Animals and experimental groups
The experimental protocol for the study was approved by the Institutional Animal Care and Use Committee of The Third Affiliated Hospital, Harbin Medical University and was performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. And the experiments reported were in compliance with the ARRIVE guidelines. Rats were euthanized by cervical dislocation after anesthetized with 2% sevofluorane.
A total of 60 adult male Sprague-Dawley rats (weight 260–360 g) were used for the study and were housed in a rodent facility under 12 h light-dark cycle with unrestricted access to food and water. Rats were allocated into three groups: sham, CA, and melatonin plus CA (Mel + CA). Melatonin (N-acetyl-5-methoxytryptamine), dissolved in vehicle (2% dimethyl sulfoxide in sterile saline), was injected intraperitoneally to the rats in the Mel + CA group with a dosage of 100 mg/kg once daily at 10:00 a.m. for 14 days before the experiment (Fig. 1A). Rats in the CA group received the same volume of vehicle. The investigators performing CA and completing the evaluations were blinded to allocation assignment throughout the study period until final analysis.
Asphyxia-induced CA and CPR
Following a 14-days administration of melatonin or vehicle, the experiment was performed at the same time of the day around 09:00 a.m.-10:00 a.m. The experimental procedures were performed as previously described with minor modification.(13) Rats in the CA group and the Mel + CA group were anesthetized with 6% sevofluorane. Mechanical ventilation was initiated using a volume rodent ventilator (Model 683, Harvard Apparatus, South Natick, MA, USA) at the rate of 40/min, I:E = 1:1, FiO2 = 0.5. The tidal volume (8–12 ml/kg) was regulated by end-tidal CO2 maintained between 35 and 45 mmHg. Anesthesia was maintained with 1.5-2% sevofluorane. The left femoral artery and vein were cannulated for blood pressure monitoring and blood sampling. Rectal and tympanic probes were used to monitor the temperature. After the rats were equilibrated on the ventilator to get the baseline mean arterial pressure (MAP) and heart rate (HR), the experimental animals were given cisatracurium (1.5 mg/kg, intravenously) 5 min before asphyxia. CA was induced via asphyxia by turning off the ventilator and clamping the endotracheal tube. CA was confirmed by a MAP sharply decreased below 20 mmHg. The rectal temperature was maintained at 37.0 ± 0.5°C during the entire asphyxia period by heating lamp. Following 8 min of asphyxia, chest compressions were performed at a rate of 200–300/min in combination with 0.01 mg/kg epinephrine, iv. Ventilation was resumed at the same time. Return of spontaneous circulation (ROSC) was confirmed by the return of HR rhythm and significant increase of MAP. If the rat does not establish ROSC after 5 min, efforts were stopped. The experimental animals maintained on mechanical ventilated after ROSC for 1 h. During this time, 1 µg epinephrine was used when MAP dropped below 50 mmHg. Rats received the same cannulation, anesthesia and infusions in the sham group. The endpoint was 24 h after resuscitation.
Survival and neurological evaluation
Survival was compared at 24 h after resuscitation among groups. Revised neurological deficit scores (NDS; 0 = normal, 500 = brain death) and overall performance categories (OPC; 1 = normal, 2 = moderate disability, 3 = severe disability but conscious, 4 = coma, and 5 = death) were evaluated at 24 h post-resuscitation.(13)
Tissue processing for histology
At 24 h after resuscitation, the survived rats were anesthetized and transcardially perfused via the left ventricle with 150 ml 0.9% iced saline followed by 100 mL of 4% paraformaldehyde. The brains were removed, post fixed in 4% paraformaldehyde overnight at 4°C, and then transferred sequentially into 20%, and 30% sucrose solution overnight prior to dissection and sectioning. The brain tissue was embedded in Tissue-Tek® O.C.T. compound (Sakura Finetek Inc, Torrance, CA) and serially sectioned into 10 µm coronal sections on a cryostat (Leica, Germany). Sections were then collected into six-well plates containing PBS for histology.
Nissl staining
Nissl staining was used to observe the morphological alterations in the hippocampus CA1 region. The sections were stained in 1% cresyl violet solution at 37°C for 10 min and then were differentiated in 2.5% iced acetate ethanol solution for 20–30 min. The severity of neuronal damage was evaluated by counting the surviving neurons under a microscope and only neurons with distinct nucleus and nucleoli were considered as healthy living cells.
Immunofluorescence staining
Immunofluorescence staining was performed as described previously. In brief, after being blocked with goat serum for 1h at 37°C, the sections were incubated with rabbit anti-SIRT3 (1:50, Santa Cruz Biotechnology) or rabbit anti-cytochrome c (1:50, Santa Cruz Biotechnology) overnight at 4°C and then with secondary antibody (CY3-conjugated rabbit anti-mouse lgG, Beyotime) for 1 h at room temperature. Nuclei was labeled with 4,6-diamidino-2-phenylindole (DAPI; 1:1000, Beyotime) for 1 min. Images were captured with a confocal fluorescence microscope (Olympus, Tokyo, Japan).
