MISCELLANEOUS Protective Effects of Colchicine on Testicular Torsion/Detorsion-Induced Ischemia/Reperfusion Injury in Rats Kerem Han Gozukara1*, Oguzhan Ozcan2, Tumay Ozgur3, Yusuf Selim Kaya1, Okan Tutuk4 Purpose: To evaluate the short-term use of colchicine on preventing ischemia-reperfusion injury after surgery in an experimental animal model. Materials and Methods: A total of 40 rats were divided into five groups (n = 8). Sham (Sh), ischemia-reperfusion (I/R), I/R and colchicine-treated for once per-operatively (I/Rc1), I/R and colchicine-treated for 5 days postoper- atively (I/Rc5), and I/R and placebo given for 5 days (I/Rp) groups. Testicular torsion was created by rotating the testicle 720o in clockwise direction and held for 3 hours. In group I/Rc1 30 minutes before detorsion, p.o. 1 mg/kg mL infusion of colchicine was given only once. In group I/Rc5, colchicine continued p.o. once daily for five days. Tissue malonyldialdehite (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) were measured for evaluating the oxidative stress. Apoptosis levels shown with Caspase-3 staining and mean sem- iniferous tubular diameter (MSTD), germinal epithelial cell thickness (GECT), and mean testicular biopsy score (MTBS) were used to evaluate the germ cell damage. Results: Decreased protein MDA levels therewithal increased SOD, CAT and GPx levels achieved in I/Rc5 group when compared to I/R group and did not differ from the I/Rp group (p < 0.05). MSTD, GECT, and JS were better in I/Rc5 than I/Rp which showed the natural course of I/R damage in testis (p < 0.005). Caspase 3 positivity, as an apoptosis indicator, were significantly lower (p < 0.05) in I/Rc5 group in comparison with I/R, I/Rc1, and I/Rp groups. Conclusion: The usage of colchicine as a complementary treatment after definitive surgery reduce early-onset ischemia-reperfusion damage and diminishes apoptosis. Keywords: colchicine; perfusion; injury; rat; torsion INTRODUCTION As a urologic emergency, testicular torsion is seen in 4,5/100000 males aged 1-25.(1) This situation may result in testicular atrophy in the absence of defini- tive surgery within hours. Detorsion of the testis causes ischemia/reperfusion (I/R) injury. The primary indica- tors of I/R injury are lipid peroxidation and apoptosis arisen from neutrophil recruitment, reactive oxygen species (ROS) and proinflammatory cytokines.(2) Low concentrations of ROS promotes sperm capacitation but excessive production acts quite the contrary.(3) High levels of ROS degrade polyunsaturated lipids which are found in the plasma membrane of spermatozoa, forming malondialdehyde (MDA) that is an indicator of oxidative stress. On the other side, several protec- tive anti-oxidant scavenger enzymes such as superox- ide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) neutralize free radicals. In the presence of excessive ROS production cellular dysfunction and related apoptosis begin. 1 Urology Department , Mustafa Kemal University School of Medicine, Turkey. 2 Biochemistry Department , Mustafa Kemal University School of Medicine, Turkey. 3 Pathology Department, Mustafa Kemal University School of Medicine, Turkey. 4 Physiology Department, Mustafa Kemal University School of Medicine, Turkey. *Correspondence: Mustafa Kemal University, School of Medicine, Tayfur Ata Sokmen Hospital Urology Department 4th floor /19, 31010, Serinyol, Hatay, TURKEY. Phone : 903262900-3379 (int.) Mobile: 905057462464. E-mail: keremgozukara@gmail.com. Received October 2018 & Accepted June 2019 Apoptosis is a physiologic mechanism used to eliminate nonfunctioning and undifferentiated cells.(4) Apoptosis occurs in two important pathways, intrinsic and extrin- sic whom the caspases are the unique markers of entire processes. The cell death cascade was initiated either with the death receptor-mediated namely extrinsic way or with the mitochondrion-mediated procaspase-activa- tion pathway known as the intrinsic way.(5) In caspase family, caspase-3 is a significant mediator of both apop- totic and necrotic cell death by inhibiting proteins that are vital for repairing DNA injury, intercellular signal transmission, cell cytoskeleton and continuation of the cell cycle.