Sprague-Dawley rat pups at postnatal day (PND) 6 and fifteen-month-old male C57BL/6 mice were used in the present study. All experimental procedures and protocols were reviewed and approved by the Animal Investigation Ethics Committee of Nanjing University and were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals from the National Institutes of Health, USA. The animals were housed in a room maintained under standard environmental condition (temperature 22-24°C, a 12 h light/dark cycle, and 50 ± 10% humidity) with free access to food and water. The rat pups were housed with their mothers till PND 20. At PND 21, the pups were weaned and housed 4-5 per cage in standard condition.
GA was performed based on our previous optimization[17, 18], in which ketamine (75 mg/kg) intraperitoneally or 3% sevoflurane 2 h inhalation daily for three consecutive days can induce neurotoxicity and cognitive impairments. For ketamine GA, rat pups at PND 6 or fifteen-month-old mice received ketamine (75 mg/kg) intraperitoneally daily for three consecutive days. For sevoflurane GA, animals were put in an anesthetizing chamber delivered with 3% sevoflurane plus 30% oxygen (O2) for 2 h daily for three consecutive days. For control experiments, 30% O2 was delivered at the same flow rate. For drug treatment, DFP (75 mg/kg, intraperitoneally, synthesized in China Peptides Co., Ltd., Shanghai, China) or DMT1i (50 mg/kg, orally, MedChemExpress, China)[19, 20] was administered to the animals 1 hour before GA daily for three consecutive days.
The composition of the chamber gas was continuously monitored using a DatexTM infrared analyzer (Capnomac, Helsinki, Finland). Animals were kept normothermic throughout the experiment. Animals were kept warm on a plate heated to 37 ± 1 °C and returned to the cage until recovery of the right reflex after GA exposure.
Rat hippocampal neuronal culture and anesthetic exposure
Primary neuronal cultures were prepared from embryonic day 16-17 (E16-17) embryos of Sprague-Dawley rats as previously described. Neurons were dissociated and seeded on Poly-Lysine pre-treated culture dishes with neurobasal medium, supplemented with B-27, glutamine, antibiotics and 5% FBS (Invitrogen GIBCO Life Technologies, Carlsbad, CA, USA). After 2 h incubation, primary cultures were maintained in neurobasal medium without FBS in 5% CO2 incubator at 37 °C. Half of the culture medium was refreshed every 3 d. After 9 d of culture (DIV 9), the neurons were treated with ketamine (10 μM) in 5% CO2 incubator or 3% sevoflurane plus 5% CO2 for six hours at 37 °C, whereas the control group was maintained in same amount of culture medium. For drug treatment, DFP (100 μM) or DMT1i (3 μM) was added to the culture medium 30 min before GAs exposure.
Cell viability assays
The neurons were seeded in 96-well plates for 9 d. Cell Counting Kit-8 (Beyotime Biotechnology) was used to assess cell viability according to the manufacturer’s instructions. Results were expressed as the percentage of reduction of absorbance at 450 nm by calibration with the absorbance of the control cells.
Lactate dehydrogenase (LDH) assays
LDH is a stable cytoplasmic enzyme present in all types of cells and released into the cell culture medium through damaged plasma membrane. The neuron cells were plated into 12-well plates for 9 d and LDH release was measured by LDH Assay Kit according to the manufacturer’s instructions (Sigma-Aldrich, US) following the treatment as needs.
Mitochondria from hippocampus or primary hippocampal neurons were isolated using Qproteome Mitochondria isolation kit (Qiagen, Cat. No. 37612, USA). In brief, tissue or cells were homogenized in ice-chilled Dounce homogenizers using isolation buffer and centrifuged at 1,000 g for 10 min at 4°C. The supernatant was then centrifuged at 6,000 g for 10 min to separate the mitochondria and cytoplasm. The later mitochondria-enriched pellets were gently resuspended and washed with isolation buffer, then pelleted by centrifugation at 6,000 g for 10 min. The later supernatant was transferred into new EP tubes and centrifuged at 1,2000 g for 10 min at 4°C. This supernatant was considered as cytosolic fraction. The protein was determined by the Micro BCA protein assay kit (Beyotime Institute of Biotechnology).
Ferrozine iron assays
Iron content was measured using a colorimetric ferrozine-based assay with some modifications. Briefly, 22 μl concentrated HCl (11.6 mol/L) was added to 100 μl cell lysate (~500 μg total protein). The mixed sample was heated at 95 °C for 20 min, then centrifuged at 12,000 g for 10 min. Supernatant was transferred very gently into fresh tubes. Ascorbate was added to reduce the Fe (III) into Fe (II). After 2 min of incubation at room temperature, ferrozine and saturate ammonium acetate (NH4Ac) were sequentially added to each tube and the absorbance was measured at 570 nm (BioTek EL x 800, Shanghai, China) within 30 min.
