Animals. In experiments we used mice of the following strains: APPswe/Psen1de9, Tau, HPPRT, C57BL/6, C57BL6/CBA, ICR1 (CD1). The animals were kept in a conventional vivarium at the Belgorod State National Research University under artificially controlled daylight hours (12 hours darks and 12 hours light) at a temperature range from + 22 to + 26°C and had free access to food and water. All experiments were carried out in compliance with the requirements of the Declaration of Helsinki on the Humane Treatment of Animals (declaration of Ethical Principles of Helsinki, 2008) and the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (ETS No. 170) and approved by the content control committee and using laboratory animals of the National Research University “BelSU” (expert opinion No 01i/23 form 2023/01/23).
Isolation and cultivation of a primary mixed culture of hippocampal neurons. The primary mixed culture of neurons obtained from the hippocampus at various stages of neurogenesis was used in the experiment. The embryonic and postnatal cultures were taken for the experiment. The embryonic culture was obtained on the 18th day of gestation (E18), the postnatal culture was taken from animals on the second day after birth (P2). Mousing general anesthesia was performed by intraperitoneal injection of Zoletil at a dose of 40 mg/kg. All manipulations were subsequently performed under cooling conditions - on ice. To obtain an embryonic culture on the 18th day of gestation, the abdominal cavity was opened and an uterineectomy with embryos in a Petri dish with a chilled Hanks solution was performed. The embryos were separated from the uterine wall and placed in a dish with cooled Hank’s solution. The embryos were sequentially transferred into a dry Petri dish which was placed under a binocular (Leica, X4). The hippocampus was isolated on a glass slide with a “well” which was immersed in a Petri dish; 2–3 drops of a cooled PBS solution to the “well” and to the edge of the slide were previously applied. The isolated hippocampus was divided into 6–8 parts with a scalpel. In the experiment, hippocampal tissue was taken from each female from six well-formed embryos which after grinding on the glass was transferred into one test tube with a 0.25% trypsin-EDTA solution. When obtaining a postnatal culture, general anesthesia was performed on a newborn 2-day-old mouse by intraperitoneal injection of Zoletil at a rate of 20 mg/kg. The isolated hippocampus was divided into 6–8 parts with a scalpel. The brain was isolated, the hippocampus was separated, placed on a glass slide with a “well” in a drop of chilled PBS, the hippocampus into 6–8 parts was crushed and the tissue into a test tube with a 0.25% trypsin-EDTA solution was transferred.
Hippocampal tissue was trypsinized in 0.25% trypsin-EDTA solution (Gibco, 25200056) for 20 minutes at 37ºC, 5% CO2 in a Binder incubator (Germany) shaking the contents every 5 minutes. The trypsin solution was inactivated with ice-cold PBS until the phenol became discolored. The supernatant was removed, and the sediment was washed three times with Dulbecco's solution (without calcium, pH 7.4). After the final washing the supernatant was removed and the cell suspension was centrifuged at 1,000 rpm for 3 min. The supernatant was removed, resuspended it in 1 ml of neurobasal medium (Gibco, 21103049) containing 10% fetal bovine serum (Paneco, FB-1001B/50m), 2% B-27 protein supplement (Gibco, 17504044), 0.5 mM L-Glutamax (Gibco, 25030081) and 1% PenStrep (PanEco).
A primary mixed culture of hippocampal neurons was cultured in 8-well plates for the Seahorse HS mini cell metabolism analyzer (Agilent, USA). The plates were prepared in advanced. The grooves around the wells were filled with 400 µl of sterile distilled water each. Culture wells B-G were coated with 10 µl of poly-D-lysine at a concentration of 0.01 mg/ml and 180 µl of distilled water was added to background correction wells A and H each. The plates were left for an hour at 37°C in a thermostat. Afterwards, the plates were washed three times with Dulbecco's medium and covered with 20 µl of neurobasal culture medium containing 2% protein B-27 supplement, 0.5 mM L-Glutamax for 10 minutes in a thermostat at 37ºC, then the medium was removed and the plates were dried.
When seeding a hippocampal culture E18 40 µl of a cell suspension containing 20,000 cells to each cell of the Cell Culture Miniplates plate was added; when working with P2 culture 80 µl of a cell suspension containing 40,000 cells was added to each cell of the plate. The total volume of the medium in the cell to 180 µl was adjusted. Cell cultivation was carried out in neurobasal medium containing 2% protein B-27 supplement, 0.25 mM L-Glutamax, 1% PenStrep. Cells were grown in a cell culture incubator (Binder, Germany) at 37ºC, 5%CO2 for three days. After 24 hours from the start of inoculation, the neurobasal medium in the plates with a similar one was replaced, but with a double content of L-Glutamax (0.5 mM). Cultivation was carried out for 14 days changing ½ portion of the medium to freshly prepared one every 2–3 days as metabolic products accumulated in the culture.
Measurement of cell respiration parameters. Basal mitochondrial respiration in E18 and P2 hippocampal cultures of various mouse strains in vitro was measured. An analytical medium DMEM Media (pH 7.4) containing 10 mM glucose, 1 mM pyruvate and 2 mM L-glutamine was prepared for measure the parameters of mitochondrial respiration, On the day of analysis, the neurobasal medium in the test plates was changed to the analytical medium and it incubated in for 1 hour at 37°C. Then, basal mitochondrial respiration using an Agilent HS Mini Cell Metabolism Analyzer (Seahorse, USA) was measured. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured on the 14th day of differentiation. Each series in four repetitions was performed. In each experimental and control cells 3 technical measurements were performed.
Energy phenotype assessment. The energy phenotype of neurons on the 14th day of differentiation using the Cell Energy Phenotype kit (Kit 103325-100, Agilent. USA) was assessed. The kit used oligomycin (ATP synthase inhibitor) at a final concentration of 100 µM and FCCP (mitochondrial uncoupler) at a final concentration of 100 µM. The concentration of the oligomycin/FCCP stress solution, which was added to the cartridge port, was 1.0/1.0 µM. Metabolic profile maps were constructed using Wave software (USA, Agilent). Each series in eight repetitions separately for the E18 culture and for the P2 culture was performed. Three technical measurements in each cell were performed. Metabolic maps were constructed using the software product Nova 1.3 (USA) and Milti-File Seahorse XF Cell Energy Phenotype (USA).
Statistical analysis. The results of experimental data were processed using Excel 10.0 descriptive statistic package. Experimental data in the tables by median (M) and standard deviation (SD) values are represented. The hypothesis of normal distribution was tested in the study. The Mann-Whitney test was used to assess the reliability of the results obtained for samples with non-normal distribution and number n ≤ 100. The total number of performed measurements of amount 24. The critical level of significance for testing the statistical hypothesis in this study was accepted at p = 0.05.