(6) Colchicine is a tricyclic alkaloid of the Colchicum Autumnale plant which is a member of Colchicaceae plant family, one of the oldest therapeutic substances known to mankind. It has been used for Gout disease, Familial Mediterranean Fever, acute pericarditis, acute arthritis, and Behçet's Disease.(7) The suppressing effect of colchicine on inflammation has not been enlightened precisely yet, but the prevailing opinion is inhibition of Urology Journal/Vol 17 No. 3/ May-June 2020/ pp. 294-300. [DOI: 10.22037/uj.v0i0.4918] the neutrophils and endothelial adhesion molecules by disrupting microtubule polymerization in leucocytes.(8) We hypothesize that the anti-inflammatory effect of colchicine may be beneficial in I/R injury which is closely related to leucocyte accumulation, apoptosis, and increased tissue ROS levels. Therefore, we aimed to evaluate the protective effect of colchicine treatment on ischemia-reperfusion damage in a testicular torsion rat model by Caspase-3 staining. MATERIALS AND METHODS Experimental Design The study was conducted in Mustafa Kemal University School of Medicine Animal Laboratory by local eth- ical committee approval (Date and decision number: 2015.02.19 / 8). A total of 40 Wistar albino adult male rats weighing 320-440 g were included in the study. Rats were caged individually in a controlled environ- ment at 200C to 220C room temperature, 50% to 55% relative humidity, and light/dark cycles of 12 hours and were fed ad libitum. They were acclimatized for ten days. In all procedures that were applied to the animals according to local ethical committee laboratory rules and rules of Guidelines for the Care and Use of Labo- ratory Animals of the US National Institutes of Health (Washington, DC) was obeyed. Rats were randomly divided into five groups (n:8). In- tramuscular ketamine hydrochloride (50 mg/kg Ketalar; Eczacıbasi, Istanbul, Turkey( and xylazine hydrochlo- ride were used for anesthesia. Rats were placed in a Effectiveness of colchicine in testis torsion-Gozukara et al. Figure 1. Caspase-3 staining in groups Figure 2. Morphology and spermatogenesis in Group 4, 5. A: Impaired and aborted spermatogenesis (→) with disorganised epitelium, some germ cells absent, necrotic or degenerated in Group 5 (Hematoxylen&Eosinx200). B: Preservation of spermatogenesis in some of the seminifer tubules with regular and compact germ cells (→) in Group 4 (Hematoxylen&Eosinx200). Vol 17 No 03 May-June 2020 295 Miscellaneous 296 dorsal recumbent position in a sterile condition. Under anesthesia the scrotal area was shaved and cleaned, dis- infected by povidone-iodine solution and a scrotal mid- line incision was done. Torsion was performed by ro- tating the spermatic cord 720 degrees clockwise along the longitudinal axis and fixed in the scrotal pouch by 5/0 nontraumatic absorbable suture.(9) The scrotal inci- sion was covered with a moist, warm sterile cover for 3 hours and the surgical technique was the same for all groups. In the Sham group, only a scrotal incision was performed. In I/R group, after 3 hours of testicu- lar torsion, testicle was detorsioned and reperfusioned for 3 hours and then orchiectomy was done. In I/Rc1, following 3 hours of ischemia; p.o. 1mg/kg mL col- chicine (Recordati Pharmaceutical Industry and Trade Inc., Esenyurt, Istanbul) was given 30 minutes prior to detorsion, and then the testicle was removed after 3 hours of reperfusion. In I/Rc5 group, p.o. 1mg/kg mL colchicine was given 30 minutes before detorsion and continued for five days postoperatively, and the testicle was removed. In I/Rp, all procedures were followed the same as the I/Rc5 group; only placebo (0,2 mL of serum physiologic; %0.9 p.o.) was given instead of colchicine. At the end of the experiment, under the general anes- thesia, for histologic and biochemical evaluation 5-6 cc blood was taken from the heart of the rats and eutha- nasia was applied by exsanguination. The biochemical examination was performed for determining the oxida- tive stress levels and studying antioxidant agents in the blood and tissue specimens over