Testicular torsion (T)/detorsion (D) can cause testicular injury due to the rotation of the spermatic cord and its vessels, therefore it represents an urological emergency that is surgically treated. Oxidative damage occurs in the testis and distant organs because of the overproduction of free radicals and overexpression of proinflammatory cytokines by reperfusion after surgery. Cerium oxide (CeO2) nanoparticles, a material also known as nanoceria, have regenerative antioxidant properties on oxidative stress. The present study aimed to investigate the effects of nanoceria on testis tissues in testicular T/D in rats. A total of 24 rats were equally and randomly divided into four groups: Control, CeO2, T/D and CeO2-T/D groups. Left inguinoscrotal incision was performed in the control group. In the CeO2 group, 0.5 mg/kg CeO2 was given intraperitoneally 30 min before inguinoscrotal incision. In the T/D group, unilateral testicular T/D was performed through an inguinoscrotal incision and rotating the left testis 720˚ clockwise, which was then left ischemic for 120 min, followed by 120 min of reperfusion. In the CeO2-T/D group, 0.5 mg/kg CeO2 was given intraperitoneally 30 min before testicular T/D. At the end of the experiment, testis tissues were removed for histopathological and biochemical examinations. The samples were histologically examined, Glutathione-s transferase (GST), catalase (CAT), paraoxonase (PON) activities and malondialdehyde (MDA) levels were measured via biochemical analysis methods, while the expression levels of p53, Bax and Bcl-2 were detected using immunohistochemistry. The present results revealed statistically significant inter-group differences in PON, CAT and GST activities and MDA levels. GST, CAT and PON activities were significantly higher, whereas MDA levels in the CeO2-T/D group were significantly lower compared with those in the T/D group. The T/D group had increased Bax and decreased Bcl-2 expression levels in their seminiferous tubules compared with the control and CeO2 groups. CeO2 treatment led to downregulation of Bax and upregulation of Bcl-2. The expression of p53 was high in the T/D group compared with that in the control and CeO2 groups, and was upregulated in all germinal cells. However, compared with that in the T/D group, p53 expression was significantly decreased in the CeO2-T/D group. The testicular injury score significantly increased in the CeO2-T/D group compared with the control and CeO2 groups. Rats in the CeO2-T/D group demonstrated significantly milder tissue lesions compared with those in T/D group. The present findings indicated that nanoceria may protect testis in rats against the harmful effects of T/D. Further studies are required to evaluate how CeO2 reduces oxidative stress and cell death in testis tissue that underwent T/D-related injury.
Testicular injury due to the rotation of the spermatic cord and its vessels caused by testicular torsion (T)/detorsion (D) represents an urological emergency (
Testicular T/D is responsible for testicular damage and necrosis due firstly to ischemic (I) injury and secondly to reperfusion (R) injury after D (
Nanotechnology is currently employed as a tool to explore the darkest avenues of medical sciences in several ways, such as in imaging (
The present study aimed to investigate the effect of cerium oxide on pathological and biochemical markers from testicular tissue after I/R injury in a testicular T and D model, based on the anti-inflammatory and antioxidant effects previously emphasized.
A total of 24 Wistar albino, male rats (12 months old, weighing 250-300 g) were used in the present study, supplied by Gazi University Experimental Animals Research Center (Ankara, Turkey), which was approved by the Gazi University Ethics Committee (approval no. G.U.ET-19-059). Rats were kept in a temperature-controlled (21±1˚C) and humidity-controlled (45-55%) room, which was maintained on a 12/12 reversed light cycle. Animals were fed with a standard pellet and allowed to drink water
Control group rats were only subjected to midline laparotomy. CeO2 group rats underwent surgical left inguinoscrotal incision and cerium oxide was given via i.p. injection (0.5 mg/kg) 30 min before the incision period.
In the T/D group, following left inguinoscrotal incision, animals underwent unilateral testicular T by 720˚ clockwise rotation of the left testis that was subsequently fixed within the hemiscrotum using a 4/0 atraumatic silk suture. After 120 min of ischemia, rats underwent a spermatic cord D procedure that was followed by reperfusion for 120 min. Sodium heparin (500 IU/kg) was administered through the peripheral vein in the tail for the maintenance of reperfusion after occlusion.
