Role of endogenous cannabinoids in ischemia/reperfusion injury following testicular torsion in rats

Objectives:   The role of endogenous cannabinoids in ischemia/reperfusion induced germ cell apoptosis in rats was investigated.
Methods:   Baseline group was for basal normal values. The Sham operated group served as a control group. The torsion/detorsion (T/D) group underwent torsion (1 h) and detorsion; AN1, AN2, and AN3 groups received anandamide (10 mg/kg) 30 min before torsion, 30 min after torsion, and just after detorsion, respectively. In the AM251 group, AM251 (0.5 mg/kg) was injected 45 min before torsion and in the AN/AM group, AM251 and anandamide were injected 45 and 30 min before torsion, respectively. Lipid peroxidation, antioxidant enzymes, and germ cell apoptosis was determined.
Results:   Malondialdehyde (MDA) levels in the T/D group were significantly higher than the control group. Moreover, MDA values in the AN1, AN2, and AN3 groups were significantly lower than T/D. There were significant decreases in catalase and superoxide dismutase activities in the T/D group versus the control group. These values in the AN1, AN2, and AN3 groups were significantly higher than T/D. It was also shown that MDA levels in the AN/AM group were significantly higher than the AN1 group. In the AN/AM group, catalase and superoxide dismutase activities were significantly lower versus the AN1 group. The mean germ cell apoptosis scores in all animals with testicular T/D were significantly higher than the control group. There was no difference between the apoptotic indices in the AN1, AN2, AN3, and T/D groups. Apoptosis scores in AM251 and AN/AM were significantly higher compared with the T/D and AN1 groups.
Conclusions:   Although anandamide increased antioxidant markers, it failed to reduce germ cell apoptosis. AM251 worsened the antioxidant defense system, which is reflected as higher germ cell apoptosis.     

Protective effects of the hydroalcoholic extract of Fumaria parviflora on testicular injury induced by torsion/detorsion in adult rats

This study was designed to determine the effects of daily oral administration (250 mg/kg) of the hydroalcoholic extract of Fumaria parviflora (FP) for 14 days on the sperm parameters, oxidative stress parameters, serum testosterone levels, expression of Bax and Bcl‐2 genes, and apoptosis index of germ cells after testicular torsion–detorsion (ischaemia–reperfusion, IR) injury model in rats. Twenty‐eight adult male Wistar rats were divided randomly into four groups of seven each: sham operation, torsion–detorsion (TD), TD plus the hydroalcoholic extract FP (TDFP) and only FP without TD application (FP). Testicular torsion was created by rotating the left testis 720° in a counterclockwise direction; then, after 4 hr, detorsion was performed. The Johnson's score, mean seminiferous tubule diameter (MSTD) and height (thickness) of seminiferous tubule epithelium (HST) were significantly increased in TDFP and FP groups as compared to TD group. The gene expression of Bcl‐2, level of serum testosterone hormone and antioxidant parameters—GPx and SOD—were significantly higher in TDFP and FP groups than TD group. The index of apoptosis, the gene expression of Bax and the level of MDA were significantly higher in TD group than TDFP and FP groups. Therefore, F. parviflora could decrease oxidative stress induced by testicular torsion–detorsion.     

茶多酚对大鼠睾丸扭转/复位模型保护作用的研究

目的:探讨茶多酚对大鼠睾丸扭转/复位模型的保护作用。方法:将24只健康雄性Wistar大鼠随机分为3组,每组8只。第Ⅰ组为假手术组(切开左侧阴囊游离睾丸,但不予扭转),第Ⅱ、Ⅲ组扭转左侧睾丸720°6h,分别于扭转复位前30min腹腔注射生理盐水和茶多酚,术后连续3d分别以低剂量维持。3组大鼠喂养至术后第5天处死,切取左侧扭转睾丸检测睾丸组织中超氧化物歧化酶(SOD)和丙二醛(MDA)含量;以原位缺口末端标记法(TUNEL)检测生精细胞凋亡指数(AI)。结果:Ⅰ、Ⅱ、Ⅲ3组左侧扭转睾丸组织SOD活力分别为(285.00±22.51)、(242.00±17.62)、(261.00±10.01)nU/mg;MDA含量分别为(1.81±0.20)、(4.34±0.34)、(2.94±0.38)nmol/mg;3组之间比较均有显著性差异。Ⅰ、Ⅱ、Ⅲ3组左侧扭转睾丸生精细胞凋亡指数(AI)分别为6.64±1.82、55.23±6.46、31.84±5.56,第Ⅲ组与第Ⅱ组相比,其生殖细胞凋亡明显减少(P<0.05)。结论:茶多酚对因睾丸扭转导致的缺血再灌注损伤具有保护作用。     