MDA, SOD, CAT, and GPx. Pathology methods A pathologist -blinded to the study protocol- evaluated prepared slides, the mean seminiferous tubular diame- ter (MSTD), germinal epithelial cell thickness (GECT), and mean testicular biopsy score (MTBS) were used to evaluate in 20 seminiferous tubules of each section. (10) The MSTD was calculated using an eyepiece mi- crometer (ZA3262, U-OCMC, 24 mm cross, 10/100X) mounted within one of the eyepiece objectives. At 400X power the field is 0.44 mm x 0.44 mm, yielding an area of approximately 0.19 mm2. The MSTD of each testis was determined in microns. GECT was determined by counting the number of epithelial cells from the basement membrane to the lumen at 90°, 180°, 270°, and 360°, and averaged. The MTBS was graded using Johnsen's score.(11) A score of 1 to 10 was given to each tubule according to epithelial maturation: 10, complete spermatogenesis, with many spermatozoa, and germi- nal epithelium organized with a regular thickness, leav- ing an open lumen; 9, many spermatozoa present but germinal epithelium disorganized with marked slough- ing or obliteration of the lumen; 8, only a few spermato- zoa (fewer than 5 to 10) present; 7, no spermatozoa but many spermatids present; 6, no spermatozoa and only a few spermatids (fewer than 5 to 10) present; 5, no sper- matozoa and no spermatids but several or many sper- matocytes present; 4, only a few spermatocytes (fewer than 5) and no spermatids or spermatozoa present; 3, spermatogonia the only germ cells present; 2, no germ cells, but Sertoli cells present; and 1, no cells present in tubular section. Table 1. The body and testicular weights of the rats for all 5 groups n Weight g Testis Weight g Sham 8 350.5 ± 26 0.72 ± 0.17 a I/R 8 379.37 ± 29 0.58 ± 0.11 I/Rc1 8 350.5 ± 26 0.57 ± 0.09 I/Rc5 8 376.63 ± 30 0.49 ± 0.15 I/Rp 8 367.37 ± 28 0.49 ± 0.08 P values > 0.005 .009 Findings of the parameters were expressed as mean ± standart de- viation. Only statistically significant groups were marked with the letter of the alphabet ap =.012 Sham vs I/Rc5; p = .014 Sham vs I/Rp n MDAprot nmol/g SODprot U/g CATprot k/mg protein GPXprot U/ g protein Sham 8 113.4 ± 22,6a 1401.5 ± 189.5e 0.12 ± 0.017 h 277.6 ± 31.6 l I/R 8 336.4 ± 62.8 b 790.9 ± 177.2f 0.067 ± 0.011i 141.37 ± 26.1m I/Rc1 8 364.1 ± 65.2 c 910.5 ± 136.4g 0.069 ± 0.01j 141.8 ± 31.6n I/Rc5 8 188.1 ± 74.9 1246.4 ± 232.1 0.11 ± 0.019 k 215.8 ± 43 I/Rp 8 359.6 ± 87.3d 978 ± 171.1 0.069 ± 0.026 151.4 ± 36.4 P values < 0.001 < 0.001 < 0.001 < 0.001 MDAprot, Tissue Malonyldialdehite; SODprot, Tissue Superoxide Dismutase; CATprot, Tissue Catalase; GPXprot, Tissue Glutathione Peroxidase. Findings of the parameters were expressed as mean ± standart deviation. Only statistically significant groups were marked with the letter of the alphabet. ap < 0.001 Sham vs I/R; p < 0.001 Sham vs I/RC1; p = .001 sham vs IRp bp = .007 IR vs IRc5 cp = .002 I/Rc1 vs I/Rc5 dp = .008 I/Rp vs I/Rc5 ep < 0.001 Sham vs I/R; p < 0.001 Sham vs I/RC1; p = .003 sham vs IRp fp = .006 IR vs IRc5 gp = .04 I/Rc1 vs I/Rc5 hp < 0.001 Sham vs I/R; p < 0.001 sham vs I/RC1; p < 0.001 sham vs IRp ip = .004 IR vs IRc5 jp = .005 I/Rc1 vs I/Rc5 kp = .006 I/Rp vs I/Rc5 lp < 0.001 Sham vs I/R; p < 0.001 Sham vs I/RC1; p < 0.001 Sham vs IRp mp = .01 IR vs IRc5 np = .0016 I/Rc1 vs I/Rc5 Table 2. Biochemical results of all 5 groups Effectiveness of colchicine in testis torsion-Gozukara et al. Immunohistochemistry Section of 3-4 mm thickness was cut from the paraf- fin blocks of these preparations and then was de-par- affinized and rehydrated through a graded series of al- cohol, microwave antigen retrieval method was used, endogenous peroxidase was blocked in 5% H2O2 at room temperature for 8 minutes, followed by washing with PBS three times. The slices were then incubated with Caspase 3 (CPP32-Ab-4) (Prediluted Polyclonal Rabbit primer antibody, Thermo Scientific, Freemont, CA, USA.) Immunohistologic staining (IHS) was ap- plied, followed by washing with PBS three times. Sub- sequently, the slices were incubated with biotinylated goat anti-polyvalent/labvision secondary antibodies at room temperature for one h, washed with PBS three times, followed by incubation with DAB reagent for 8 min at room temperature. Nuclear staining in tonsil tissue was accepted as the positive control. Caspase-3- positive cells were count in one mm2 of the tissue mod- ified from the study of Mosadegh et al.