Animals and grouping
In-vivo: Sixty-four seven-day-postnatal (P7) Sprague-Dawley (SD) male rats (12-15g) were purchased from the Department of Zoology of Kunming Medical University. Experimental procedures were reviewed following the standard biosecurity and institutional safety procedures and approved by the Ethic Committees at Kunming Medical University in Yunnan province, China (reference number: kmmu2018031). All animals were raised with their mother in plastic cages with soft wood and free access to food and water in a temperature (21-25℃) and humidity (50-60%)-controlled room. Experimental operations and data analysis can’t be done by the same researchers, and data analysis must be dealt by at least two researchers blinded to the experimental design. Rats were randomly divided into the sham and HI group, n=32 per group, with 10 rats in each group were used to perform the zea-longa score test. The morphological tests included Triphenyte-trazoliumchloride (TTC) staining (n=3), Terminal DeoxynucleotidylTransferasedUtp Nick End Labeling (tunel) staining (n=3), immunohistochemistry staining (n=3) and immunofluorescent analysis (n=3). The expression of COX5A was detected by quantitative real-time polymerase chain reaction (qRT-PCR) (n=10) and western blotting (WB) (n=10). In-vitro: Twenty SD new-born rat pups (1 day of age) were used to collect and culture the primary cortical neurons. Neurons were seeded on poly-d-lysine and laminin coated dishes at a density of 7×105 cells/ml for qRT-PCR and WB (n=6 wells) and 5×105 cells/ml for tunel staining and immunoflurscent analysis (n=3 wells). Primary cultured neurons were divided into 4 groups: normal group; OGD group; OGD+ negative control (NC) group; OGD + COX5A group. Molecular biomarker in this study represented as the primary experimental outcomes.
Rat model of neonatal HI injury
In this experiment, we employed the classic Rice-Vannucci method to establish the HI model [23, 24]. Briefly, P7 SD rats were anesthetized with isoflurane (4% for induction, 2% for maintainence, inhalation anesthesia) after they were weighed and numbered. The hypoxic chamber was set at 37℃ with humidity 50-80% before the operation. Briefly, the midline of the ventral cervical skin was cut followed by a blunt dissection of parenchyma, to expose the right common carotid. Subsequently, the right common carotid was ligated by an electric coagulator. Then, the wound was stitched and animals were returned to their mother for 1h before being kept in a hypoxic chamber under 8% O2 and 92% N2 (flow rate 3 L/ min) for 2 h. Rats in the sham group underwent the same procedures, apart from ligation of the right carotid artery.
Zea-longa score
All rats underwent the zea-longa score test to evaluate the neurological dysfunction at 0 h, 2 h, 4 h, 8 h, and 16 h after HI. The specific scoring criteria were as follows: 0 points - no signs of nerve injury; 1 point – the contralateral forepaw lost ability to become fully stretched; 2 points - animals turns to the side while walking; 3 points - walking is unstable, falling to the a side; 4 points - loss of consciousness [25].
Tissue acquisition
For morphological analysis, rats in the sham (n=3) and HI groups were anesthetized with 3% isoflurane (inhalation anesthesia) at 16 h after the surgery. Then, after the perfusion of 0.9% normal saline followed by 4% paraformaldehyde, the rats were killed and the brain was harvested and put in 4% paraformaldehyde for more than 72 h. With paraffin embedded, the brain tissue was prepared for immunohistochemical staining and immunofluorescent staining.
For molecular biology analysis, rats in the sham (n=10) and HI (n=10) groups were anesthetized with 3 % isoflurane at 16 h after the surgery followed by perfusion with 0.9% normal saline. Thereafter, the cortex from both hemispheres of harvested brains were removed and stored at -80ºC for further WB and qRT-PCR.
Triphenyte-trazoliumchloride (TTC) staining
The whole brain from the sham (n=3) and HI (n=3) rats was quickly removed at 16 h after rats being humanely killed by 5% CO2 inhalation, brain tissues was taken out (operating on ice) and frozen in a refrigerator at -20 °C for 10 min before being cut into five pieces (2 mm each). Afterwards, the sections were placed in 1% TTC solution at 37 °C and incubated for 30 min. The sections were then captured using the digital camera. The non-ischemic necrotic area was pale red, and the ischemic necrotic tissue was white, the ratio of infarct was analyzed using Image J software [26].