Determination of mitochondrial swelling
To initiate mitochondrial swelling by Ca2+ uptake, freshly isolated mitochondria were suspended in mitochondrial suspension buffer [120 mM KCl, 25 mM sucrose, 5 mM KH2PO4, 0.1 mM EGTA, 20 MOPS (pH 7.2)] in the presence of 5 mM malate and 5 mM pyruvate as substrates. Swelling was recorded as the decrease of the density of the mitochondrial matrix at 540 nm with a UV/Vis spectrophotometer after adding 0.5mM Ca2+ into the medium.
Determination of mitochondrial membrane potential (MMP) levels and ATP contents
Levels of MMP and ATP contents were determined in hippocampal neurons using a 5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolylcarbocyanine iodide (JC-1) mitochondrial membrane potential detection kit (Solarbio Science & Technology Co. Ltd.) and ATP content assay kit (Beyotime Biotech) according to the manufacturer’s instructions.
Mitochondrial morphology imaging.
Mitochondrial morphology was observed under the confocal microscopy following staining with the mitochondria targeting dye MitoTracker Red CMXRos (Beyotime Biotech). After GA treatment for 6 h, the neurons were washed and stained with 20 nM MitoTracker Red CMXRos in neurobasal medium for 30 min at 37˚C in dark in a 5% CO2 incubator. The cells were then washed with HBSS and immersed in neurobasal medium to prevent cell damage. Images were obtained with confocal microscopy (Fluoview FV 10i, Olympus) and analysed using FV10-ASW 2.1 Viewer software.
Measurement of cellular ROS, MDA and GSH levels
Intracellular reactive oxygen species (ROS) levels were estimated using a fluorescence-labelled probe DCFH-DA (Beyotime Biotech). Cellular malondialdehyde (MDA) and glutathione (GSH) levels were measured following the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Western blot analysis
Proteins were obtained from hippocampal tissue or cultured neurons or isolated mitochondria. For western blotting, 35-50 µg protein was added per lane of 12% SDS-PAGE. Primary antibodies were diluted in primary antibody dilution buffer (Beyotime Biotech., Shanghai, China). Antibodies used included: anti-GAPDH (Abgent Biotech. Co. Ltd., Suzhou, China), anti-ferritin, superoxide dismutase 2 (SOD2), Mitoferrin1, Drp1, Mfn2, RASD1, NMDAR1, and NMDAR2A (Abcam, Cambridge, MA, USA), anti-TfR1 (Zymed, San Francisco, CA, USA), anti-IRP2 (polyclonal, raised from rabbit), and anti-DMT1 (Alpha Diagnostic International, San Antonio, TX, USA). Detection was performed using peroxidase-conjugated secondary antibodies (Thermo Fisher Scientiﬁc, Waltham, UK). Quantiﬁcation of the density of the western bands was done with programme ImageJ (http://rsb.info.nih.gov/ij/).
Morris Water Maze (MWM) tests
To investigate spatial learning and memory function, we subjected the rats (n = 12 for each group) to the MWM tests (XR-XM101; Shanghai Xinruan Information Technology Co., Ltd., Shanghai, China) at PND 60. The MWM was a black metal tank (120 cm in diameter, 60 cm in depth) equipped with a platform (10 cm in diameter) 1-2 cm below the surface of the water. The MWM task was performed according to our previous study . Briefly, it consists of two phases, training phase for five consecutive days and probe trial phase on day 6. In the training phase, the rat was allowed to face to the pool wall in four random places (N, S, E, W) in the pool to find the fixed platform. Release positions were randomly predetermined. The trial was terminated once the rat reached the platform. If the rat failed to reach the platform within 60 s, it would be guided to the platform and allowed to stay for 15 s, then the latency was recorded for 60 s. In the probe test, single-probe trial was conducted with the original platform removed 24 h after the last training session. The rat was released at a random start position and allowed to swim for 60 s in the pool.
Fear conditioning tests
To measure the abilities of learning and memory, we employed the fear conditioning paradigm (30 cm long × 26 cm wide × 22 cm high, XR-XC404, Shanghai Softmaze Information Technology Co. Ltd.) to the aged mice 24 hours after GA. Each mouse was exposed in the conditioning chamber for 3 min of accommodation then one tone-foot-shock pairing (tone, 30 s, 65 dB, 3 kHz; foot-shock, 3 s, 0.75 mA) was delivered. The contextual fear conditioning test was performed 24 h later by placing mice back to the same test chamber for 5 min without any stimulation. Two hours later, each mouse was placed in a novel chamber altered in shape, color, and smell, and the same tone was presented for 3 min without the foot shock to evaluate tone fear conditioning. Cognitive deficits in the test were assessed by measuring the length of time of “freezing behavior”, which is defined as a completely immobile posture except for respiratory efforts. Freezing behavior was automatically recorded by the video tracking system.
Data were presented as mean ± SEM and analysed by the Statistical Product for Social Sciences (SPSS; version 18.0, IL, United States). The difference between the groups was determined by one-way analysis of variance followed by the Bonferroni test. Comparisons for the spatial training sessions of MWM were performed by repeated two-way ANOVA followed by the LSD test. A p-value <0.05 was considered statistically significant.