2.1 Animal care
Timed pregnant female Sprague-Dawley (SD) rats were bought from Animal Centre of Kunming Medical University and placed in individual cages. Postnatal pups were put in their cage with food and water available as libitum throughout the study in a 12 h light/dark cycle. Then the postnatal day 7 SD rat pups (weighing 10-15g) were employed in this study. All procedures for the use of animal were approved by University Committee on Animal Use and Care. Rat pups were randomly divided into four groups, as shown in Table. The animal study was legally approved by the Animal Care & Welfare Committee of Kunming Medical University.
Table Animal grouping
Animal grouping
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HI12 h
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HI24 h
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Sham group (Sham)
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n = 6
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n = 6
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Hypoxia ischemia group (HI)
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n = 6
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n = 6
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Hypoxia ischemia with no-targeting lentivirus (Negative Control)
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n = 6
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n = 6
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Hypoxia ischemia with IL-6-targeting lentivirus (IL-6-RNAi-LV )
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n = 6
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n = 6
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2.2 Establishment of HI model
Before experiments, the neonatal rats were performed to the anesthetized with 3% isoflurane. After that, the right common carotid artery (CCA) was exposed with a 0.5 cm skin incision in the midline of the neck and permanently fused by Monopolar Microsurgery Electrocoagulato (Spring Medical Beauty Equipment co., LTD, Wuhan, China). The rats were returned to their mother to recovery for 1 h after surgery. Subsequently, the pups were taken into a hypoxic chamber (8% O2, 92% N2) for 2 hours (h) to produce HI injury, then returned to their maternal rats. While in the sham group, the rats were anesthetized with 3% isoflurane and their CCA was exposed without hypoxia and ischemia [19].
2.3 Behavioral Analyses
The neurologic deficits of rats were recorded according to the standard of Longa scores. The higher the score, the more serious the neurological deficit. Briefly, the neurobehavioral scores of the sham group were evaluated at 0, 2, 4, 6, 12, 24, 48 h after hypoxia-ischemia treatment [20]. The scoring criteria: 1) 0 points: the behavior is completely normal, no symptoms of neurological deficit; 2) 1 point: The bending of the left forelimb could not be fully extended and the neurological function was mild; 3) 2 points: cannot go straight and walk forward when the body continues to turn to the side, moderate neurological deficit; 4) 3 points: Rats inability to stand, fell to the left when standing, severe neurological deficit; 5) 4 points: unable to self-issued, loss of consciousness.
2.4 Brain Water Content
At the indicated time points after HI, the brains of rat pups were removed after deep anesthesia and separated into ipsilateral, contralateral and cerebellum portions to measure the water content. Afterwards, wet weight was weighed on electronic balance at once after the brain was removed, then the dry weight was obtained after the tissues dried in an oven at 105 ℃ for 24 h to 36 h to reach a constant weight (the last two times weighing < 0.2 mg). Finally, the brain water content was calculated by Elliot formula: the percentage of brain water content = (wet weight – dry weight)/wet weight × 100%.
2.5 Tissue harvest
The tissues of HI groups were taken at 6 h, 12 h and 24 h after surgery, while the sham group were obtained at 6 h post-surgery for Hematoxylin and Eosin Staining (HE staining) and immunohistochemistry. To be specific, the sterile saline was infused from the left ventricular to right auricle until liquid was cleared, then 20 ml of 4% paraformaldehyde (pH 7.4, 0.01mol / L PBS) was infused again. Subsequently, the brain was obtained immediately after perfusion and stored in 4% paraformaldehyde solution for a fixation more than 72 h. The brain and lung were fetched for quantitative real-time polymerase chain reaction QRT-PCR) and western blot (WB). Then the membrane of brain was peeled under a dissecting microscope and the whole hippocampus was quickly taken out on the ice. Finally, the tissues can be stored at -80 ℃ for following experiments.
