Experimental Animals
This experimental study was conducted at Gazi University Animal Experiments Laboratory on 2nd September 2021, and carried out in accordance with ARRIVE guidelines. The study protocol was approved by the Animal Research Committee of Gazi University (G.Ü.ET—21.063), Ankara, Turkey. All animals were maintained in accordance with the recommendations of the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
A total of 36 adult female Wistar Albino rats (weighing 270 to 320 g) were used. The rats were kept at 20–21 oC in cycles of 12 hours daylight and 12 hours dark environment and had free access to food until two hours before the initiation of the study.
Experimental Design
The rats were randomly divided into six equal groups each containing six rats: sham group (Group S, n = 6), the fullerenol C60 group (Group FC60, n = 6), the ischemia-reperfusion group (Group IR, n = 6), and the ischemia-reperfusion-sevoflurane group (Group IR-Sevo, n = 6), ischemia-reperfusion -fullerenol C60 group (Group IR-FC60, n = 6), and ischemia-reperfusion-fullerenol C60-sevoflurane group (Group IR-FC60-Sevo, n = 6).
All rats were anesthetized with 50 mg/kg intramuscular ketamine (Ketalar®; 1 mL = 50 mg; Pfizer, Istanbul, Turkey) and 10 mg/kg xylazine hydrochloride (Alfazyne® %2, Ege Vet, Turkey). They were placed on an electric heating pad under a warming light.
Sham Group (Group S): Only midline laparotomy was performed without any additional surgical intervention.
Fullerenol C60 group (Group FC60): Thirty minutes after the administration of 100 mg/kg fullerenol C60 intraperitoneally (i.p.), midline laparotomy was performed without any additional surgical intervention.
Ischemia-reperfusion group (Group IR)
Midline laparotomy was performed, followed by 120 minutes of ischemia by clamping the porta hepatis by placing an atraumatic microvascular clamp, and then 120 minutes of reperfusion was performed.
Ischemia-reperfusion-sevoflurane group (Group IR-Sevo): Midline laparotomy and IR were performed, and sevoflurane (Sevorane, AbbVie, England) was applied at a 2,3% concentration for the target minimum alveolar concentration (MAC) 1 and a rate of 4 L/min in 100% O2 concurrently with the onset of the ischemia period and extending for 240 minutes. The anesthesia protocol was administered in a wide transparent plastic box. The box was integrated into a semi-open anesthesia device with static hoses.
Ischemia-reperfusion-fullerenol C60 group (Group IR-FC60): Thirty minutes after the administration of 100 mg/kg fullerenol C60 i.p., IR was performed.
Ischemia-reperfusion-fullerenol C60-sevoflurane group (Group IR-FC60-Sevo)
Thirty minutes after the administration of 100 mg/kg fullerenol C60 i.p., IR were performed, and sevoflurane (Sevorane, AbbVie, England) was applied at a 2,3% concentration for the target minimum alveolar concentration (MAC) 1 and a rate of 4 L/min in 100% O2 concurrently with the onset of the ischemia period and extending for 240 minutes.
At the end of the experiments, the rats were anesthetized with i.p. ketamine (50 mg/kg) and then sacrificed by exsanguination from the abdominal aorta. Serum samples were drawn for biochemical assays, and the liver tissues were removed for histopathological studies.
Histopathological Evaluation
Histopathological assessment was studied in the Department of Histology at Kirikkale University. After the fixation process, specimens were prepared with paraffin blocks. Tissue sections of 5 µm were stained via hematoxylin and eosin (H&E). The histopathological assessment and scoring were performed under light microscopy. The same pathologist performed the histologic evaluations in a blinded manner.
In the histopathological examination, each preparation was examined for hepatocyte degeneration, sinüsoidal dilatation, prenecrotic cell, mononuclear (MN) celluler infiltration in the parenchyma. Histological testing Semiquantitative evaluation technique used by Abdel-Wahhab et al’s (10) was applied for interpreting the structural changes investigated in hepatic tissues of control and research groups. According to this, (–) (negative point) represents no structural change, while (+) (one positive point): mild, (++) (two positive points): medium and (+++) (three positive points): severe structural changes.
Biochemical Evaluation
The biochemical examination was conducted in the Department of Medical Biochemistry at Gazi University. Oxidative stress and lipid peroxidation in liver tissue, was evaluated by measuring the Thiobarbituric acid reactive substance (TBARS) level, Catalase (CAT), and Glutathione-S-transferase (GST) enzyme activities.
The TBARS assay was carried out to determine lipid peroxidation using the thiobarbituric acid method (11). TBARS measurements were conducted based on the reaction of MDA with thiobarbituric acid (TBA), which form a pink pigment with an absorption maximum at 532 nm in acid pH, and 1,1,3,3-tetraethoxypropane was used as a standard MDA solution.
The catalase (CAT) activity is based on the measurement of absorbance decrease due to H2O2 consumption at 240 nm as described by the Aebi H method (12).
Glutathione-s-transferase (GST) enzyme activity was measured using the method described by Habig et al. (13) The GST activity method is based on the measurement of absorbance increase at 340 nm due to the reduction of dinitrophenyl glutathione (DNPG). The results were expressed in international unit per milligram of protein.
Statistical analysis
The Statistical Package for the Social Sciences (SPSS, Chicago, IL) 20.0 program was used for statistical analysis. The Shapiro-Wilk test was used for comparisons to determine the distribution of all variable groups. Variations in CAT and GST activities and TBARS levels, as well as histopathologic parameters, were assessed using the one-way ANOVA test. The Bonferroni posthoc test was used after significant ANOVA test to determine the differences among groups. The results are expressed as mean ± standard deviation, and statistical significance was set at p < 0.05.