2. Materials and Methods

The study was approved by the Ethical Committee of Yeditepe University, Medical Faculty, Experimental Animals Research Laboratory (Atasehir, Istanbul, Turkey 34755) on 27th of August in 2018 with the registry number 675. Authors declare here that all applicable international, national and/or institutional guidelines for the care and use of animals were followed. All procedures and practices involving animals in the study were in compliance with the ethical standards of the respective institution at which the study was conducted and is in accordance with the ARRIVE guidelines (16).2.1. Experiment:32 adult male Spraque-Dawley rats weighing 250-300 g were used for the study. All experimental animals were adapted to the environment by keeping 50-70% relative humidity at ambient temperature at 22 ± 2 ° C for 12-hour night and 12-hour daytime until the beginning of the experiment. Rats were given standard diet and water during follow-up. For the study, 32 Spraque-Dawley rats were randomly divided into 4 groups; Group 1 (Control): 12,4 ml/kg normal saline IV (n=8) Group 2 (ILE): 12,4 ml/kg intralipid emulsion IV (n=8) Group 3 (PIO): ½ LD50 (1000mg/kg) pioglitazone PO + 12,4 ml/kg normal saline IV (n=8) Group 4 (PIO+ILE): ½ LD50 (1000mg/kg) pioglitazone PO + 12,4 ml/kg intralipid emulsion IV (n=8) Before all drug applications and interventional procedures, inhaler isoflurane (Izofluran, Isofludem 100 ml, Dem Ilac, Istanbul, Turkey) was provided by anesthesia machine (Animal Anesthesia Machine) to all rats. After providing sufficient depth of anesthesia, tail vein cannulation was performed with 26 G catheter (Bicakcilar -B-CAT2, İstanbul, Turkey) to each group to administer the drugs used in the study. Pioglitazone tablets (Glifix tablet 45 mg, Bilim Ilac /Turkey) were crushed in mortar and mixed with normal saline in vortex to prepare the solution. The prepared solution was given to the rats in the 3rd (PIO) and 4th (PIO+ILE) groups by a gastric tube at a dose of 1000 mg/kg pioglitazone. Toxicity was induced by administering pioglitazone at a dose of ½ LD50 with an average lethal dose (LD50) of 2000 mg/kg (8,17). ILE (ClinOleic 20% lipid 500 ml, Eczacibasi, Baxter/Belgium) was administered to the 2nd (ILE) and 4th (PIO+ILE) groups at the dose of 12.4 ml/kg IV from the tail vein of the rat. 20% intralipid emulsion dose was applied with reference to previous studies (18–20). All rats were anesthetized prior to decapitation using inhaler isoflurane (Izofluran, Isofludem 100 ml, Dem Ilac, Istanbul, Turkey). After decapitation liver, heart and kidney tissue samplings were performed. Tissue specimens were taken for histopathological examination.2.2. Histopathological studies:The kidney, liver and cardiac tissues of each rat identified in 10% formol and they were sampled by pathologist. Tissue specimens were fixed in 10% neutral buffered formalin and embedded in paraffin by rising alcohol series (70%, 90%, 96% and 100%) and xylene. 5 μm thick sections were taken with microtome and stained with Hematoxylin & Eosin method for histopathological evaluation. Tissues were examined by light microscope (Carl Zeiss, AxioZoom). Liver tissue morphology was evaluated microscopically for vacuolization, vascular dilatation, inflammatory cell infiltration, necrosis and hemorrhage. Heart tissue morphology was evaluated microscopically for vascular dilatation, connective tissue edema, inflammatory cell infiltration, hemorrhage and transverse streaking in cardiomyocytes. Renal tissue morphology was evaluated microscopically for glomerular structure, vacuolization, hemorrhage, tubular dilatation and inflammatory cell infiltration. Each evaluation criterion was scored as 0 = normal, 1 = mild, 2 = moderate, and 3 = high. Scores of these parameters were combined on the basis of organs and histopathological scores of each animal were formed.2.3. Statistical Examinations: Nonparametric Kruskal-Wallis test and Dunn’s multiple comparison test were used to compare histopathological scoring results between the groups. Statistical significance level was taken as p <0.05. Statistical analysis of histopathological findings was performed using GraphPad (Prism 6.0, GraphPad Software, San Diego, California, USA).