Animals and drug treatments.
Female C57BL/6 6 weeks young (19 ± 3 g, Orient Bio Inc., Seongnam, Gyeonggi-do, Republic of Korea) and female C57B216J 17-month-old (40 ± 10 g, Korea Basic Science Institute, Buk-gu, Gwangju, Republic of Korea) were used for the present study. Animals were housed in a place where water and food were freely consumed, and the environment was regulated (12 h light/dark cycle; 23 ± 2°C; the humidity of 50 ± 10%). This study was approved by the Animal Care Committee of Kyung Hee University (KHSASP-20-113), and the authors complied with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and Korean Academy of Medicine Animal Care Guidelines. Aptamers are short strands of oligonucleotides (DNA or RNA) or peptide molecules that can bind to specific target molecules and modulate their activity. By utilizing Nexmos Co., Ltd. self-developed SELEX (Systematic Evolution of Ligands by Exponential enrichment) technology, it specifically binds to vitamin C (ascorbic acid) as a single-stranded DNA aptamer, allowing its influx into the brain and prolonging the antioxidant effect in the brain. The purified DNA aptamer NXP032 (ascorbic acid/DNA Aptamin C320 complex) is dissolved in distilled water at 95°C for 5min and then cooled slowly at room temperature to form a tertiary structure. Aptamin C320 was mixed with ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA) in a ratio of 1:50 (w/w) to produce final NXP032 (200 mg/kg of ascorbic acid and 4 mg/kg of Aptamin C320).
In the experimental design, 6-week-old female mice and 17-month-old female mice were randomly divided into 5 groups (n = 40). The Young group (6-week-old mice), the A + Vehicle group (17-month-old mice + vehicle treatment), the A + Vit C group (17-month-old mice + Vitamin C treatment), the A + Apt C320 group (17-month-old mice + Aptamin C320 treatment), and the A + NXP032 group (17-month-old mice + NXP032 treatment). Mice were treated with vehicle, 200 mg/kg of vitamin C, 4 mg/kg of Aptamin C320, and NXP032 by oral gavage for 8 weeks simultaneously every day. After 8 weeks of treatment, cognitive behavioral experiments were conducted for 2 weeks. All mice were sacrificed the next day after the cognitive behavioral experiment test was finished.
Passive avoidance test
The passive avoidance test is a fear-aggravated test used to assess learning and memory in rodent models of CNS disorders. The manual evacuation system consists of two chambers separated by a wall with a guillotine door. During the training phase (1 day), mice acclimatize to the environment in the dark chamber for 60 seconds, then when the light is turned on, they will naturally move to the dark chamber according to their instincts. When it moves, the guillotine closes and receives an electric shock (0.5 mA for 2 seconds) through the stainless grid floor. In the retention test (2 day), the same procedure is performed and the waiting time for the mice to enter the dark room is measured for 300 seconds.
Y-maze test
The Y-maze test is a test to evaluate short-term memory and spatial perception. In the Y-maze test apparatus, each arm made of wood is divided into three branches, and each branch is 25 cm long, 14 cm high, and 5 cm wide, at the same angle (120° apart). After designating each arm as A, B, and C, the mice were allowed to move freely for 6 minutes starting from center, and then the movements of the mice were recorded. Mice were considered to have entered only when their hind legs had passed more than half of the maze when entering each arm. Changed behavior was assessed by recording the order in which each arm was entered. Only one point was recognized when entering each arm consecutively (alternation, ABC, ACB, BAC, BCA, CAB, or CBA). However, no points were awarded if each arm did not enter consecutively (for example, ABA, ACA, BAB, BCB, CAC, or CBC). The spontaneous alternation (%) was calculated by the formula: Spontaneous alteration (%) = [(total of number of alternations) / (total arm entries − 2)] × 100.