(12) Biochemical methods Testis tissues were weighed and homogenized in ice- cold phosphate-buffered saline at pH 7.4 (10% w / v). After centrifugation at 10 000 rpm for 20 minutes, all supernatants were removed for biochemical analysis. Protein levels of supernatants and homogenates were measured by the Bradford method using bovine serum albumin as a standard.(13) The MDA levels of homogenates were measured by the double heating method of Draper and Hadley.(14) MDA equivalents (1,1,3,3-tetramethoxypropane, Lot no, MK- BP9901V, Sigma-Aldrich) were used as standards, and MDA results were expressed as nmol / g-protein. Catalase activities were assayed by the Aebi method. (15) The decomposition of the substrate H 2 O 2 was spec- trophotometrically monitored at 240 nm (Shimadzu UV 1601, Japan). Absorption reduction was measured and expressed as k / mg protein. GSH-Px activity was measured by Paglia and Valentine method.(16) The enzymatic reaction was initiated by the addition of H 2 O 2 to the reaction mixture contain- ing reduced glutathione, reduced nicotinamide adenine dinucleotide phosphate and glutathione reductase. A spectrophotometer monitored the absorbance change at 340 nm. One unit of GSH-Px is defined as NADPH mi- cromoles oxidized per minute. The activity was given as units per g protein. Total (Cu-Zn and Mn) SOD activity values were ob- tained from Sun et al.(17) and Durak et al.(18) A unit of SOD, nitro blue tetrazolium (NBT), was defined as the amount of enzyme causing 50% inhibition at the re- duction rate. The results are expressed in units per g protein. Statistical Analysis Kolmogorov-Smirnov test was performed to determine whether the distribution of the data obtained was nor- mal or not. Weigth, testicular weight GECT, MSTD, Caspase 3 staining, MDAprot, SODprot, CATprot and GPXprot values were analysed with one-way analysis of variance and as a post-hoc test Tukey’s was used to determine the group that caused statistical difference between groups. Kruskall Wall Test was used to ana- lyse Johnsen score values. Bonferonni corrected Mann- Whitney U test was used to compare two by two groups regarding to Johnsen scores to determine statistically different groups. Each test group was compared with the appropriate control groups and P values less than 0.05 were con- sidered as significant except Johnsen groups where p = 0.005 were considered as significant. The results were expressed as mean + standard error mean or median and 25-75 percentile values. SPSS v21.0 program was used for statistical analysis. RESULTS The mean body and testicle weights of the rats were not statistically different between groups. (Table 1) The bi- ochemical and histopathological findings were given in Table 2, and 3. In the 5-day colchicine given the group, the protein MDA levels were significantly lower than I/R and I/Rp groups (p < 0.002) which were reached the significantly highest levels. Table 3. Histopathologic results of all 5groups n Johnsen Score GECT MSTD Sham 8 9.6 ± 0.52 b 8.5 ± 0.53 f 12.5 ± 1.93 j I/R 8 7 ± 0.54 c 7.1 ± 0.84 g 9.1 ± 1.36 k I/Rc1 8 6.6 ± 0.52 d 6.9 ± 0.64 h 9.4 ± 0.91l I/Rc5 8 6.4 ± 0.74 e 6.6 ± 0.74 i 7.1 ± 0.84 m I/Rp 8 4.3 ± 0.52 4.5 ± 1.07 4.9 ± 0.84 P values < 0.001 < 0.001 < 0.001 GECT, Germinal Epithelial Cell Thickness; MSTD, Mean Seminiferous Tubular Diameter Findings of the parameters were expressed as mean ± standart deviation. Only statistically significant groups (p=0.005) were marked with the letter of the alphabet bp< 0.001 Sham vs I/R; Sham vs I/RC1; Sham vs I/Rc5; Sham vs IRp cp< 0.001 I/R vs I/Rp dp< 0.001 I/Rc1 vs I/Rp ep< 0.001 I/Rc5 vs I/Rp fp = .018 Sham vs I/R; p = .001 Sham vs I/Rc5; p< 0.001 Sham vs IRp gp = .001 I/R vs I/Rp hp< 0.001 I/Rc1 vs I/Rp ip = .005 I/Rc5 vs I/Rp jp = .013 Sham vs I/R; p = .017 sham vs I/RC1; p < 0.001 sham vs I/Rc5; p < 0.001 Sham