Immunohistochemistry in-vivo
The collected brain was paraffin embedded and cut into 5µm sections, then fixed on the glass slides. Before immunohistochemical staining, the sections were deparaffinised and a circle was drawn around the tissue with a PAP pen. Thereafter, sections were washed for three times with 0.01M PBS buffer (PH=7.45), 5 min each. Next, 3% hydrogen peroxide was added onto the sections followed by incubating at 37℃ for 15min and washed as described above. In order to block the non-specific binding in the tissue, a drop of 5% goat serum (Solarbio, S9070) was added on the section and incubated at 37℃ for 30 min. Subsequently, the primary antibodies (COX5A, Zhongshanjinqiao, rabbit, 1:50) were added and 2% goat serum was used as the negative control and left overnight in a refrigerator at 4℃. The next day, sections were washed with 0.01M PBS buffer for three times, followed by addition of enhancement solution, incubation at 37℃ for 20min and washing with 0.01M PBS three times. Subsequently, the secondary antibody (Zhongshanjinqiao, goat anti-rabbit IgG) was added and the sections were incubated at 37℃ for 1 h, and washed with PBS. Next, DAB staining was performed for 5 min following the manufacturer’s protocol. To perform nuclei staining, the sections were treated with hematoxylin solution for 5 min, and soaked in 1% HCl-ethanol solution for 10 s followed by 5% ammonia treatment for 1 min. Afterwards, they were dried with serial dilutions of (75%, 80%, 85%, 95%, and 100%) and treated with xylene solution for 2 min, finally sealed with neutral gel. The immune-positive pictures were captured using a light microscope (Leica DMI 6000 B inverted microscope, Germany), and each slice was randomly collected in 200x, and the quantitative analysis in each group was determined by the positive cells in each field via Image-Pro Plus 6.0 software (MediaCybernetics, Silver Spring, MD, USA).
Primary cortical neuron cultures and COX5A-over-expression HSV vector transfections
The primary cortical neurons were isolated and cultured from freshly dissected brains of rat pups at postnatal day one. In brief, the cortical tissues were dissociated to disperse the cells in a nerurobasal medium (Gibco, California-USA) supplemented with 2% B27 and 0.5 mM of glutamine. The cells were plated on poly-L-lysine and laminin coated vessels at the density as described above and were incubated at 37°C with 5% CO2 incubator. The media were then replaced on alternating days as described before [27]. Thereafter, a low toxicity HSV-COX5A virus was purchased from Sky Bio (Beijing-China) to explore the function of COX5A according to the manufacturer’s protocol. Briefly, HSV-COX5A virus was transfected into the neurons after five days of incubation at a MOI=10. Detection of the green fluorescence and qRT-PCR were employed to demonstrate the successful transfection and over-expression of COX5A.
Oxygen-glucose deprivation (OGD)
After culturing for 7 days, OGD was established to mimic the HI condition in vivo. In detail, following three washes with PBS, the neurons were incubated using glucose-free medium (Gibco, USA) in an anaerobic chamber containing 5% CO2 and 95% N2 at 37°C for 90 min as described earlier [27]. Subsequently, cells were returned to original medium, then placed in a normoxic chamber with 95% air and 5% CO2 for 16 h before further testing.
Terminal Deoxynucleotidyl Transferased Utp Nick End Labeling (tunel) in-vivo and in-vitro
The brain slices and cells were rinsed three times with 0.01M PBS buffer for 5 min, and 50 μl of 0.01% sodium citrate and 0.1% TritonX-100 was added to each slice and incubated for 30 min at 37°C. Then, the slices were incubated with the tunel mixture reagent (tunel label solution: Tune enzyme solution = 9:1) for 1h at 37°C. For the negative control, only the tunel label solution was added dropwise. After that, counterstaining of the nuclei was performed using DAPI staining. Five fields on each slip were randomly chosen for imaging with a fluorescence microscope (Leica, CM1860, Germany). The positive rate was quantified by Image-Pro Plus 6.0 software (tunel positive cells / DAPI cells).