Cortex and hippocampus slice preparation and immunohistochemistry
Mice were sacrificed under deep anesthesia, perfused with 50 mM phosphate buffered saline (PBS), fixed with 4% paraformaldehyde (PFA), and brains removed. Brain tissues were extracted and fixed in 4% PFA for 1 day and then fixed in 30% sucrose solution in PBS for 4 days. Using a freezing microtome (CM3050S, Leica Biosystems GmbH, Nussloch, Germany), the brain was frozen sectioned to a thickness of 30 µm in the coronal direction. After incubation of endogenous peroxidase with 3% H2O2 in PBS for 20 minutes at room temperature. Brain tissues were then processed using a Mouse on Mouse detection kit (M.O.M. kit®, Vector Laboratories, Burlingame, CA, USA) to remove non-specific labels using mouse monoclonal antibodies on mouse brain tissue. Brain tissues were blocked with 1% bovine serum albumin (BSA) and 10% normal goat serum in PBS for 2 hours. Brain tissues were then incubated overnight with a PECAM-1 (1:1,000, LSBio, LifeSpan BioSciences, Seattle, WA, USA) at 4°C. Brain tissues were incubated with anti-mouse secondary antibody (1:200, Vector Laboratories, Burlingame, CA, USA) for 2 hours at room temperature. The bound secondary antibody was amplified using the antibody-biotine-avidin-peroxidase complex solution (Vector Elite ABC kit®; Vector Laboratories, Burlingame, CA, USA) for 1 hours at room temperature. Brain tissues were visualized using 3.3′-diaminobenzidine tetrahydrochloride (DAB kit; Vector Laboratories, Burlingame, CA, USA) for 5 min. Brain tissues were mounted onto gelatin-coated slides and air dried overnight at room temperature. Coverslips were mounted using Permount® (Vector Laboratories, Burlingame, CA, USA). Quantitative data were analyzed using Image-Pro® Plus software (Media Cybernetics, Inc., Rockville, MD, USA).
Immunofluorescence
Lycopersicon esculentum (Tomato) Lectin, DyLight 488 (Thermo Fisher Scientific, Waltham, MA, USA) was slowly injected with intracardiac perfusion (100 µg/mL) for 20 seconds. Then, after waiting for 90 seconds, it was perfused with PBS for 9 minutes, and then fixed with 4% PFA for 9 minutes. Brain tissues were washed and blocked with 3% BSA for 2 h at room temperature. They were then incubated overnight at 4°C with goat antibody to GFAP (1:500; Santa Cruz Biotechnology, CA, USA), rabbit antibody to PDGFR-β (1:500, Abcam, Cambridge, UK), and rabbit antibody to Iba-1 (1:500, Abcam, Cambridge, UK). Brain tissues were washed and incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG and Alexa Fluor 594-conjugated goat anti-mouse IgG (1:1,000; Molecular Probes, Eugene, OR) for 90 minutes at room temperature. Brain tissues were then washed and mounted onto gelatin-coated slides. The coverslips were mounted using 4′, 6-diamidino-2-phenylindole. Fluorescence staining was taken using confocal microscopy (LSM 700, Zeiss, Oberkochen, Germany).
Western blot
Proteins were extracted from mouse brain tissue using RIPA (ThermoFisher Scientific, Waltham, MA, USA) buffer. Proteins were separated on 8–10% SDS gels and transferred to PVDF membranes. The membrane was blocked with 5% skim milk at room temperature for 1 hour, then anti-ZO-1 (1:200, Lifespan Biosciences, WA, USA), anti-laminin (1:1,000, Abcam, Cambridge, UK), PECAM-1 (1:1,000, LSBio, LifeSpan BioSciences, Seattle, WA, USA) and β-actin (1:100,000, Santa Cruz, CA, USA) were incubated overnight at 4°C. The membrane was washed with PBST, and Membranes were incubated with a 1:2,000 dilution of horseradish peroxidase-conjugated anti-rabbit and mouse secondary antibodies for 2 hours. Band detection was performed using chemiluminescence (Clarity™ Western ECL Substrate, Bio-Rad, Hercules, CA, USA) on the membrane. Analysis was performed using image J (U.S. National Institutes of Health, Bethesda, MD, USA) to compare relative protein expression.