In the T/D + CeO2 group, cerium oxide (Sigma Aldrich; Merck KGaA) was given (i.p 0.5 mg.kg-1) 30 min prior to the ischemic procedure. Following left inguinoscrotal incision, animals underwent unilateral testicular T by 720˚ clockwise rotation of the left testis that was subsequently fixed within the hemiscrotum using a 4/0 atraumatic silk suture. After 120 min, rats underwent spermatic cord D procedure that was followed by reperfusion for 120 min. Sodium heparin (500 IU/kg) was administered through the peripheral vein in the tail for the maintenance of reperfusion after occlusion.
Following reperfusion, blood samples were collected from the abdominal aorta. Subsequently, rats were anesthesized using ketamine (100 mg/kg) and xylazine (10 mg/kg) i.p. injection and sacrified by taking intracardiac blood with an injector. After heartbeat and respiration ceased, these were monitored for further 2 min to confirm death. Testicular tissue samples were obtained for subsequent biochemical and histopathological analyses.
Testicular tissue samples were fixed in 10% neutral formaldehyde for 48 h at room temperature (RT), dehydrated and embedded in paraffin. Cross-sections of 4-µm thickness were sliced from the paraffin blocks using a microtome (Thermo Fisher Scientific, Inc.). The sections were deparaffinized in xylenes using three changes for 10 min each at RT and rehydrated in a descending ethanol series. Tissue specimens were stained with H&E for 10 min at RT and examined using a Nikon Eclipse 80i light microscope (Nikon Corporation). Histopathological changes in the testicular specimens were evaluated according to a four-level grading system proposed by Cosentino
The paraffin embedded sections were deparaffinized and rehydrated in a descending alcohol series. For heat-induced antigen retrieval, the sections were placed in citrate buffer (pH 6.0) and boiled 3 times for 5 min each using a microwave oven at 700 W. Endogenous peroxidase activity was blocked with 3% H2O2 and the epitopes were stabilized using serum blocking solution (Ultra V Block) for 5 min at RT (Thermo Fisher Scientific, Inc.). Sections were then incubated overnight at 4˚C with PBS containing primary antibodies against Bax (1:100; cat. no. E-AB-33819; Elabscience Biotechnology, Inc.), Bcl-2 (1:100; cat. no. E-AB-60012; Elabscience Biotechnology, Inc.), caspase-3 (1:100; cat. no. E-AB-63602; Elabscience Biotechnology, Inc.) and p53 (1:100; cat. no. E-AB-60866; Elabscience Biotechnology, Inc.). Following incubation with primary antibody, the sections were incubated with biotinylated goat anti-polyvalent secondary antibody and streptavidin peroxidase (cat. no. TP-125-HL; Thermo Fisher Scientific, Inc.) for 10 min each at RT. PBS was used to wash the sections between each step. The binding sites of antibody were visualized using 3,3'-diaminobenzidine (Thermo Fisher Scientific, Inc.). The sections were counterstained with Harris's hematoxylin for 30 sec at RT, evaluated under a Nikon Eclipse 80i light microscope (magnification, x100; Nikon Corporation). ImageJ analysis software (version 1.52; National Institutes of Health) was used to assess staining intensity of the antibodies in testis tissues (
Testicular tissues were washed with cold NaCl solution (0.154 M) to discard blood contamination and then homogenized in a Diax 900 (Heidolph Instruments GmbH and Co KG) at 1,000 rpm for ~3 min. After centrifugation at 10,000 x g for ~60 min at 4˚C, the upper clear supernatant was subjected to further analysis.
Malondialdehyde (MDA) levels were measured using the spectrophotometric thiobarbituric acid reactive substances method developed by Van Ye
Catalase (CAT) activity was measured using the method developed by Aebi (
Glutathione S-transferases (GST) activity was measured using the method described by Habig
SPSS statistical software, version 24.0 (IBM Corp.) was used for statistical analyses. The distribution of data was analysed with the Shapiro-Wilk test and Q-Q plot test. The results were analysed using the Kruskal-Wallis test followed by Dunn's test or one-way ANOVA followed by Tukey's test. All quantitative data are expressed as means±standard deviation. P<0.05 was considered to indicate a statistically significant difference.
