Animals and grouping
Forty seven-day-old postnatal (P7) Sprague-Dawley (SD) male rats (12-15 g) used for in vivo experiments and twenty one-day-old rats for primary cortical neurons cultures 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.
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 sustained 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 1 h 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
Zea-longa scores were used to evaluate the neurological function in neonatal rats subjected to HI injury, verifying the successful modeling. 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 one side while walking; 3 points - walking is unstable, falling to one side; 4 points - loss of consciousness [25].
Tissue acquisition
For morphological analysis, rats in the sham (n=5 for TTC staining, n=3 for immunohistochemical and immunofluorescent staining) and HI (n=5 for TTC staining, n=3 for immunohistochemical and immunofluorescent staining) groups were euthanatized at 16 h after HI under deep anesthesia with 4 % isoflurane (sustained inhalation anesthesia) for 2 min. Then, after the perfusion of 0.9 % normal saline followed by 4 % paraformaldehyde, the brain was harvested and put into 4 % paraformaldehyde for more than 72 h. With paraffin embedded, the brain sections (5 μm) were prepared for immunohistochemical staining and immunofluorescent staining, and the core infarction regions were the key observations.
For molecular biology analysis, rats in the sham (n=6 for qRT-PCR and n=6 for WB) and HI (n=6 for qRT-PCR and n=6 for WB) groups were sacrificed at 16 h after the surgery under deep anesthesia by continuously inhaling 4 % isoflurane for 2 min followed by perfusion with 0.9 % normal saline. Thereafter, the white infarcted area and non-infracted area of the contralat-eral hemisphere were removed and stored at -80 ºC for further WB and qRT-PCR.
Triphenyte-trazoliumchloride (TTC) staining
To evaluate the brain damages after HI, TTC staining was performed to observe the infarction of brain tissues in neonatal HI rats. The whole brain from the sham and HI rats was quickly removed at 16 h after rats being deeply anaesthetized with 4% isoflurane, 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 immunohistochemistry was employed to detect COX5A expression. The collected brain was paraffin-embedded and cut into 5 µm sections, then fixed on the glass slides. Before immunohistochemical staining, the sections were deparaffinized 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 15 min 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, lefting 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 20 min 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 then 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 ethanol (75 %, 80 %, 85 %, 95 %, and 100 %) and treated with xylene solution for 2 min, finally sealed with neutral resins. The immune-positive pictures were captured using a light microscope (Leica DMI 6000 B inverted microscope, Germany). For quantification, three sections of each brain with 50 μm apart were selected, and five fields of each slice were randomly collected at 100 x. The positive cells (dark brown labeled) in each field were quantified via Image-Pro Plus 6.0 software (MediaCybernetics, Silver Spring, MD, USA).
Primary cortical neuron cultures and COX5A-over-expression HSV vector transduction
COX5A overexpression transduction was conducted for the further functional verification of COX5A’s role in the process of HI. 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 neuro-basal 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. 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 transducted into the neurons after five days of incubation at a MOI=10. GFP, emitting green fluorescence, expressed from a co-infection with a GFP-expressing virus in neurons after transduction. Then the infection rate was calculated (GFP -positive cells / total neurons). Detection of the green fluorescence and qRT-PCR were employed to demonstrate the successful transduction and over-expression of COX5A.
Oxygen-glucose deprivation (OGD)
After culturing for 7 days, OGD was established to mimic the HI condition in vitro. 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.
Immunofluorescent staining in-vivo and in-vitro
In order to examine the neuronal viability after HI injury in vivo and in vitro, immunofluorescence staining was carried out. In vivo, the brain slices (n=3 brains, and three sections of each brain with 50 μm apart) were subsequently deparaffinized and washed three times with 0.01 M 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 primary antibodies 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. As NeuN was located on the cytoplasm and nucleus of neurons, so the positive staining was the nuclei of neurons labeled with green fluorescence and co-stained with DAPI, which showed blue fluorescence. Finally, the slices were observed under a fluorescent microscope (Leica, CM1860, Germany) at 200 x. In detail, five fields of each slice were randomly selected for the estimation of relative number of NeuN positive cells (green fluorescence labeled) using Image-Pro Plus 6.0 software.
In vitro, 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 cell sections (n=6 in each group) were fixed with 4 % formalin and blocked with 0.3 % TritonX-100 in 5 % normal goat serum for 30 min at 37 °C. The slips were then incubated with anti-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 1 h. Then DAPI was used to counterstain the nuclei. Five fields in each well were randomly collected at 400x with a fluorescence microscope (Leica, CM1860, Germany). The COX5A positive cells and average cell number of Tuj+ cells, average length of the neurite (about calculating 15 neurons) were quantified by Image-Pro Plus 6.0 software.
Terminal Deoxynucleotidyl Transferased Utp Nick End Labeling (Tunel) in-vivo and in-vitro
Tunel staining was performed to determine the apoptotic occurrence in the cortical infarction region of neonatal HI rats and cultured primary cortical neurons subjected to OGD. Briefly, the brain slices (n=3 brains, and three sections of each brain with 50 μm apart ) and cell sections (n=6 in each group) were rinsed three times with 0.01 M PBS buffer for 5 min, and 50 μl of 0.01 % sodium citrate and 0.1 % TritonX-100 was added onto 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. Finally, tunel positive cells were labeled by red fluorescence and can be co-stained with DAPI staining, which showed blue fluorescence. For quantification, five fields on each slice around the core infarction regions were randomly chosen for imaging with a fluorescence microscope (Leica, CM1860, Germany) at 200 x. Then, about 200 cells in each field were quantified by Image-Pro Plus 6.0 software. Then, apoptosis rate was calculated as (tunel positive cells / DAPI cells) %.
Quantitative real-time polymerase chain reaction (qRT-PCR)
QRT-PCR was performed to detect the mRNA expression of COX5A and other candidate 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 (n=6 per group) 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 Table 1. Briefly, each reaction was performed in a volume of 20 μl mixture consisting of 10 μl of SYBR Green master mix, 1 μl of cDNA, 7.8 µl of water, and 0.6 µl 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)
WB was employed with the intent to detect the protein expression of COX5A in the cortex of neonatal HI rats and of TPI in OGD neurons after COX5A overexpression. 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 (n=6 wells in each group) were collected and lysed with RIPA buffer containing a proteinase inhibitor cocktail (Roche) on ice for 30 min, then the lysates were centrifuged at 12000 rpm for 10 min at 4 °C. Protein concentrations were determined 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, membranes were incubated 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.
Bioinformatics prediction
GeneMANIA provides many large, publicly available biological datasets to find related genes. These include protein-protein, protein-DNA and genetic interactions, pathways, reactions, gene and protein expression data, protein domains and phenotypic screening profiles. Data is regularly updated. The relationship between Gstp1, Sod2, Rho-GDIa, TPI and COX5A was predicted using the GeneMANIA website (http://genemania.org/).
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.