Animals

The Cre-lox recombination system was used to create the AIF overexpression mice, and the breeding pattern was consistent with our previous study [30]. The floxed mice with the insertion of the exogenous Aif gene at the Rosa26 gene locus were crossed with beta-actin-Cre mice (Fig. 1a). Both sexes of AIF-Tgflox/flox-actin-Cre (homozygous AIF hTg) and AIF Tg+/+-actin-Cre (wild-type, WT) mice with reasonable body weight (4.0–5.5 g) at postnatal day (P) 9 and with 5–8 pups per litter were used in this study. A total of 9 pups, including 6 males (3 WT) and 3 females (2 WT), were excluded because of death during hypoxia, and a total of 111 mouse pups were used for analysis. No statistical methods were used to predetermine sample size, and instead we based our experimental design on numbers reported in previous studies. All animals were allocated to the different experimental groups according to the different genotypes and were housed in a controlled temperature and pathogen-free environment under a 12-h light/dark cycle. All experimental procedures conformed to guidelines established by the Swedish Board of Agriculture (SJVFS 2019: 10) and were approved by the Gothenburg Animal Ethics Committee (112/2014). All animal experiments were performed in the Laboratory for Experimental Biomedicine of Gothenburg University and followed the guidelines of ARRIVE (Animal Research: Reporting in vivo Experiments). Genomic DNA was isolated from the tail sample, and PCR was performed for the genotyping. The primers for the AIF transgenic flox gene were 5′-GAG TTC TCT GCT GCC TCC TG-3′ (forward), 5′-AAG ACC GCG AAG AGT TTG TC-3′ (reverse for flox band, 215 bp), and 5′-CGA GGC GGA TAC AAG CAA TA-3′ (reverse for WT band, 322 bp), and the primer pair for the beta-actin-Cre gene was 5′-CTG CCA CGA CCA AGT GAC AGC AAT G-3′ (forward) and 5′-GCC TTC TCT ACA CCT GCG GTG CTA A-3′ (reverse) to produce an amplicon of 326 bp.

Fig. 1

Determination of AIF overexpression in AIF hTg mice. a Schematic figure of AIF-Tgflox/flox-actin-Cre mice. b Based on the FPKM data from the RNA-seq analysis, Aif expression was significantly increased in AIF hTg mice compared to WT mice under physiological conditions (n = 6/group). c The mRNA expression of different variants was determined at 24 h post-HI using RT-qPCR (n = 6/group). The total transcription of the Aif gene in AIF hTg mice was significantly higher than in WT mice, and this upregulation only existed for Aif1 and not for Aif2. Data are presented as the mean ± SEM and were analyzed using two-way ANOVA followed by Sidak’s post hoc test. ***p < 0.001

HI surgery

Unilateral HI was induced in mice of both sexes on P9, which is roughly equivalent to the brain development of a term newborn infant, according to the Rice–Vannucci model [17]. Mice of both sexes were anesthetized with isoflurane (5% induction, 1.5–2.0% maintenance), and the duration of anesthesia and surgery was < 5 min. The right common carotid artery was permanently ligated. The wounds were infiltrated with xylocaine after the surgical procedure. Following a 1-h recovery in their dam’s cage, the pups were placed in a chamber perfused with a humidified gas mixture (10% ± 0.01% oxygen in nitrogen) for 40 min at 36 °C. After the hypoxic exposure, the pups were returned to their dams until sacrifice. Control pups were subjected to all procedures except HI.

BrdU Administration

Bromodeoxyuridine (BrdU) (Roche, Mannheim, Germany, 5 mg/mL dissolved in 0.9% saline) was prepared fresh before use and injected intraperitoneally (50 mg/kg) on P8 and P9. The pups were continuously maintained without further disruption until P30 to collect brain tissues.

Tissue Collection and Histology

Brain tissues were harvested at 24 h or 72 h after HI. For the AIF hTg mice under physiological conditions, brain tissues were harvested at P30. The sample harvesting and histological procedures were as described in our previous study [30]. These slides were used for immunofluorescence staining for BrdU and doublecortin (DCX).