In the histopathological examination of testicular tissues of both control (
The results of Johnsen's scoring demonstrated that spermatogenesis was normal in both control (9.33±0.71) and CeO2 groups (9.17±0.83) (
When seminiferous tubular diameter measurements were evaluated among the groups, a significant decrease was revealed in T/D group (289.21±25.68) compared with the control (329.05±32.36) and CeO2 (327.06±33.88) groups (P<0.0001;
Immunohistochemical analysis of the Bax/Bcl-2 expression demonstrated that rats in the control and CeO2 groups had low Bax expression in a few spermatogonia and spermatozoa and high Bcl-2 expression in all germinal cells including spermatogonia, spermatocytes, spermatids and spermatozoa (
MDA level was significantly increased in the T/D group compared with the control (P=0.003) and CeO2 (P=0.004) groups in the testicular tissue (
The CAT enzyme activity in the T/D group was significantly higher compared with that in the control and CeO2 groups (P<0.0001;
The PON-1 enzyme activity in the T/D group was significantly lower compared with that in the control (P=0.011) and CeO2 (P=0.029) groups (
GST enzyme activity was revealed to be significantly increased in the T/D group compared with the control group in the testicular tissue (P=0.027). A significant decrease in GST enzyme activity was observed in the T/D + CeO2 group compared with T/D group (P=0.012;
Testicular T can produce germ cell damage, resulting in subfertility or infertility (
Nanotechnology is making striking developments in different areas of human life (
In the present study, nanoceria was injected in rats because of the very low absorption of nanoparticles by inhalation or oral administration (
Although SOD and CAT enzymes, which are in the antioxidant enzyme group, generally show similar trends in previous I/R studies, there are also studies with contradictory results. Islekel
Several biomarkers have been identified to accurately assess the apoptotic process (
Apoptosis is an active form of cell death considered to occur in adult tissues in a wide range of physiological settings such as metamorphosis, tissue removal and several other conditions (
Another important gene involved in these specific alterations is Bax (
In the histopathological evaluation, rats in the T/D group had severe testicular degenerative changes characterized by loss of cohesion in germinal cells, shedding of germinal cells within the seminiferous tubules and coagulative necrosis. Testicular injury score was significantly increased in this group compared with the control and CeO2 groups. Rats in the T/D + CeO2 group demonstrated milder tissue lesions compared with the T/D group and a near-normal appearance was observed. Seminiferous tubules were relatively intact and germinal cells are more adherent. Germinal cells with degenerated shells and multinucleated giant cells were present in some tubules. Tubular atrophy was decreased and marginal irregularities were observed in only a few tubules. Haemorrhage and vascular oedema were also reduced. Johnsen's scoring results demonstrated that spermatogenesis was normal in both the control and CeO2 groups, while in the T/D group this was significantly decreased compared with the control and CeO2 groups. A significant increase was observed in the Johnsen's score in the T/D + CeO2 group compared with the T/D group. Histopathological results and Johnson's scores are consistent with with the studies from Saleh
In the present study CeO2 significantly reduced testicular damage after testicular T/D and increased the Johnsen's score in histopathological examination. The increased MDA levels, SOD and GST activities along with decreased PON-1 activities in testicular tissues may reflect cellular oxidative stress or an involvement of these enzymes in compensatory mechanisms. CeO2 treatment significantly reduced the expression of caspase-3 and p53 in the seminiferous tubules. In the biochemical analysis, a significant decrease in MDA level and CAT activity, along with a significant decrease in GST enzyme activity, were detected after CeO2 treatment. The present results demonstrated that CeO2 had a positive effect after testicular T/D.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
AA, SY and MK were responsible for designing the study, and analyzing and interpreting the data. CO performed the study in the laboratory in accordance with the methodology. MA was responsible for the acquisition, analysis and interpretation of the data. MA and SY confirm the authenticity of all the raw data. ACG and MK provided scientific and technical assistance to the experiments, and critically revised the article for important intellectual content. SY collected samples and was responsible for the execution of the project. ACG was responsible for the cellular and molecular experiments. All authors read and approved the final manuscript.
Ethical approval for the study was obtained from Gazi University Experimental Animals Ethics Committee (Ankara, Turkey; approval no. G.U.ET-19-059).
