Ethics Statement
The protocols of animal experiments were approved by the Local Ethics Committee of Application and Research Center of Experimental Medicine, Necmettin Erbakan University, No. 2016-050, on 30.09.2016.
Experimental Protocol
Animals
In this study, 50 male Wistar albino rats, 12 months old, were used. The care and feeding of the rats were done at Necmettin Erbakan University Experimental Medicine Application and Research Center. Rats were housed in plastic cages where they could move freely with food and water containers, their food and water were given as ad-libitum. Cages were cleaned weekly. The animals were stored at room temperature (22 ± 1 ˚C) for 12 hours light/dark period under standard laboratory conditions.
Animals were divided into 5 groups: 1) Control group (C, n = 10), 2) Alzheimer’s disease group (AD, n = 10), 3) Alzheimer’s disease + NK3R agonist (Senktide) administered group (ADS, n = 10), 4) Control + NK3R agonist administered group (CS, n = 10), 5) Alzheimer’s disease + NK3R agonist + NK3R antagonist (Osanetant) administered group (ADSO, n = 10).
Drug Administration
Aβ 1-42 (Sigma-Aldrich, USA) was used to construct the experimental Alzheimer's model in rats. The Aβ peptide was dissolved in saline. It was left for incubation at 37oC for 72 hours and kept waiting for ICV injection. NK3R agonist senktide ([succinyl-Asp6-N-Me-Phe8] SP6-11; Bachem, Germany) was dissolved with 5% dimethylsulfoxide in phosphate buffered physiological saline (PBS). NK3R antagonist osanetant (SR142801, Sigma Aldrich, USA) was dissolved in 0.01% Tween 80 (Sigma-Aldrich, Steinheim, Germany) in ultrapure water. Senktide and SR142801 were prepared in a volume of 1 mg/kg body weight. Senktide was administered subcutaneously at a dose of 0.2 mg/kg and osanetant was administered intraperitoneally at a dose of 6 mg/kg. In the antagonist group, senktide was injected 1 minute after administration of osanetant. Control animals were injected with solvent. Doses were determined taking into account previous studies that showed a healing effect in learning and memory tests [20, 21, 25, 26, 27, 28].
Intracerebroventricular Aβ injections and neurosurgery
Anesthesia was administered before ICV injection by the combination of ketamine (100 mg/kg) and xylazine (5 mg/kg). After the anesthetic agent was administered, the head of the rats placed in the stereotaxic device was fixed with the ear sticks of the device. After the scalp was cut from the midline, Bregma, the reference point, was identified. The Aβ 1-42 peptide was bilaterally injected into the CA3 region of the hippocampus (AP: -4.36mm, ML: -4.4mm, and DV: -8.0mm, Paxinos and Watson 2009) (Figure 3.1) using a micro injector (1μl Hamilton) (2.2 nmol / 10μl) to AD model groups (AD, ADS and ADSO). Rats of C and CS groups were injected with the same amount of saline as ICV (Figure 3.2). On the 15th day of behavior and memory experiments, the animals were first injected with the NK3R agonist senktide and the NK3R antagonist until the end of the experiment. On the 15th day, when the behavioral and memory experiments started, the animals were injected subcutaneously with the NK3R agonist senktide, and the NK3R antagonist osanetant was administered to ADSO group until the end of the experiment.
Behavioral and Memory Tests
OF and MWM tests were performed to evaluate the locomotor activity, anxiety-like behaviors, spatial memories of animals. OF test and MWM were carried out between 09:00-12:00 am. Animals were brought to the room where the test would be performed at least one hour before the test and adaptation to the environment was ensured.
Open Field Test (OF)
OF test was performed at the beginning and end of the experiment in order to evaluate the locomotor activities, some behaviors such as anxiety and depression of animals. OF test apparatus used is made of 80x80x30cm square black plexiglass material. All test applications were recorded with the video recording system associated with the special software program (Ethovision Video Monitoring System XT11, The Netherlands) (Figure 3.4). With software program, two regions are determined in the test apparatus, center and edge. The parameters of the distance (cm), speed (cm/s) and the number of entries from the edge to the center for 5 minutes were calculated with a software program. The rearing and grooming behaviors of the animals were scored manually. The test apparatus was cleaned with 10% ethyl alcohol solution after each animal [29, 30].