Mitochondrial isolation and extraction
The brain was harvested at 24 h post-resuscitation. The hippocampal mitochondria were isolated from the brain. The highly purified mitochondria were used for western blot and transmission electron microscopy (TEM) analyses. For the mitochondrial function assay, the hippocampal mitochondria were extracted using a tissue mitochondria isolation kit (Beyotime Institute of Biotechnology, China). The homogenate of hippocampus was centrifuged at 1000 g for 5 min at 4°C to remove nuclei and any unbroken cells. The supernatant was collected and centrifuged at 35,000 g for 10 min at 4°C to obtain the mitochondrial fraction.(17)
TEM
TEM analyses of mitochondria were performed by an expert from the Electron Microscopy Laboratory of Harbin medical University as described previously.(5) The standards for the evaluation of mitochondrial injury from grade 0 to 4 were as follows: grade 0, normal mitochondria (mitochondria appeared highly dense with well-organized cristae); grade 1, early swelling as manifested by early clearing of matrix density and separation of cristae (a large amorphous matrix density and a linear density are present); grade 2, more marked swelling as manifested by further clearing of matrix density and separation of cristae; grade 3, more extensive mitochondrial swelling with disruption of cristae; grade 4, severe mitochondrial swelling with disruption of cristae and rupture of inner and outer mitochondrial membranes.(5) Normal (grade 0–1) and swollen mitochondria (grade 2–4) were counted with the Image J software (10 neuropil areas per group).
Detection of ΔΨm
ΔΨm was monitored using the JC-1 Mitochondrial Membrane Potential Detection Kit (Beyotime Institute of Biotechnology, China). ΔΨm was determined using the ratio of JC-1 aggregates (red) to JC-1 monomers (green). Mitochondrial depolarization was expressed as the decrease in the intensity ratio of red/green fluorescence.(32)
Detection of ROS
Mitochondrial ROS was measured with a ROS assay kit (GENMED Bioengineering Institute, Shanghai, China). The chromogenic reaction mixture was added to equal amounts of mitochondria (50 µg/50 µL) in each sample at 37°C for 15 min. All steps were performed in the dark. Production of ROS was observed at an excitation and emission of 490 nm and 530 nm with an epifluorescence microscope.(32)
Mitochondrial oxygen consumption
Oxygen consumption by mitochondria (1 mg/mL) was measured with a Clark-type oxygen electrode at 25°C (Hansatech Oxygraph, Hansatech, Norfolk, UK). The baseline oxygen consumption rate (OCR) was measured at 2 min following the addition of mitochondria until the recorded curve stabilized. State 4 respiration was initiated by 20 µL disodium succinate (4 mM). Then, 20 µL adenosine diphosphate (ADP, 50 mM) was added to the incubation medium to initiate state 3 respiration. The respiratory control rate (RCR) was calculated from the rate of state 3 to state 4 respiration.(35)
Western blot analysis
The frozen hippocampus samples in the each group were homogenized and protein was quantified by BCA Protein Assay kit (Beyotime Biotechnology). Western blot was performed as previously described.(34) Images of blots were captured with an Image Quant ECL Imager (GE Healthcare, Chicago, IL), and the bands were quantified with Image J software. The following primary antibodies were used: cleaved caspase-3 (1:500, Cell Signaling Technology), caspase-9 (1:500, Cell Signaling Technology), Cyclophilin D (CypD; 1:500, Abcam), mitofusin 2 (Mfn2; 1:1000, Abcam), mitochondrial dynamin-related protein 1 (Drp1; 1:500, Abcam), PTEN-induced putative kinase 1 (PINK1; 1:500, Abcam), Parkin (1:500, Abcam), LC3 (1:400, Abcam), and β-actin (1:2000, Abcam). Acetylation of CypD was determined by immunoprecipitation followed by western blot analysis. Acetylated CypD was quantified by normalization with IgG.
Statistical analysis
Sample size for CPR outcome measurements was at least twelve rats per group with an alpha error of 0.05 and a power of > 80%. The physiological parameters, the mitochondrial integrity, the mitochondrial swelling, ΔΨm, ROS, and immunofluorescence were analyzed by a one-way ANOVA followed by Bonferroni post hoc analysis or a Student’s t-test. The chi-square test was used to test the rates of ROSC, the survival, the differences in proportions of OPC values between groups (favorable vs. unfavorable outcome, OPC 1–2 vs. OPC 3–5). Kruskal-Wallis test followed by Mann-Whitney U test were used to compare NDS among groups. One-way ANOVA followed by LSD post hoc tests was performed to identify differences between groups in the neuronal cell loss, mitochondrial oxygen consumption parameters and to evaluate the western blot quantification analysis. A P < 0.05 was considered a significant difference. Statistical tests were performed with SPSS 19.0 software (SPSS Inc., USA).