Co-administration of Salvia miltiorrhiza and verapamil inhibits detrimental effects of torsion/detorsion on testicular tissue in rats

Testicular torsion/detorsion is one of the important emergencies that requires fast surgical intervention. This study aimed to investigate the effects of Salvia miltiorrhiza hydroalcoholic extract combined with verapamil on testicular ischaemia/reperfusion damage in Wistar albino rats. All animals were distributed in 3 groups (n = 8), including the sham-operated group, torsion/detorsion (TD) group and torsion/detorsion + pretreatment with 200 mg/kg Salvia miltiorrhiza extract combined with 0.3 mg/kg verapamil (SMV) group. Oxidative stress biomarkers (MDA, GPx, CAT and TAC) both in plasma and testicular tissue, sperm parameters (motility, vitality, concentration and morphology) and histopathological parameters (MSTD, GECT, Johnson's score, Cosentino's score and testicular cell thickness) were assessed in all groups. Ischaemia/reperfusion significantly increased MDA and decreased GPx, CAT and TAC levels (p < .05). Pretreatment with SMV significantly increased GPx, CAT and TAC levels (p < .05). SMV group increased progressive sperm motility and vitality and reduced non-progressive motility of spermatozoon (p < .05). Testicular torsion significantly decreased all histopathological parameters compared to the sham group (p < .05). SMV pretreatment remarkably increased MSTD, GECT and Cosentino's score in comparison with the TD group (p < .05). A combination of Salvia miltiorrhiza with verapamil could reduce damages triggered by testicular torsion detorsion and improve sperm functionality parameters and oxidative stress defence systems.     

Protective effect of erythropoietin on torsion/detorsion injury in rat model

Purpose

The aim of the study is to investigate the effects of erythropoietin on torsion/detorsion injury in rats.

Methods

Forty rats were divided randomly into 5 groups: group I (sham, S), sham operation; group II (torsion/detorsion 1, T/D₁), 3 hours ischemia and 1 hour reperfusion; group III (torsion/detorsion 2, T/D₂), 3 hours ischemia and 48 hours reperfusion; group IV (erythropoietin 1, EPO₁), 3 hours ischemia, 1 hour reperfusion, and a single dose of EPO; and group V (erythropoietin 2, EPO₂), 3 hours ischemia, 48 hours reperfusion, and 2 doses of EPO. Malondialdehyde (MDA) and nitric oxide (NO) levels and activities of superoxide dismutase and catalase were measured. Tissue damage to ovarian tissue was scored by histologic examination. Data were compared among groups with parametric tests.

Results

The MDA levels in the S and EPO groups were significantly lower than the T/D groups (P < .001). Catalase and superoxide dismutase activities, and NO levels in the S and EPO groups were significantly higher than in the T/D groups (P < .05). Ovarian tissue damage in the S and EPO groups was significantly less than in the T/D groups (P < .05). Levels of all biochemical markers and ovarian tissue damage scores were similar among the S, EPO₁, and EPO₂ groups (P > .05).

Conclusion

Erythropoietin attenuates ischemia-reperfusion injury when given during the acute phase of ovarian torsion-detorsion in a rat model.     

Dose-dependent protective effect of sildenafil citrate on testicular injury after torsion/detorsion in rats