vs IRp kp = .036 I/R vs I/Rc5; p < 0.001 I/R vs I/Rp lp = .001 I/Rc1 vs I/Rc5; p < 0.001 I/Rc1 vs I/Rp mp = .001 I/Rc5 vs I/Rp Effectiveness of colchicine in testis torsion-Gozukara et al. Vol 17 No 03 May-June 2020 297 Miscellaneous 298 GPx, CAT, and SOD levels were higher in Sh and I/ Rc5 groups (p < 0.05). Also, antioxidant levels (SOD, CAT) in I/Rc5 group did not differ from the Sh group statistically (p > 0.05). As a positive indicator of apoptosis, Caspase-3 positiv- ity was higher in group I/R, I/Rc1, and I/Rp than group Sh and I/Rc5. I/Rc5 group had lower Caspase-3 positiv- ity than group I/R (p < 0.05). (Figure 2) When comparing Johnsen’s scores in I/Rc5 group with I/R and I/Rc1 groups, there was no statistical difference (p = .211 and p = .905, respectively) that implies to an alleviation in tissue deterioration. Also, I/Rp group’s Johnsen’s scores were significantly lower than the oth- ers (P < 0.001). The mean seminiferous tubular diameter and germinal epithelial cell thickness were affected negatively in all groups but group Sh. With the use of colchicine in the postoperative period, in I/Rc5 group, MSTD and GECT measurements were significantly better than group I/Rp (P < 0.005) which showed the natural course of ischem- ic events and not statistically differed from the group I/R. (Figure 1) DISCUSSION Testicular torsion is a urological urgency that's inci- dence peaks in 2 periods of life (neonatal and prepu- bertal), resulting in testicular infarct if not immediately treated. The first-line treatment is surgery(19), but tes- ticular salvage and postoperative changes do not only depend on operation. Because atrophy can be seen in one-fourth of the torsion cases that are treated in time. (20) Logically an additional medical salvage therapy should be considered in the postoperative period. In this respect, numerous substances like vardenafil, sildenafil, rosuvastatin, Coenzyme Q10, different surgical, and in- terventional techniques showed positive results on pre- serving testis from ischemia-reperfusion damage.(221-23) However, none of these studies discuss their subjects for postoperative usage. From this point of view, our study revealed that oxidative stress was reduced in rats which colchicine usage was started peroperatively and continued for five days after the operation. Addition- ally, the levels of anti-oxidative agents such as SOD, CAT, and GPx kept their levels in 5-day colchicine group postoperatively. Total antioxidant capacity and total oxidative stress findings were also significantly better with the short term colchicine usage (p < 0.005). This favorable effect of colchicine may be explained with leucocyte interaction. Chappey et al.(24) determined the colchicine deposition dynamics in leucocytes and revealed that colchicine rises to the plasma peak lev- el at 1-hour after application and accumulates mostly in leukocytes. Mitsui et al.(25) showed increased major proinflammatory cytokines which lead to leucocyte ac- cumulation in the tissues in the first hours of the torsion. So colchicine could be easily accumulated in migrated leucocytes. After this, the main anti-inflammatory ef- fect of colchicine occurs by inhibiting the assembly and polymerization of microtubules which are the keystones in cell migration, secretion of cytokines, maintenance of the cytoskeleton and cell shape.(26) Additionally, colchicine suppresses the TNF-alpha pro- duction from macrophages that generated after tissue necrosis induced by lipopolysaccharides.(27,28) In the present study, short term colchicine treatment group; SOD, CAT, and GPx molecules which are endogenous antioxidants commissioned to protect the steady-state of the cell against oxidative stress were found signifi- cantly higher as the sham group (p < 0.005). The high levels of endogenous anti-oxidants can be explained by the suppression of inflammation, which is triggered by many stress pathways, rather than increasing the gener- ation of these molecules. The other possible protective mechanism of colchicine may be related to lipid peroxidation, cytosolic Ca+2, and oxidative stress. Testicular injury increases linearly with the degree and duration of torsion.