This research was approved by the Ethics Committee of Animal Experiments at Sadat City University, Sadat City, Egypt, funded by Taif University (Supporting Project number: TURSP-2020/99), and performed based on the Guide for the Care and Use of Laboratory Animals (8th edition, National Academies Press). In the present study, 36 male Wistar rats (weighing 150-220 g) were kept in the standard temperature (23+1°C), 12/12 h light/ dark cycle with ad libitum access to food and water.
The study protocol was designed as demonstrated in Figure 1. Rats were randomly allocated to three groups (n = 12 in each group): Control (healthy animals), Model: with rats subjected to restraint stress and IVC injected with phosphate-buffered saline (PBS) , Model+T3: rats subjected to restraint stress and injected IP with T3. The rats in the Model and Model+T3 groups were exposed to stress. For this purpose, each rat was placed in a plastic cylinder (3 h/day from 8:00 am to 11:00 am) for 14 consecutive days . Twenty-four hours after model induction, the animals in the Model+T3 group were injected with IVC T3 (a single dose of 25 ug/kg body). T3 was dissolved in dimethyl sulfoxideand diluted with PBS (a total volume of 5 ul) to be injected into the right ventricle via a Hamilton syringe (bregma: ML = -1.8 mm, AP = -0.9 mm, and DV = 3.5 mm deep from the dura) . The equal volume of PBS was injected into the right ventricle of rats in the Model group. The rats were examined via forced swimming (FST), tail suspension (TST), and open field (OFT) tests. In the end, the brains of rats were used for various molecular (n=8 in each group) and histological (n=4 in each group) studies. The fresh samples of the hippocampus were extracted immediately after sacrificing and put in a freezing tube and kept at -80̊ C. Also, for histological studies, the total brain was isolated after prefixation. The prefixation procedure was done via the transcranial perfusion of normal saline to remove the blood from the brain, followed by 4% paraformaldehyde perfusion (PFA, Sigma) via the same route. The post-fixed was done by using a 10% formalin at 4° C for 72 h.
Forced swimming test (FST)
A glass cylinder with a height of 80 cm and a diameter of 30 cm filled with 40 cm of water (25°C) was used to evaluate the rats’ behavior for two consecutive swimming sessions of training and test. The cylinder was filled with tap water (23 ± 1 °C), and water depth was adjusted according to the animal size. Therefore, rats could not touch the bottom of the cylinder. At first, for training, each rat was placed in the cylinder for 10 minutes and forced to swim. After 24 h, the procedure was repeated for 5 min period as a test session. In this session, the animal behavior was video-recorded by a blind observer, and the immobility, latency, swimming, and climbing times were recorded . An enhanced time of immobility time was considered as depressive-like behavior.
Tail Suspension Test (TST)
One day after FST, TST was performed. Briefly, each rat was suspended via its tails using adhesive tape to a horizontal bar for 6 min (2 min for adaptation and 4 min for test). The test was performed by a blind observer, and the immobility time was recorded.
Open Field Test (OFT)
The OFT was performed to evaluate the depressive-like behavior, as reported previously . Briefly, the open-field apparatus (80 cm×80 cm×50 cm2) is consists of floor divided into 25 equal squares. Each rat was placed in the center of the open field individually and allowed to freely explore for 5 min, and the behavior was recorded. The time of immobility was evaluated for a 5 min period. After finishing the test, the rat was placed in the home cage by the experimenter. After each test, the apparatus was cleaned with 90% ethanol to remove olfactory cues.
RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)
The fresh samples were used for quantitative real-time PCR (qRT-PCR) assay (n= 4 in each group) of NF-κB, NLRP3, Caspase-1, and ASC. In summary, total RNA was isolated from samples by using the Tripure Isolation Reagent (Roche Applied Science, Peuzberg, Germany) according to the instruction. NanoDrop™ 2000/2000c spectrophotometer (Thermo Fisher Scientific) was used for evaluation of its purity. For complementary DNA synthesis, PrimeScript RT Reagent Kit (Takara Bio Inc., Otsu, Shiga, Japan) was used for reverse transcription. The PCR primers used are shown in Table 1. The StepOnePlus Real-Time PCR machine was carried out to run the reactions in triplicates. beta-actin (β-actin) was used to normalize the gene expression, and the relative fold change expression of genes was calculated via the 2-ΔΔCt method .
The isolated hippocampal samples were used for western blot (n = 4 in each group) to determine the synthesis of inflammasome proteins. The samples were lysed via a lysis buffer (RIPA) and centrifuged. Total Protein Kit, Micro (Sigma, USA) was used to detect the total protein concentration. After protein denaturation, 5 μg of protein was loaded on 10% SDS-PAGE, and separated proteins were put on polyvinylidene difluoride transfer membranes (Sigma, USA) and then incubated for one hour with specific primary antibodies (Novus Biologicals, USA). Then, the procedure was followed by incubation of Membranes for one hour with anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam, Germany). Protein bands were detected by the luminescent substrate solution (Sigma, USA). Finally, to quantify the specific bands, ImageJ software (NIH, USA) was used. GAPDH (Thermoscientific, USA) was used for normalization .
Nissl Staining (Cresyl Violet Staining)
After fixation and tissue processing (n = 4 samples in each group), 5-um-thick coronal sections cut by microtome (Leica Biosystems, Milan, Italy) and mounted on the slides. After subjecting to Nissl staining, four random sections were picked for light microscopy observation. The photographs were prepared under an optical microscope (Labomed, USA). The photomicrographs were prepared from the CA1 region, and the number of dark neurons (intensely stained neurons) were calculated in each photomicrograph.
Data were analyzed via SPSS software (V22.0) and expressed as mean ± SD. The analyses were carried out using the one-way analysis of variance (ANOVA) and followed by post hoc Tukey's comparison. P value less than 0.05 was considered as significant.