This experiment was designed to investigate the effect of sildenafil citrate on testicular injury after unilateral testicular torsion/detorsion (T/D). Thirty-seven adult male Wistar albino rats were divided into four groups: sham operated group (group 1), T/D+ saline (group 2), T/D+ 0.7 mg sildenafil citrate (group 3) and T/D+ 1.4 mg sildenafil citrate (group 4). Testicular torsion was created by rotating the right testis 720° in a clockwise direction for 2 h in other groups, except for group 1, which was served as sham group. The level of GSH (P < 0.05) in the testis in the group 2 were significantly lower (P < 0.05) and the levels of MDA and NO (P < 0.01 for both) in the testis were significantly higher when compared with those of the group 1. Administration of low dose sildenafil citrate prevented the increases in MDA and NO levels and decreases in GSH values induced by testicular torsion. However, administration of high dose sildenafil citrate did not have any effect on these testicular tissue parameters (P > 0.05). Also, mean values of seminiferous tubules diameters, germinal cell layer thicknesses and mean testicular biopsy score were significantly better in group 3 than groups 2 and 4. These results suggest that T/D injury occurred in testis after unilateral testicular T/D and that administration of low dose sildenafil citrate before detorsion prevents ischemia/reperfusion cellular damage in testicular torsion. Sildenafil citrate probably acts through reduction of reactive oxygen species and support antioxidant enzyme systems.     

Milrinone ameliorates ischaemia-reperfusion injury in experimental testicular torsion/detorsion rat model

This experimental study aims to evaluate the efficacy of milrinone against ischaemia-reperfusion injury due to testicular torsion/detorsion. Group 1 was defined as the control group. Testicular torsion/detorsion model was performed in Group 2. Group 3 had similar procedures to the rats in Group 2. In addition, 0.5 mg/kg of milrinone was administered intraperitoneally immediately after testicular torsion in Group 3. Histopathological examinations indicated a dramatic improvement in terms of inflammation, haemorrhage, oedema, congestion, Cosentino and Johnson scores in Group 3 compared to Group 2 (p = .037, p = .045, p = .018, p = .040, p = .033 and p = .03 respectively). Blood biochemical analyses, superoxide dismutase (SOD), glutathione peroxidase (GSH-px) activity and total antioxidant status (TAS) levels increased significantly in Group 3 compared to Group 2 (p = .001, p = .024 and p < .001). Malondialdehyde (MDA), protein carbonyl (PC), interleukin 1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and total oxidant status (TOS) levels decreased in Group 3 compared to Group 2 (p = .001, p = .018, p < .001, p = .036 and p = .002 respectively). Tissue biochemical analyses determined an increase in SOD and GSH-px activity in Group 3 compared to Group 2, while PC and MDA levels were reduced (p = .001, p < .001, p = .038 and p < .001 respectively). Milrinone attenuates ischaemia-reperfusion injury that causes highly harmful effects due to testicular torsion/detorsion.     

低温对大鼠睾丸扭转复位后生殖细胞凋亡的影响

目的 探讨低温对大鼠睾丸扭转复位后生殖细胞凋亡的影响。方法 24只健康青春期SD雄性大鼠随机分为三组:A组为睾丸扭转组,B组为睾丸扭转加低温组,C组为对照组。建立单侧睾丸扭转模型。术后第14天采集睾丸。原位缺口末端标记法(TUNEL)检测其生殖细胞凋亡指数(AI),光镜下观察睾丸组织学变化。结果 B组AI明显低于A组(P<0.01),而高于C组(P<0.01)。结论 低温能够提高扭转睾丸耐缺血能力,减少睾丸扭转复位后生殖细胞凋亡。     

Dehydroepiandrosterone treatment attenuates reperfusion injury after testicular torsion and detorsion in rats

Purpose

We aimed to evaluate the effects of dehydroepiandrosterone (DHEA) on antioxidant enzyme activities, lipid peroxidation, and histopathologic changes in both testes after unilateral testicular torsion and detorsion.

Methods

Twenty-four adult male Sprague-Dawley rats were randomly divided into 4 groups (n = 6 for each group): sham operation, torsion/detorsion (T/D), T/D + vehicle, and T/D + DHEA. Three hours before detorsion, 50 mg/kg DHEA was given intraperitoneally to the T/D + DHEA group. Testicular ischemia was achieved by twisting the left testis 720° clockwise for 3 hours, and reperfusion was allowed for 24 hours after detorsion. In all groups, bilateral orchiectomies to determine the testicular tissue catalase (CAT) and superoxide dismutase activities and malondialdehyde (MDA) levels and histopathologic examination were performed.