(29) Due to is- chemia, new acidic environment induces Ca+2 influx into cells in various ways. Increased intracellular Ca+2 levels trigger the activation of inflammatory markers and cell death especially over mitochondria.(30,31) At this point, Korkmaz et al.(32) reported that colchicine has a reducing effect on cytoplasmic Ca+2 release in neutro- phils. In this way, colchicine reduces the oxygen radi- cals generated by mitochondria which are most damag- ing cellular macromolecules, lipids in particular. One of the most critical indicators of lipid peroxidation and the secondary index of oxidative stress is MDA.(33) MDA interacts with DNA and proteins per the aldehyde com- ponent, which is toxic to the structure at high levels and causes irreversible damage linked to DNA fragmenta- tion, protein denaturation. We found that MDA levels were significantly lower in the colchicine-treated group compared to other untreated groups (p < 0.005). Caspase is one of the cysteine endoprotease families and plays a regulatory role in cell death and inflamma- tion.(6) They have classified into two groups; apoptot- ic and inflammatory, and apoptotic caspases work as initiator or executioners. Caspase 3 is a well-known executioner apoptotic caspase and proteolytically pro- cessed to the active form that is essential for apoptosis. (34-36) The active form of caspase 3 causes cell death by DNA damage and protein degradation.(37) In our study, we found that Caspase 3 staining in the 5-day colchi- cine-treated group was significantly lower (P < 0.005) when compared to groups without colchicine treatment and this score was not statistically different from the sham group, and even lower scores were obtained. This finding also emphasizes the less DNA fragmen- tation and cell death with short term colchicine usage like forementioned MDA. Colchicine may trigger con- trolled apoptosis by stopping the cell cycle in the G2/M phase and contribute to planned cell death by microtu- bule depolymerization.(26) Because of this feature, it has been tried for some cancer treatments(26) but not preferred due to its high side-effect profile. However, with the secondary necrosis or apop- tosis which is seen in testicle torsion, the inflammatory, and immunogenic activity of colchicine become promi- nent; thus uncontrolled apoptosis is precluded(38). To our knowledge, long term usage of colchicine harms sperm cell maturation.(39) However, the sus- tained benefit of short-term colchicine treatment on torsioned-detorsioned testis survival is unknown and side effects arising from short term usage are still pre- sumptive. In our study, when compared with the pla- cebo group the Johnsen score was better with 5-day colchicine treatment group. Similar results were found with the GECT and MTBS scores. The most significant damage to testicular tissue was created with the activa- tion of inflammatory and apoptotic cascades.(40) These results have allowed us to concentrate on the potent an- Effectiveness of colchicine in testis torsion-Gozukara et al. Vol 17 No 03 May-June 2020 299 ti-inflammatory effect of colchicine. In an overall per- spective, the benefits outweigh the possible danger of short-term colchicine treatment in patients with torsion. Experimental design and relatively small sample size are the major limitations of our study. However, this is one of the few studies related to colchicine treatment on testicular torsion. CONCLUSIONS Colchicine is a highly active and fructuous molecule on ischemia-reperfusion injury in testicle torsion animal model with short-term usage and may be a convenient option for this patient group over its noticeable anti-in- flammatory effect. But further experimental animal studies are required to determine the drug dose, dura- tion, and way of administration of colchicine in patients with testicular torsion. REFERENCES 1. Karaguzel E, Kadihasanoglu M, Kutlu O. Mechanisms of testicular torsion and potential protective agents. Nat Rev Urol. 2014;11:391- 9. 2. Cuzzocrea S, Riley DP, Caputi AP, Salvemini D. 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