Immunofluorescent staining in-vivo and in-vitro
Sections harvested from the brain were deparaffinized and washed three times with 0.01M PBS and pre-incubated for 30 min with 0.3% TritonX-100 in 5% normal goat serum. Then they were incubated overnight at 4°C with a primary antibody against NEUN (mouse, Bioss, 1:100). The next day, the primary antibodies were removed and sections were rinsed with 0.01M PBS buffer for three times before being incubated with the fluorescence-labeled secondary antibodies of Alexa 488 (anti-mouse, Invitrogen, 1:100) for 1 h at 37°C. Counterstaining of the nuclei was performed using DAPI staining. Finally, the slides were analyzed with a fluorescent microscope (Leica, CM1860, Germany) to observe the change of NEUN after HI injury, in detail, five fields with at least 10 cells per field were randomly selected for the estimation of relative number of NEUN positive cells using Image-Pro Plus 6.0 software (NEUN positive cells) .
To study the expression of COX5A in OGD condition and the effect of COX5A on Neuronal Class III β-Tubulin (Tuj1+) in neurons, the primary neuronal cells from the normal, OGD, OGD+NC, OGD+COX5A groups were cultured on glass cover slips. In brief, the cells fixed with 4% formalin and blocked with 0.3% TritonX-100 in 5% normal goat serum for 30min at 37°C. The slips were then incubated with COX5A (Mouse, Santa Cruz, 1:100) and anti-beta III Tubulin antibody (Mouse, abcam, 1:200) respectively at 37°C for 16-18h. Then, the cells were rinsed three times with PBS before being incubated with fluorescence-labeled secondary antibodies of Dylight 594 (anti-mouse, abbkine, 1:200) at 37 °C for 1h, then DAPI was used to counterstain the nuclei. Five fields in each well was randomly collected in 200x with a fluorescence microscope (Leica, CM1860, Germany). The COX5A positive cells, neurite lengths, average cell number and neuron area of Tuj+ cells quantified by Image-Pro Plus 6.0 software.
Quantitative real-time polymerase chain reaction (qRT-PCR)
The relationship between Gstp1, Sod2, Rho-GDIa, TPI and COX5A was predicted using the GeneMANIA website (http://genemania.org/), then qRT-PCR was performed to detect the mRNA expression of these genes after COX5A over-expression. The primers were designed using Premier 5.0 software, then verified via the BLAST software and synthesised by Takara Bio Inc. (Takara, Japan). Total RNAs from the fresh cortex and cultured neurons were extracted using the RNAiso plus kit (TaKaRa, JAPAN) and cDNA Synthesis was performed using the ReviertAid Kit (Thermo Fisher Scientific Inc.). The levels of Gstp1, Sod2, Rho-GDIa, TPI, COX5A and β-actin mRNA expression in the samples were estimated by qRT-PCR (CFX-96, Bio-Rad, USA) according to the manufacturer’s instructions using the primers described in Table1. Briefly, each reaction was performed in a volume of 20 μl consisting of 10 μl of SYBR Green master mix, 1 μl of cDNA, 7.8µl of water, and 0.6µl each of forward and reverse primers. Finally, the data were analyzed using a comparative critical threshold (Ct) method where relative expression was calculated as 2-△△Ct method.
Western blotting (WB)
The right and left cortex from rats of the sham and HI group and primary neuronal cells from the normal, OGD, OGD+NC, OGD+COX5A groups were collected and lysed with RIPA buffer containing a proteinase inhibitor cocktail (Roche) on ice for 30 min, then the lysates were centrifuged at 12000rpm for 10 min at 4 °C. Protein concentrations were estimated by BCA protein quantification kit. The protein samples were boiled and denatured with a loading buffer and 40 μg of protein was added to each well and run at 120 V for 90 min. After that, the proteins in the gel were transferred to a PVDF membrane at a current of 200 mA. The PVDF membrane was then blocked with 5% skim milk powder for 90 min at room temperature, and further incubated with related primary antibodies (seen in Table 2) at 4 °C overnight, β-actin was used as an internal control. Thereafter, proteins were visualized by incubation with the secondary antibodies for 2 h (seen in Table 2). Finally, the membranes were developed and the bands were visualized with the ECL (ECL Western blotting kit) luminescence solution. The quantitative analysis was carried out by Image J software. The data were expressed as a ratio of the protein of interest band to β-actin band optical density values.
Statistical Analysis
Data were expressed as mean ± standard deviation (SD). The comparison between two groups was performed using a Student’s t test. For comparison of multiple groups in-vitro experiments, ANOVA with least-Significant Difference (LSD) or Dunnett’s T3 post hoc test was applied, if equal variances were found, LSD was performed; otherwise, Dunnett’s T3 was used [28]. All statistical analyses were performed with SPSS18.0 software (IBM Corporation, NY, USA). p<0.05 was considered statistically significant.