Results

Compared with those from the sham group, CAT activities in the ipsilateral testis obtained from the T/D group were significantly lower and MDA levels were significantly higher (P < .05 for all).Administration of DHEA prevented increases in MDA levels and decreases in CAT and superoxide dismutase activities when compared to the T/D group. Specimens from the T/D and the T/D + vehicle groups had a significantly greater histologic injury than the specimens from the sham and the T/D + DHEA groups had (P < .01 for both).

Conclusions

The results suggest that DHEA may be a protective agent for preventing biochemical and histopathologic changes related to oxidative stress in testicular injury caused by testis torsion.     

大鼠睾丸扭转生殖细胞凋亡与抗氧化酶和Bcl-2基因表达的关系

目的　探讨睾丸扭转复位后生殖细胞凋亡及其发生机制。方法　建立单侧睾丸扭转复位大鼠模型(72 0° ,2h)。术后 72小时取手术侧睾丸 ,采用TUNEL法检测生殖细胞凋亡 ,免疫组化SP法检测Bcl 2基因表达 ,并测定睾丸组织中超氧化物歧化酶 (SOD)和过氧化氢酶 (CAT)活性。结果　实验组手术侧睾丸生殖细胞凋亡增多 ,Bcl 2基因低表达 ,SOD和CAT活性下降 ,与对照组相比 ,两组差别具有极显著性意义 (P <0 .0 1)。结论　睾丸扭转复位后生殖细胞凋亡增多与Bcl 2基因低表达和抗氧化酶活性下降有关。     

Propofol attenuates reperfusion injury after testicular torsion and detorsion

Propofol, which is widely used as an intravenous anesthetic, has been shown to have an antioxidant activity on several tissues. This study was designed to investigate the prevention of reperfusion injury with propofol after testicular torsion. Five groups of rats (seven in each group) were used. Animals in the control group (group I) did not received any treatment, while animals in the sham group (group II) underwent scrotal incision and testicular fixation only. After 2 h of 720° left testicular torsion in groups III, IV and V, subsequent detorsion was done for 2 h in groups IV and V. Propofol (50 mg/kg) was injected transperitoneally 30 min prior to detorsion in group V. Both testicles in all rats were retrieved and tissue malondialdeyhde (MDA) level, which is a measure of the amount of free oxygen radicals, and enzymatic activity of xanthine oxidase (XO), which converts hypoxanthine to xanthine and uric acid were studied. In addition, tissue catalase (CAT) and glutathione peroxidase (GSH-Px) activities, which are endogenous scavenger enzymes, protecting tissues against free radicals, were studied. Additionally, histological evaluations were performed after hematoxylin and eosin staining. Testicular MDA levels, and XO and CAT activities were higher in the torsion group compared to sham control group (P<0.05). Detorsion caused a further increase in MDA levels, contrasting with a decrease in the levels of XO activity, while CAT activity was not changed. Pretreatment with propofol prevented a further increase in MDA levels and significantly decreased CAT activity following detorsion. GSH-Px activities were not effected either by torsion/detorsion or propofol pretreatment. Histologically, torsion caused some separation between germinal cells in the seminiferous tubules, which became much more prominent in the detorsion group and attenuated with propofol pretreatment. There was no significant change in any of the abovementioned enzymatic activities nor were there histopathological changes in the contralateral testicle in any groups. It is concluded that biochemically and histologically reperfusion injury occurs in the ipsilateral testis following detorsion up to 2 h. Preference of propofol for anaesthesia during the detorsion procedure may attenuate such reperfusion injury.     

生脉注射液对不同周龄大鼠睾丸扭转复位后睾丸损伤影响的研究

目的:探讨生脉注射液对不同周龄大鼠睾丸扭转复位后睾丸损伤影响的差异。方法:3、6、12周龄健康雄性SD大鼠各16只,随机分成睾丸扭转复位+注射生脉注射液组(实验组)和睾丸扭转复位+注射生理盐水组(对照组),每组8只,建立睾丸扭转动物模型(左侧阴囊切开,绕精索顺时针扭转睾丸720°2h),并于手术后24h处死,测定各组大鼠睾丸组织内总抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)活性与丙二醛(MDA)含量。结果:与各自对照组比较,3、6周实验组大鼠左侧睾丸组织中T-AOC、SOD活性和MDA含量均无显著性改变(P>0.05);12周实验组大鼠左侧睾丸组织中SOD、T-AOC明显升高,而MDA含量显著降低,差异均具有显著性(P<0.05)。结论:生脉注射液对睾丸扭转复位后的缺血再灌注急性损伤有保护作用,但对不同周龄大鼠的再灌注损伤保护作用不同,存在年龄相关性差异,对较大周龄大鼠的急性保护作用较为明显。     