Fluoro-Jade Staining

After deparaffinization and rehydration, the sections were incubated with freshly prepared 0.06% potassium permanganate for 15 min and then rinsed in distilled water for 2 min. The sections were then incubated with 0.0004% Fluoro-Jade B (Merck Millipore, AG310-30MG) for 30 min in the dark at 21 °C and then washed with distilled water and mounted with ProLong Gold anti-fade reagent with DAPI (Invitrogen, P36931).

Immunohistochemistry

Brain sections were deparaffinized in xylene and rehydrated in graded ethanol, and antigen retrieval was performed by heating the sections in 10 mM boiling sodium citrate buffer (pH 6.0) for 10 min and allowing them to cool for 30 min. Non-specific binding was blocked by incubating with 4% donkey or goat serum in PBS for 30 min, and endogenous peroxidase activity was blocked with 3% H2O2. The primary antibodies were as follows and were diluted in PBS and incubated with the sections overnight at 4 °C: monoclonal mouse anti-MAP2 (1:1,000 dilution, clone HM-2, Sigma, M4403), monoclonal mouse anti-MBP (1:500 dilution, clone SMI94, BioLegend, 836,504), polyclonal rabbit anti-cleaved caspase-3 (1:200 dilution, Asp175, Cell Signaling, 9661), and monoclonal rabbit anti-AIF (1:500 dilution, E20, Abcam, ab32516) antibodies. After the primary antibody incubation, the appropriate biotinylated secondary antibodies (1:200 dilutions, all from Vector Laboratories, Burlingame, CA, USA) were added to each section for 60 min at room temperature. The sections were visualized with a Vectastain Elite ABC HRP Kit (Vector Laboratories, PK-6100) and 0.5 mg/mL 3,3′-diaminobenzidine enhanced with ammonium nickel sulfate, β-D glucose, ammonium chloride, and β-glucose oxidase. After dehydrating with graded ethanol and xylene, the sections were mounted on coverslips with Vector mounting medium.

Immunofluorescence and Microscopy

The deparaffinization, rehydration, and antigen recovery were performed as above. After blocking with 4% donkey serum in PBS for 30 min, the monoclonal mouse anti-3-NT (1.5:1000 dilution, 7A12AF6, Abcam, ab110282), polyclonal rabbit anti-DCX (1:500 dilution, Abcam, ab18723), or monoclonal mouse anti-BrdU (1:200 dilution, IIB5, Abcam, ab8955) antibodies were incubated with the sections overnight at 4 °C. After the primary antibody incubation, the donkey anti-mouse Alexa Fluor® 488 (1:500 dilution, Life Technology, A21202) or donkey anti-rabbit Alexa Fluor® 488 (1:500 dilution, Life Technology, A21206) secondary antibodies were added to each section for 120 min at room temperature. After washing, the sections were mounted on coverslips with ProLong Gold anti-fade reagent with DAPI (Invitrogen, P36931). For BrdU-DCX double staining, the mixed mouse anti-BrdU and rabbit anti-DCX primary antibodies, as well as mixed donkey anti-rabbit Alexa Fluor® 488 and donkey anti-mouse Alexa Fluor® 594 secondary antibodies, were used following the same staining procedure above.

Fluorescent staining was visualized using a Zeiss Axio Scan.Z1 digital slide scanner (Carl Zeiss, Germany), and panoramic images were processed using Zen software. For measuring the number of 3-NT-positive cells and the relative fluorescence units (RFUs) of 3-NT-positive areas in different brain regions, the whole brain sections, including both hemispheres, were scanned to obtain panoramic images using a 20 × objective lens. Three different sections with 50 interval sections were scanned for each brain sample. Three different regions of each section, including the cortex, cornus ammonis 1 (CA1), and striatum, were further captured within a defined area using Zen software. For measuring the number of BrdU-positive cells, the number of BrdU-DCX-positive cells, the DCX-positive area, and the RFUs of this area in the hippocampus, the whole hippocampal area of three sections was scanned at 250-μm intervals using the same scanner settings.