Not applicable.
The authors declare that they have no competing interests.
Representative photomicrographs of H&E stained testicular sections. (A and B) Control and (C and D) CeO2 groups showing regular seminiferous tubule morphology with orderly arranged germinal cells (arrowheads) between Sertoli cells (white arrows) resting on intact basement membrane (black arrows). Tubules are separated by normal interstitial tissue containing Leydig cells (yellow arrows) located around blood vessels (red arrows). Scale bars, 100 µm. H&E, hematoxylin and eosin.
Representative photomicrographs of H&E stained testicular sections of T/D group. (A) Haemorrhage and oedema (asterisk) in the interstitial area. (B) Indistinctive impaired borders of the seminiferous tubules (black arrows). (C) Disordered sloughed germinal cells with shrunken pyknotic nuclei (black arrow) and hyalinization of the seminiferous tubules (white arrow). (D) Detachment of the germinal cells from the basement membrane (black arrow), shrunken tubules (white arrow) and atrophy of germinal epithelium (arrowhead). Scale bars, 100 µm. T, torsion; D, detorsion. H&E. hematoxylin and eosin.
Representative photomicrographs of H&E stained testicular sections of T/D + CeO2 group. (A) Intact seminiferous tubules with cohesive germinal cells attached to the basement membrane and regressed haemorrhage (arrow). (B) Tubules displaying sloughed germinal cells (arrow). (C) degenerated germinal cells (arrow). (D) Multinucleated giant cells (arrow). Scale bars, 100 µm. T, torsion; D, detorsion; H&E, hematoxylin and eosin.
Quantitative evaluation of tubular damage. Comparisons among groups in terms of (A) Cosentino's score, (B) Johnsen's score and (C) seminiferous tubule diameter indicate that CeO2 treatment reduced the T/D related tissue lesions in rat testis. *P<0.0001 vs. control group and CeO2 group. #P<0.0001 vs. T/D group. T, torsion; D, detorsion; CeO2, Cerium oxide.
Immunohistochemical analysis of Bax and Bcl-2 proteins in testicular tissue. T/D group shows higher Bax and lower Bcl-2 protein expression compared with the control and CeO2 treated group. T/D + CeO2 group shows lower Bax expression and higher Bcl-2 protein expression compared with the T/D group. Black, yellow and white arrows indicate the Bax- and Bcl-2-positive spermatogonia, spermatocytes and spermatozoa, respectively. Scale bars, 100 µm. T, torsion; D, detorsion; CeO2, Cerium oxide.
Immunohistochemical analysis of caspase-3 and p53 proteins in testicular tissue. T/D group shows higher caspase-3 and p53 protein expression compared with the control and CeO2 groups. T/D + CeO2 group shows caspase-3 and p53 expression in respect to the T/D group. Black, yellow and white arrows in the enlarged micrographs indicate the caspase-3- and p53-positive spermatogonia, spermatocytes and spermatozoa, respectively. Scale bars, 100 µm. T, torsion; D, detorsion; CeO2, Cerium oxide.
Cosentino's
Score | Features |
---|---|
Grade 1 | Normal testicular structure with an orderly arrangement of germinal cells |
Grade 2 | Less orderly, non-cohesive germinal cells and closely packed seminiferous tubules |
Grade 3 | Disordered sloughed germinal cells with shrunken pyknotic nuclei and impaired borders of the seminiferous tubules |
Grade 4 | Seminiferous tubules tightly surrounded by coagulative necrosis of germinal cells |
Johnsen scoring system (
Score | Features |
---|---|
10 | Complete spermatogenesis with several spermatozoa and regular tubules |
9 | Slightly impaired spermatogenesis with several late spermatids and disorganized germinal epithelium |
8 | Less than five spermatozoa per tubule with a few late spermatids |
7 | No spermatozoa and late spermatids, several early spermatids |
6 | No spermatozoa and late spermatids, few early spermatids |
5 | No spermatozoa or spermatids, several spermatocytes |
4 | No spermatozoa or spermatids, few spermatocytes |
3 | Only spermatogonia |
2 | No germinal cells, Sertoli cells only |
1 | No seminiferous epithelium |
Comparison of staining intensity of the apoptosis-related proteins between groups.