Metformin decreases testicular damages following ischaemia/reperfusion injury in rats

The effects of metformin on a testicular torsion injury in adolescent rat testis after I/R were evaluated in the present study. Forty adolescent rats were divided into five groups with eight rats per group: a control group; a sham-operated group; an ischaemia group, where torsion was applied for 4 hr and testis was examined immediately after detorsion; an I/R group, where torsion was applied for 4 hr and the testis was examined 4 hr after detorsion; and an I/R + M group, where the metformin (300 mg/kg) administration was added to the identical procedures used for the I/R group. Spermatogenesis, basal membrane integrity and cleaved caspase-3 expression were assessed. The I/R + M group had a significantly higher Johnsen score than the I/R group (7.9 ± 0.1 vs. 7.5 ± 0.2; p < .001; F-value = 14.2). Failure of basal membrane integrity was highest in the ischaemia group (45 ± 5) compared to the other groups (control group, 20 ± 5; sham-operated group, 16.6 ± 2.8), but not different between the I/R + M (31.6 ± 12.5) and the I/R groups (25 ± 3.5). Cleaved caspase-3 expression was highest in the ischaemia group (73.5 ± 0.7), and significantly lower in the I/R + M group (33.4 ± 0.9) than the I/R group (58.5 ± 0.2; p < .05; F-value = 7.6). Metformin decreases testicular damage by exerting protection against the harmful effects of I/R on spermatogenesis and alleviating apoptosis in adolescent rat testis.     

大鼠单侧睾丸扭转复位后对对侧睾丸的影响

目的研究大鼠单侧睾丸扭转复位后对对侧睾丸的影响。方法16只成年健康SD雄性大鼠随机分为实验组(n=8)和对照组(n=8)；建立单侧睾丸扭转复位模型。术后30d取扭转对侧睾丸,采用原位缺口末端标记法(TUNEL)检测生殖细胞凋亡,光镜下计数精子数。结果与对照组相比,实验组对侧的睾丸重量和日产精子量都有显著性差异(P＜0.05),实验组生殖细胞凋亡显著增多(P＜0.01)。结论大鼠单侧睾丸扭转后,对侧睾丸生殖细胞凋亡增多可能是导致不育的原因之一。     

Reperfusion injury following testicular torsion and detorsion in prepubertal rats

Summary Acute testicular torsion is a surgical emergency which requires immediate intervention. Although damage to the gonad has been well documented, it remains unknown whether the majority of injury occurs during the period of torsion (ischemia) or following detorsion (reperfusion). The aims of this study were to determine: (1) whether damage following testicular torsion-detorsion has a reperfusion component similar to that described in other tissues, and (2) whether iron-catalyzed oxygen radical formation or altered calcium homeostasis plays a role in this injury. To study this, anesthetized prepubertal rats underwent 720° intravaginal testicular torsion and were divided into groups of torsion only (ischemia) and torsion with reperfusion (ischemia/reperfusion). Reperfusion groups were treated prior to detorsion with either deferoxamine (iron chelator), diltiazem (calcium channel blocker), or saline vehicle. The results indicated that detorsion produces a qualitatively distinct reperfusion injury from that of non-reperfused testicles; however, such damage was not ameliorated by deferoxamine or dilitiazem. Thus, testicular torsion-detorsion appears to have a significant reperfusion component that appears to not be mediated by iron-catalyzed oxygen radical formation or calcium injury.     