2.6 Triphenyl tetrazolium chloride (TTC) Staining
TTC staining was performed to detect the infract volume of the brain. After the rats were deeply anesthetized and sacrificed after HI for at least 6 h, the brains were promptly removed. Afterwards, the tissues were frozen at -20℃ for 15 min and sliced into coronal sections of 1-3mmthick using a rat brain matrix (Seino Co., Ltd. Beijing, China). Immediately, the above sections were put into 2% triphenyl tetrazolium chlorides (Sigma Co., St Louis, MO, USA) in the dark and incubated for 10 to15 min at 37 ℃. Then the stained brain slices were taken out and fixed in phosphate buffered solution by 4% formaldehyde. Finally, Image J software (National Institutes of Health, USA) was used to trace and measure the infarction area of every slice. In addition, the following formula: corrected percentage of infarct volume = (contralateral hemispheric volume-ipsilateral non-infarcted volume)/contralateral hemispheric volume, was used to calculate infarct volume [21].
2.7 Hematoxylin and Eosin (HE) Staining
For HE staining, the obtained brain tissues were fixed with 4% paraformaldehyde for more than 72 h and chopped into 0.5 cm×0.5 cm×1 cm pieces. Then, samples were dehydrated until transparent, immersed in wax, and embedded in paraffin. The paraffin-embedded tissues were cut into 3µm slices using a Rotary Microtome YD-1508R (Jinhua YIDI Medical Appliance Co., Ltd, China). Then, the slices were routinely stained with HE. Next, the stained sections were observed under a light microscope (CX40, Shunyu, Ningbo, China) to detect the pathological changes.
2.8 Immunohistochemistry
For immunohistochemical assay, the paraffin embedded sections including hippocampus, cortex and lung were routinely de-paraffinized and rehydrated. Sections were washed four times in PBS and incubated with 10% goat serum for 1 h at room temperature to block non-specific binding. Subsequently, sections were incubated with primary antibodies (Abcam, M, ab9324) overnight at 4 ℃. Next, the sections were incubated in biotin-conjugated secondary antibody (Goat anti Rabbit IgG, HRP/IgG) for 30 min at 37 ℃, then incubated with horseradish peroxidase conjugated streptavidin avidin for 20 min at 37 ℃. After that, sections were washed with PBS three times for 5 min each, and continually incubate with diaminobenzidine (DAB), followed by dehydration, transparentization and mounting. Counterstaining of sections by hematoxylin was also performed. While in the sham group, PBS was used instead of primary antibody. Images were taken with a laser scanning confocal microscope (Nikon, Tokyo, Japan). Quantification of the immune labeled brain sections was performed separately. For each slice, ×200 magnification photomicrographs were taken to measure the density of IL-6. The mean density was presented as IOD over the area of interest using Image-Pro plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA), which was described previously.
2.9 Primary culture of rat cortical neurons and oxygen–glucose deprivation (OGD) model
Primary cortical neurons were obtained from one-day-postnatal SD rats. Briefly, the cortexes of rats were harvested and cut into approximately 1 mm3 small pieces, then digested with 0.25% trypsin (Gibco) at 37 ℃ for 10 min and eluted with 10% fetal bovine serum (BSA, Gibco). The tissue suspension was centrifuged at 1000 rpm for 10 min. Subsequently, the tissue suspension was centrifuged at 1500 rpm for 5min, and the pellet in the bottom was resuspended at the bottom using a complete medium (Hyclone) containing DMEM / HIGH GLUCOSE, 10% fetal bovine serum and 1% penicillin-streptomycin solution. Next, neurons were seeded at a density of 5×105 cells/ml in 6 well plates (Corning, USA) coated with poly-d-lysine and laminin (Sigma-Aldrich, St. Louis, MO), then incubated under the condition of 37 ℃ and 5% CO2.Thecomplete medium was replaced with 2% B27 (Invitrogen, CA) at 4h after incubation. The medium was changed in the next day and then one-half change was made every 3 days.
2.10 Construction of IL-6 siRNA Lentivirus
Not-targeting and targeting siRNA were obtained from GeneCopoeia Company (GuangZhou, China). Three 19-nucleotide sequences were designed corresponding to the IL-6 reference sequence (NCBI, NM_031512.2) to specific silencing of IL-6. Non-targeting siRNA was constructed using a 19-nucleotide sequence which is no homology to any mammalian gene sequence as negative control. IL-6 interference lentivirus (MSH028938-HIVU6) was purchased from GeneCopoeia Company (GuangZhou, China) to construct IL-6 siRNA expression vector. In brief, IL-6 siRNA lentivirus was produced by co-transfected IL-6 expression vector and viral packaging system (gag, pol and env) into 293 T cells according to the manufacturer’s protocol. Forty-eight hours after transfection, virus medium was harvested, filtered through a 0.45 µm cellulose acetate filter, purified and concentrated by concentration solution (GeneCopoeiaCompany). Finally, the IL-6 siRNA lentivirus was frozen at -80 ℃ for following experiment. Then, IL-6 siRNA lentivirus (5 µl for each position) was injected into right side of the lateral ventricle hemisphere, and the injection coordinates were vertical depth 4 mm, right 1.0 mm relative to bregma and 1.5 mm from the lambdoid suture. Additionally, the rate of injection was 0.2 µl /min.