Brain Injury Evaluation

Brain injury was evaluated based on microtubule-associated protein 2 (MAP2) and myelin basic protein (MBP) immunostaining. Both hemispheres of each section were measured using Micro Image (Olympus, Japan) after the staining. The MAP2-positive and negative tissue volume, the MBP-positive tissue volume of the cerebral subcortical white matter (SWM) area, and the neuropathological scores of the gray matter in different brain regions were assessed as described previously [30]. Briefly, the cortical injury was graded from 0 to 4 with 0 indicating the absence of observable injury and 4 confluent infarction. The injury in the hippocampus, striatum, and thalamus was assessed both with respect to hypotrophy (scored from 0 to 3) and injury/infarction (scored from 0 to 3), resulting in a total possible score of 22. All evaluations were carried out by an experienced investigator blinded to group assignment.

Cell Counting, Positive Staining Area, and RFU Measurement

Area contours with fixed locations were drawn and measured in every 50th section. The section thickness was 5 μm. The active caspase-3-positive cells, AIF-positive nuclei, and Fluoro-Jade-positive cells were counted within a defined area (one visual field) of the cortex (100 × magnification), striatum (200 × magnification), CA1 (200 × magnification) (Fig. S1), and habenular nuclei (200 × magnification). The 3-NT-positive cells were counted in a fixed and same-sized area of the cortex (100 ×), CA1 (200 ×), and striatum (200 ×), and RFUs of the 3-NT-positive area were measured using the ImageJ software. BrdU-positive cells in the granular layer of the dentate gyrus were counted at 200 × magnification. The DCX-positive area and RFUs in the granular layer of the dentate gyrus (200 × magnification) were measured using ImageJ software. For the measurements using ImageJ, images were split into channels to transfer into gray mode. The positively stained area was determined by manually setting a threshold to include stained tissue, the integrated density of this positive area was measured, and the result was expressed as RFUs. All of the countings and measurements were carried out by investigators blinded to group assignment.

Sample Preparation for Immunoblotting and ELISA Assay

The pups were sacrificed by decapitation at 24 h after HI. Tissue from the parietal cortex (including the hippocampus) in both hemispheres was rapidly dissected out and homogenized immediately on ice using a 2-ml Dounce tissue grinder set (Sigma, D8938), and an isolation buffer was added (15 mM Tris–HCl, pH 7.6, 320 mM sucrose, 1 mM dithiothreitol, 1 mM MgCl2, 3 mM EDTA-K, and 0.5% protease inhibitor cocktail (Sigma, P8340)). The procedures for cellular fraction isolation, including nuclear, cytosolic, and mitochondrial fractions, were as described in our previous study [30]. All fractions were kept at – 80 °C.

Immunoblotting

Protein concentration was determined using the bicinchoninic acid method. A total of 65 µl of each sample was mixed with 25 µl of NuPAGE LDS 4 × sample buffer (ThermoFisher Scientific, NP0007) and 10 µl of NuPAGE Sample Reducing Agent (ThermoFisher Scientific, NP0004) and heated at 70 °C for 10 min. Samples were run on 4–12% NuPAGE Bis–Tris gels (Invitrogen) and transferred to reinforced nitrocellulose membranes (Bio-Rad). After blocking with 5% fat-free milk in TBST buffer (20 mM Tris, 150 mM NaCl, and 0.1% Tween 20, pH 7.6) for 60 min at room temperature, the membranes were incubated overnight with the following primary antibodies: rabbit anti-phospho-DRP1 (1:1000 dilution, Ser637, Cell Signaling, 4867), mouse anti-OPA1 (1:1000 dilution, BD Bioscience, 612,606), rabbit anti-FIS1 (1:500 dilution, FL-152, Santa Cruz, sc-98900), and mouse anti-VDAC1 (1:500 dilution, B-6, Santa Cruz, sc-390996). After washing, the membranes were incubated with peroxidase-labeled goat anti-rabbit IgG antibody (1:2000 dilution, Vector, PI-1000) or peroxidase-labeled horse anti-mouse IgG antibody (1:4000 dilution, Vector, PI-2000). Immunoreactive species were visualized using the SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific, 34,580) and an LAS 3000 cooled CCD camera (Fujifilm, Japan).