Protective effect of liraglutide on experimental testicular ischaemia reperfusion in rats

This study was performed to evaluate the effect of liraglutide on experimental testicular ischaemia reperfusion in rats in terms of biochemistry, histopathology and immunohistochemistry. A total of 28 male Wistar-Albino rats were divided randomly into 4 groups: control (7), sham (7), ischaemia-reperfusion (7) and ischaemia-reperfusion + liraglutide (7). Biochemically, Nitric Oxide, Malondialdehyde, Superoxide dismutase, Glutathione peroxidase and Catalase levels were measured in the testis. Apoptosis protease activating factor-1 and inducible nitric oxide synthase activity were evaluated immunohistochemically as well. Statistical analyses were made via the Kruskal–Wallis and Mann–Whitney U tests. In the reperfusion group, CAT and SOD values were increased (p > .05), NO and MDA values were decreased (p < .05) after administration of liraglutide. In addition, GPx values were significantly increased in ischaemia reperfusion + liraglutide administered group compared to reperfusion group (p < .05). Apaf-1 and iNOS activity were significantly decreased with the addition of liraglutide treatment to the ischaemia-reperfusion group (p < .05). First of all, we would like to say that liraglutide treatment is moderately preventive against I/R injury in testicular torsion. The anti-inflammatory, antioxidant and antiapoptotic properties of liraglutide are create a moderately protective effect as we show in this study.     

葡萄籽原花青素对小鼠睾丸扭转复位后生精功能的保护作用

目的:探讨葡萄籽原花青素(GSP)对小鼠睾丸扭转复位后生精功能的保护作用。方法:24只健康雄性昆明小白鼠(8周龄,25～27 g)随机分为3组:对照组、扭转组、治疗组,每组8只。扭转组及治疗组建立单侧睾丸扭转复位动物模型,治疗组于扭转复位前30 min腹腔注射GSP(50 mg/kg),术后采用腹腔注射方式连续给药3 d,每天1次,每次50 mg/kg。扭转组方法同治疗组,治疗同体积生理盐水。术后第4天取扭转侧睾丸,检测组织病理学参数和生精细胞凋亡指数(AI),并检测睾丸组织超氧化物歧化酶(SOD)和丙二醛(MDA)含量。对照组行假手术。结果:治疗组与扭转组相比,Johnsen评分上升[(7.38±0.92)分vs(5.00±1.85)分,P<0.05],生精小管直径略增大[(178.75±1.58)μm vs(176.50±1.60)μm,P>0.05],生精细胞层数增加[(5.75±0.71)层vs(3.75±1.03)层,P<0.05],生精细胞凋亡指数AI明显降低[(16.25±1.67)%vs(40.50±1.60)%,P<0.05)],SOD活性明显上升[(52.67±3.57)U/mg prot vs(29.04±4.46)U/mg prot,P<0.05],MDA含量明显下降[(2.91±0.04)nmol/mg prot vs(4.63±0.05)nmol/mg prot,P<0.05]。结论:GSP对小鼠睾丸扭转复位后生精功能损伤有明显的保护作用,其作用机制可能与其能清除氧自由基、抑制脂质过氧化、提高机体抗氧化能力有关。     

“生精散”对大鼠睾丸扭转复位后生精功能的影响及其机制研究

目的:研究"生精散"对大鼠睾丸扭转复位后生精功能的影响及其机制。方法:40只雄性SD大鼠随机分为假手术组(A组),对照组(B组),生精散组小(C组)、中(D组)、大剂量组(E组),每组8只。建立左侧睾丸扭转模型,B组自扭转前1 h开始给予每日灌胃生理盐水1 ml/d,C组(0.01 g/kg.d)、D组(0.02 g/kg.d)、E组(0.03 g/kg.d)分别按体重给予灌服生精散,连续35 d后处死大鼠,对大鼠进行精液常规分析,用RT-PCR检测大鼠精子CatSper1的表达。结果:a+b级精子百分率、精子存活率、精子浓度,CatSper1基因表达,与A组[(51.30±6.60)%、(69.01±7.20)%、(40.53±7.01)×106/ml、2.04±0.77]相比,B组[(15.30±6.30)%、(44.42±6.36)%、(21.00±6.14)×106/ml、1.12±0.50),均显著降低(P<0.01);与B组相比,D组[(51.63±3.20)%、(72.09±2.20)%、(55.30±5.90)×106/ml、2.11±0.20]、E组[(55.93±3.17)%、(73.01±2.11)%、(58.33±4.90)×106/ml、2.31±0.17]均显著升高(P<0.01),而C组[(18.02±0.23)%、(48.04±7.01)%、(22.87±2.10)106/ml、1.19±0.51]升高不明显(P>0.05)。结论:生精散可以促进睾丸扭转复位后精子质量的恢复,其机制可能与调节生精功能,提高精子细胞CatSper1基因的表达有关。