Animals
In this research, 24 male Wistar rats (weighing 250 ± 20 g; 8–10 weeks old) were used. The animals were purchased from Urmia University of Medical Sciences and were maintained at 22 ± 2℃ and 50 ± 10% humidity using a 12-h light/12-h dark cycle. Animals had ad libitum access to food and water. This study was approved by the ethics committee of Urmia University of Medical Sciences (Code: IR.UMSU.REC.1400.126).
Experimental design
The animals were randomly assigned to 4 groups ( n = 6 per group): Untreated control (C), ivermectin ( 2 mg/kg) treated control, control ischemia/reperfusion (IR), and ivermectin treated rats with IR (ivermectin + IR). Rats in the ivermectin group received ivermectin 2 mg/kg ip for three days, while those in the IR group underwent ligation of bilateral common carotid arteries for 20 min and received normal saline ip for three days thereafter. Rats in the ivermectin + IR group were treated for three days with ivermectin (2 mg/kg/day ip) starting 1 h after inducing the cerebral IR. The Morris water maze test was used to determine memory function in rats. Animals were anesthetized for blood sampling from the portal vein and then euthanized for collection of the brain, which was kept frozen at -80℃ until later analyses.
Surgery
Global cerebral ischemia is a compact, quick procedure that briefly reduces blood flow to the brain (Camarata 1994). We induced transient global cerebral ischemia-reperfusion (IR) by bilateral common carotid artery occlusion. In brief, animals were anesthetized with a mixture of ketamine (60 mg/kg, ip), xylazine (10 mg/kg, ip), and acepromazine (2 mg/kg, ip). After the animals had no response to painful stimuli, they were placed in a supine position, and the surgical site was disinfected with a solution of povidone-iodine (Betadine ®). An incision (~ 3 cm) was made to expose both carotid arteries, separated from the vagal nerves, and then occluded with clamps for 20 min. The clamps were removed to resume cerebral blood flow, and the skin was sutured with 3 − 0 surgical thread (Salehi et al. 2021; Zamani et al. 2013).
Infarct size measurement
Rat brains were excised at the end of the experiment and then cut into 2 mm coronal slices. The slices were incubated in 2, 3, 5-triphenyl tetrazolium chloride (TTC) solution (2%) for 20 min at 37℃ in the dark. The TTC solution was replaced with 10% formaldehyde to fix the tissue slices for 6 h. Finally, brain slice images were captured and analyzed by ImageJ software (Kamat et al. 2015; Karimipour et al. 2018).
Myeloperoxidase Assay
Myeloperoxidase (MPO) activity was used to measure polymorphonuclear leukocyte accumulation in the brain tissues (Bradley et al. 1982). Briefly, brain tissues were homogenized (1KA homogenizer, Staufen, Germany) in a solution containing 0.5% hexadecyltrimethylammonium bromide (HTAB) in 50 mM potassium phosphate buffer (pH = 6). After homogenization, the samples were centrifuged at 4,500 rpm for 30 min at 4℃ and then subjected to three cycles of freezing/thawing with sonication for 10 s during each freezing/thawing cycle. The samples were then centrifuged at 4,500 rpm for 30 min. A 0.1 ml aliquot of the supernatant was added to a test tube containing 2.9 ml of 50 mM potassium phosphate buffer at pH = 6, containing 0.167 mg/ml o-Dianisidine dihydrochloride and 0.0005% H2O2. Afterward, it was allowed to react for 5 min before stopping the reaction with 0.1 ml of 1.2 M hydrochloric acid. The absorbance of each sample was measured with a spectrophotometer (Cecil 9000, Cambridge, UK) at 450 nm. MPO activity is reported as mU/g wet tissue (Bradley et al. 1982).
Malondialdehyde Assay
The thiobarbituric acid (TBA) reactive assay was used to determine malondialdehyde (MDA) concentrations. Brain tissue was added to a KCl solution (1.15%) and homogenized (1KA homogenizer, Staufen, Germany) to produce a 10% homogenate. The resulting mixture was centrifuged at 3,000 rpm for 15 min, and 250 µl of the supernatant was added to a tube containing 1% phosphoric acid (3 ml) and TBA (1 ml). The samples were incubated at 90℃ in a water bath for 45 min and then chilled on ice. Then, 3 ml of n-butanol was added, and the solution was centrifuged at 3,000 rpm for 15 min. The absorbance of each sample was measured at 532 nm using a spectrophotometer (Cecil 9000, UK). The level of lipid peroxidation was expressed as nanomoles of MDA per mg protein (Satoh 1978; Salehi et al. 2021)
Histopathology
The apical part of the brain was cut, fixed in 10% formalin, and sectioned at 5 µm slices. Hematoxylin and eosin (H&E) staining was used to determine the degree of inflammation and leukocyte infiltration into brain tissue using a morphometric point-counting procedure. Two trained persons (one of which was a pathologist) scored histopathological changes and leukocyte accumulation using a scale of 1–4: 1 (low), 2 (moderate, with small multifocal degeneration and a slight degree of leukocyte infiltration), 3 (high, with extensive degeneration and/or diffuse leukocyte infiltration), and 4 (intensive, with necrosis and diffuse leukocyte infiltration) (Hussein et al. 2013; Azarbaijani et al. 2021).
Morris Water Maze
The Morris water maze test was used to study spatial memory and learning over 5 days. The rats were initially trained for 4 consecutive days with four daily trials. Animals were given 60 s to find the rescue platform and 10 s as rest time between trials. In each trial, the rat was placed in a different part of the pool facing the water tank wall and allowed to explore for 60 s. On day 5, the rescue plate was removed, and the rat was allowed to explore for 60 s. The time spent by each rat in the target quadrant was used as an indicator of memory (Salehi et al. 2021).
Western Blot
Western blot analysis was carried out as previously described with minor modifications (Rameshrad et al. 2015). Briefly, brain tissues were homogenized in lysis buffer (50 mM Tris base, 150 mM NaCl, 1 mM EDTA, 1% Triton, 0.1% SDS, 1 mM dithiothreitol, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, and 0.5% sodium deoxycholate and protease Inhibitor). A Bradford Protein Assay kit was used to determine protein concentrations. Equal amounts of protein (50 µg) were loaded into wells and separated by 12% SDS polyacrylamide gel electrophoresis. After transfer, PVDF membranes were blocked in 5% non-fat milk in Tris-buffered saline Tween-20 (TBST) for 1 h at 25℃. Gels were incubated with primary antibodies of phospho-AMPK (Thr172) (Cell Signaling Technology, #2535) and AMPK (Cell Signaling Technology, #5831) at 4℃ overnight, followed by incubation with a secondary goat anti-rabbit HRP conjugated antibody (Cell Signaling Technology, Danvers, MA). Immunoreactive bands were visualized with a BM Chemiluminescence kit (Roche, Mannheim, Germany). Finally, densitometric analysis of the immunoblots was performed using image J software.
Statistical analysis
The normal distribution of data was verified using the Kolmogorov-Smirnov test. Two-way repeated measure analysis of variance (ANOVA) was used to analyze the Morris water maze test data, whereas one-way ANOVA was used in other experiments. In addition, a Tukey post-test analysis was performed to determine the significance between groups. Statistical analyses were performed with GraphPad Prism 8.0, the results were expressed at mean ± standard error of the mean (S.E.M), and the significance level was set at P < 0.05.