Total Antioxidant Capacity, Lipid Peroxidation, and Protein Carbonyl Content Assay

The homogenate of cortical brain tissue was used to measure total antioxidant capacity, lipid peroxidation, and protein carbonylation. The concentrations of total antioxidant capacity (Sigma-Aldrich, MAK187), malondialdehyde (MDA, Sigma-Aldrich, MAK085), and protein carbonyl content (Abcam, ab126287) were measured according to the manufacturer’s instructions. The concentration of total antioxidants was calculated as nmol per μL, MDA was calculated as nmol per mg protein, and the protein carbonyl content was calculated as nmol per mg protein. All measurements were performed by a person who was blinded to the grouping.

RNA-seq Analysis

Cortical samples from both sexes of P9 WT and AIF hTg mice were prepared for RNA sequencing. Total RNA from each sample was extracted using an RNeasy Mini kit (Qiagen, 74,104), and the library preparation was done using an MGI Easy mRNA Library Prep Kit (BGI, Wuhan, China) following the manufacturer’s instructions. The sequencing library was used for cluster generation and sequencing on a BGISEQ-500 system (BGI) [31]. To explore the potential molecular mechanisms underlying the effects of AIF overexpression, Gene Set Enrichment Analysis (GSEA) was performed using the clusterProfiler package to identify enriched terms predicted to have a correlation with the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The criterion of p < 0.05 was considered statistically significant.

RT-qPCR

Total RNA concentration and purity were determined using a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, USA). One microgram of total RNA was reverse transcribed using the QuantiTect Reverse Transcription kit (Qiagen, 205,311). According to the manufacturer’s instructions, RT-qPCR was performed using the LightCycler 480 instrument (Roche Diagnostics, Mannheim, Germany) and the SYBR green (ThermoFisher Scientific, 0253) technique. The primers used in the qPCR reactions with the mitochondrial fission and fusion genes were designed by Beacon Designer software (PREMIER Biosoft) and were as follows: Aif (sense: 5′- TAT TTC CAG CCA CCT TCT TTC-3′, anti-sense: 5′-TTC ACC ATG TTG CCT CTT AC-3′), Aif1 (sense: 5′-AGT CCT TAT TGT GGG CTT ATC-3′, anti-sense: 5′-GCA ATG GCT CTT CTC TGT T-3′), Aif2 (sense: 5′-TTC TTA ATT GTA GGA GCA ACA GT-3′, anti-sense: 5′-CCC ATC ACT CTT TCA TTG TAT CT-3′), Drp1 (sense: 5′-TGC TCA GTA TCA GTC TCT TC-3′, anti-sense: 5′-GGT TCC TTC AAT CGT GTT AC-3′), Fis1 (sense: 5′-ATG AAG AAA GAT GGA CTG GTA G-3′, anti-sense: 5′-GGA TTT GGA CTT GGA GAC A-3′), Opa1 (sense: 5′-CCT GTG AAG TCT GCC AAT-3′, anti-sense: 5′-TTA GAG AAG AGA ACT GCT GAA AT-3′), and Sdha (the reference gene) (sense: 5′-TTG CCT TGC CAG GAC TTA-3′, anti-sense: 5′-CAC CTT GAC TGT TGA TGA GAA T-3′). The relative expression levels of mRNAs were calculated according to the formula of 2−(ΔΔCT).

Statistical Analysis

GraphPad Prism 8.01 Software (GraphPad Software, San Diego, CA, USA) was used for all analyses. Comparisons between groups were performed by Student’s t-test, and data with unequal variance were compared with the Mann–Whitney U-test. Two-way ANOVA followed by Sidak’s post hoc test was used for multiple comparisons of data from more than two groups. Results are presented as means ± standard error of the mean (SEM), and p < 0.05 was considered statistically significant.