Protein | Control (n=6) | CeO2 (n=6) | T/D (n=6) | T/D + CeO2 (n=6) | Multiple comparison | P-value |
---|---|---|---|---|---|---|
Bax | 24.61±3.67 | 21.81±3.21 |
128.68±7.27 |
65.99±6.39 |
Control vs. CeO2 | 0.0026 |
Control vs. T/D | <0.0001 | |||||
Control vs. T/D + CeO2 | <0.0001 | |||||
T/D vs. CeO2 | <0.0001 | |||||
T/D vs. T/D + CeO2 | <0.0001 | |||||
Bcl-2 | 117.77±8.01 | 107.59±12.32 |
32.20±4.43 |
63.16±6.21 |
Control vs. CeO2 | 0.0001 |
Control vs. T/D | <0.0001 | |||||
Control vs. T/D+CeO2 | <0.0001 | |||||
T/D vs. CeO2 | <0.0001 | |||||
T/D vs. T/D+ CeO2 | <0.0001 | |||||
Caspase-3 | 19.34±2.27 | 21.81±2.80 |
78.07±5.45 |
35.60±3.39 |
Control vs. CeO2 | 0.1098 |
Control vs. T/D | <0.0001 | |||||
Control vs. T/D+CeO2 | <0.0001 | |||||
T/D vs. CeO2 | <0.0001 | |||||
T/D vs. T/D+ CeO2 | <0.0001 | |||||
p53 | 36.97±4.77 | 35.42±4.45 |
104.75±4.63 |
80.79±6.18 |
Control vs. CeO2 | <0.0001 |
Control vs. T/D | <0.0001 | |||||
Control vs. T/D+CeO2 | <0.0001 | |||||
T/D vs. CeO2 | <0.0001 | |||||
T/D vs. T/D+ CeO2 | <0.0001 |
Values are expressed as mean ± standard deviation.
aStatistically different from the control group (P<0.05).
bStatistically different from the T/D group (P<0.05). CeO2, cerium oxide; T, torsion; D, detorsion.
MDA level and CAT, GST and PON-1 enzyme activities.
Features | Control (n=6) | CeO2 (n=6) | T/D (n=6) | T/D + CeO2 (n=6) | Multiple Comparison | P-value |
---|---|---|---|---|---|---|
MDA, nmol/mg protein | 3.98±1.03 | 4.10±0.48 |
7.06±2.82 |
4.31±0.82 |
Control vs. CeO2 | 0.894 |
Control vs. T/D | 0.003 | |||||
Control vs. T/D + CeO2 | 0.720 | |||||
T/D vs. CeO2 | 0.004 | |||||
T/D vs. T/D + CeO2 | 0.007 | |||||
CAT, IU/mg | 238.04±43.40 | 299.19±65.49 |
1,201.68±243.47 |
642.18±117.14 |
Control vs. CeO2 | 0.762 |
Control vs. T/D | <0.0001 | |||||
Control vs. T/D + CeO2 | 0.056 | |||||
T/D vs. CeO2 | <0.0001 | |||||
T/D vs. T/D + CeO2 | 0.011 | |||||
GST, mIU/mg | 36.66±8.73 | 38.36±4.52 | 45.65±8.09 |
35.28±2.75 |
Control vs. CeO2 | 0.657 |
Control vs. T/D | 0.027 | |||||
Control vs. T/D + CeO2 | 0.719 | |||||
T/D vs. CeO2 | 0.067 | |||||
T/D vs. T/D + CeO2 | 0.012 | |||||
PON-1, U/mg | 3.06±0.77 | 2.72±0.51 |
1.03±0.25 |
2.15±0.56 | Control vs. CeO2 | 0.659 |
Control vs. T/D | 0.011 | |||||
Control vs. T/D + CeO2 | 0.243 | |||||
T/D vs. CeO2 | 0.029 | |||||
T/D vs. T/D + CeO2 | 0.171 |
Values are expressed as mean ± standard deviation.
aStatistically different from the T/D group (P<0.05).
bStatistically different from the control group (P<0.05). CeO2, cerium oxide; T, torsion; D, detorsion; MDA, malondialdehyde; CAT, catalase; GST, Glutathione S-transferases; PON-1, serum paraoxonase-1.