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).