralateral testicular sections after detorsion. Scale bars, 50 μm. D. The level of immunoreactivity of Bcl-2. n = 5 per group. ***P<0.001, versus sham; ##P<0.01, versus TI/R (by one-way ANOVA). E. The level of immunoreactivity of Bax. n = 5 per group. ***P<0.001, versus sham; ###P<0.001, versus TI/R (by one-way ANOVA). F. Bcl-2/Bax ratio. n = 5 per group. ***P<0.001, versus sham; ##P<0.01, versus TI/R. G. The immunostaining for IL-6 and TNF-α in contralateral testicular sections after detorsion. Scale bars, 50 μm. H. Testicular immunoreactivity levels of IL-6 protein. n = 5 per group. ***P<0.001, versus sham; ###P<0.001, versus TI/R (by one-way ANOVA). I. Testicular immunoreactivity levels of TNF-α protein. n = 5 per group. **P<0.01, ***P<0.001, versus sham; ##P<0.01, versus TI/R (by one-way ANOVA).
Fig 8.
Remote ischemic preconditioning enhances testicular ERK1/2 phosphorylation.
A. Left, western blots of ipsilateral testicular phosphorylated ERK1/2 and total ERK1/2. Right, mean band densities of p-ERK1/2/ERK1/2. n = 4 per group. *P<0.05, ***P<0.001, versus sham; ##P<0.01, versus TI/R (by one-way ANOVA). B. Left, western blots of contralateral testicular phosphorylated ERK1/2 and total ERK1/2. Right, mean band densities of p-ERK1/2/ERK1/2. n = 4 per group. *P<0.05, ***P<0.001, versus sham; ###P<0.001, versus TI/R (by one-way ANOVA).
Fig 9.
Pharmacological inhibition of ERK1/2 abolishes RIPC-induced ipsilateral testicular protection.
A. Left, Typical images of ipsilateral testes after TI/R injury. sham: sham-operated; TI/R: testicular ischemia/reperfusion; RIPC (liver): remote liver ischemic preconditioning; RIPC (limb): remote limb ischemic preconditioning. U: U0126. Right, TW/BW ratio of the ipsilateral testicular tissues postI/R injury. TW: testicular weight; BW: body weight. †P<0.05, ††P<0.01, versus RIPC (liver) rats. ‡P<0.05, ‡‡P<0.01, versus RIPC (limb) rats. n = 6–7 per group. Values for sham, TI/R and RIPC rats are repeated from Fig 2B for comparison (by one-way ANOVA). B. Typical H&E-stained ipsilateral testicular sections after TI/R injury. Scale bars, 50 μm. n = 5 rats per group. C. Johnsen score in the ipsilateral testicular tissues postI/R. n = 5 rats per group. †††P<0.001, versus RIPC (liver) rats. ‡‡‡P<0.001, versus RIPC (limb) rats. n = 5 per group. Values for sham, TI/R and RIPC rats are repeated from Fig 3B for comparison (by one-way ANOVA). D. Consentino score in the ipsilateral testicular tissues postI/R. n = 5 rats per group. †††P<0.001, versus RIPC (liver) rats. ‡‡‡P<0.001, versus RIPC (limb) rats. n = 5 per group. Values for sham, TI/R and RIPC rats are repeated from Fig 3C for comparison (by one-way ANOVA). E. Apoptotic index (TUNEL-positive nuclei*100%/total nuclei). n = 5 per group. †††P<0.001, versus RIPC (liver) rats. ‡‡‡P<0.001, versus RIPC (limb) rats. Values for sham, TI/R and RIPC rats are repeated from Fig 4B for comparison (by one-way ANOVA). F. TUNEL-stained testicular sections. Scale bars, 20 μm. n = 5 rats per group.
Fig 10.
Pharmacological inhibition of ERK1/2 abolishes RIPC-induced contralateral testicular protection. A. Left, Typical images of contralateral testes after TI/R injury.
sham: sham-operated; TI/R: testicular ischemia/reperfusion; RIPC (liver): remote liver ischemic preconditioning; RIPC (limb): remote limb ischemic preconditioning. U: U0126. Right, TW/BW ratio of the contralateral testicular tissues postI/R injury. TW: testicular weight; BW: body weight. ††P<0.01, versus RIPC (liver) rats. ‡P<0.05, ‡‡P<0.01, versus RIPC (limb) rats. n = 6–7 per group. Values for sham, TI/R and RIPC rats are repeated from Fig 5B for comparison (by one-way ANOVA). B. Typical H&E-stained contralateral testicular sections after TI/R injury. Scale bars, 50 μm. n = 5 rats per group. C. Johnsen score in the contralateral testicular tissues postI/R. n = 5 rats per group. †††P<0.001, versus RIPC (liver) rats. ‡‡‡P<0.001, versus RIPC (limb) rats. n = 5 per group. Values for sham, TI/R and RIPC rats are repeated from Fig 6B for comparison (by one-way ANOVA). D. Typical TUNEL-stained contralateral testicular sections (upper) and apoptotic index (TUNEL-positive nuclei*100%/total nuclei, lower). n = 5 per group. †††P<0.001, versus RIPC (liver) rats. ‡‡‡P<0.001, versus RIPC (limb) rats. Values for sham, TI/R and RIPC rats are repeated from Fig 7B for comparison (by one-way ANOVA). Scale bars, 20 μm.
Fig 11.
U0126 diminishes RIPC-induced testicular ERK1/2 phosphorylation.
A. Western blots (upper) and band densities (lower) of p-ERK1/2/ERK1/2 in rat ipsilateral testes with or without U0126 (U). n = 4 per group. †††P<0.001, versus RIPC (liver) rats. ‡‡‡P<0.001, versus RIPC (limb) rats (by one-way ANOVA). B. Western blots (upper) and band densities (lower) of p-ERK1/2/ERK1/2 in rat contralateral testes with or without U0126. n = 4 per group. †P<0.05, ††P<0.01, †††P<0.001, versus RIPC (liver) rats. ‡P<0.05, ‡‡P<0.01,‡‡‡P<0.001, versus RIPC (limb) rats (by one-way ANOVA).