Morris Water Maze Test (MWM)
MWM consists of a circular water tank with a diameter of 180 cm and a depth of 60 cm and a 10x10 cm hidden platform (Figure 3.5). The water tank was darkened with black paint to prevent the platform from being seen. The room where the experimental setup is located was illuminated with the light source. Different shapes were hanged on the walls of the room where the experiment was made, which could provide clues for the subjects. Attention was paid to making the experiment with the same team, to wear the same clothes during the experiment, to be quiet in the room and not to create an extra stimulus for the subjects. All rats were given a habituation swim the day before their experiment. On the day of the habituation, the platform was kept visible 1 cm above the water. The animals were floated for 60 seconds and the animals that could not find the platform were left on the platform and kept there for 20 seconds. The platform was held at a fixed point on the days of training, and the platform was removed from the pool on the day of the memory test (probe). In the training of the experiment, the rats were left to the pool 4 times in different directions consecutively every day. Rats were expected to find the hidden platform within 60 seconds. After staying on the platform for about 15 seconds, they were removed from the pool. In the memory test, the platform was removed from the pool and the rats swam for 60 seconds. All tests were recorded with a special software program (Figure 3.6). Distance moved, latency to the platform, number of passes through the target quadrant and time spent in the target quadrant parameters were analyzed.
Ending the Experiment and Removing Tissues
After the injections and behavioral tests were completed, the animals were decapitated under anesthesia, and the hippocampus, cortex and brainstem parts of the brain tissue on dry ice were separated according to the Paxinos and Watson rat brain atlas and placed in tubes. All tissues were kept at -80 ˚C until analysis.
Enzyme-linked immunosorbent assay (ELISA) analysis
Rat hippocampus and cortex tissues were placed separately in tubes filled with cold phosphate buffer. Each hippocampus and cortex tissue sample of the rats in the same group was weighed and combined three by one to provide sufficient study sample.
In the homogenization buffer containing the protease inhibitor cocktail. All homogenization steps are made in accordance with the cold chain. Homogenates were centrifuged at 10,000 g and +4 ° C for 15 minutes using Hettich Rotina 46R (Hettich Zentrifugen, Tuttlingen, Germany) brand-cooled centrifuge and portioned to -80 °C. Rat Choline Acetyltransferase (Catalog number: MBS161266, Sensitivity: 0.01 ng/mL, % CV: <10) and Rat Acetylcholinesterase (Catalog number: MBS725468, Sensitivity: 1 pg/mL, % CV: <10) levels were measured using the double antibody sandwich ELISA method in rat hippocampus and cortex tissue (MyBioSource, San Diego, CA, USA). Protein levels in rat hippocampus and cortex tissues were measured by spectrophotometric method using Pierce bicinchoninic acid-BCA kits (Thermo Scientific, Illinois, USA). Parameter levels in rat hippocampus and cortex tissues were standardized by dividing the results into protein concentrations. In spectrophotometric measurements, results were calculated according to the absorbance concentration calibration charts using the Bio-rad Microplate absorbance readerxMark system (Bio-rad Laboratories, California, USA).
High performance liquid cromotography (HPLC) Analysis
The brainstem tissues were weighed and mixed with 1 ml 0.1 M hydrochloric acid to determine the NA and DA monoamines. The homogenates were centrifuged at 14000 rpm at +4 °C for 15 minutes and their supernatants were separated. The supernatants were made ready for analysis in HPLC (Agilent Technologies 1260 brand; 250x4.6 mm C18 ODS analytical HPLC column) by passing them through a 0.2 µm microfilter. HPLC analysis interval was set to 5. The temperature of the column oven was fixed at 40 °C. With the cell section of the electrochemical detector (Waters 2465 electrochemical) in the open position, the analysis reference line was waited for 1 hour (equilibration process). Injections were carried out in 20 µl volume (with Hamilton injector). Between sample injections, the injection unit of the device was washed with methanol.
Statistical analysis
Data analysis of the study was carried out with SAS University Edition 9.4 program. Descriptive statistics about variables are given. Mean ± standard error (AM±SE) for numerical variables, frequency and percentage values for qualitative variables were given. p <0.05, p <0.01, p <0.0001 was considered significant. The least squares averages were compared for the significant effects. Tukey-Kramer correction was used when necessary. Standard error plots of the mean squares of the relevant variables were drawn.