2.11 Screen for effective fragment of IL-6 siRNA
At first, the gene database of IL-6 from NCBI was gathered. Four silencing RNAs (siRNAs) specifically inhibiting IL-6 gene expression and one nonsense siRNA as negative control (NC) were designed, which were purchased from Ribobio Company (Guangzhou, China). The target messenger RNA (mRNA) sequence of fragment 1 is CCAAGTCCGTC TTCTACAT; fragment 2: CAGGTGCACTTTACGAG TA; fragment 3: CAGCATGAATCCAGCTCGA. In brief, when the PC12 cells were 40% confluence, three candidate target fragments of IL-6 siRNA were added into cells. There were four groups in this experiment as follows: the normal group, reagent group, negative control group and IL-6 siRNA group. Transfection was performed using SuperFectin™ II in vitro transfection reagent (Pufei Biotech, China). Briefly, a mix of transfection stock Buffer and siRNA was prepared and 3 µl of SuperFectin™ II reagent was added to the mixture. Mixture of IL-6 siRNA (100 nM) was added drop-wise to the appropriate wells, respectively. Another1.2 ml of fresh culture medium was added at 37 ℃ for 24 h after incubation. Quantitative real-time polymerase chain reaction (QRT-PCR) was performed to validate the inhibition rate of three fragments. Then the siRNA of the highest inhibition rate was transfected into neurons of HI to explore the function of IL-6. In addition, the morphologic changes of neurons after IL-6 siRNA treatment was observed and collected by a phase contrast microscopy. Non-targeting siRNA was transfected into neurons as negative control.
2.12 Immunofluorescence Staining (IF)
To examine the neurons outgrowth, the location and expression of IL-6 following IL-6-RNAi-LV injection, comparative analysis of immunofluorescence staining was performed in the brain tissue section. Briefly, sections were washed three times (5 min for each time) in PBS and incubated with 5% goat serum for 2 h at room temperature to block non-specific binding. Subsequently, sections were incubated overnight at 4 ℃ with the following primary antibodies: rabbit anti-GFAP antibody (1:100; Zhongshan Jinqiao, China) together with mouse anti-IL-6 antibody (1:100; Abcam, Cambridge, MA, USA). Then, the sections were washed 3 times with PBS for 5 min each time and incubated with the appropriate secondary antibodies: CY3 anti-rabbit IgG (red), Alexa Fluor 488 anti-mouse IgG (green) and Alexa Fluor 488 anti-rabbit IgG (green) for 2 h at 37 ℃. The sections were washed 3 times for 5 min each with PBS, and stained with DAPI and mounted. Section image was collected using a confocal microscope (Zeiss LSM).
2.13 TUNEL staining
A Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) reaction mixture of enzyme solution and labeling solution was added at a ratio of 1:9 (v/v), and the slices were stored at 4 ℃ overnight in the dark. After washing with PBS, the neurons were stained with DAPI for 5 min at room temperature, and photographs were obtained using fluorescence microscopy (Leica, CM1860, Germany). The nuclei of apoptotic neurons were stained in red by TUNEL and the all nuclei of neurons were stained in blue by DAPI. Apoptosis was quantified by determining the percentage of TUNEL/DAPI using Image-Pro Plus 6.0 software.
2.14 Western Blot (WB)
WB technique was used to detect the expression of IL-6 protein in cortex and the expression of β-actin and IL-6 in vitro. The samples were lysed and treated with RIPA lysis buffer (Beyotime, Jiangsu, China). All the samples were centrifuged at 12000 ×g for 10 min at 4 ℃. The total supernatant protein was collected and its concentration was determined by BCA protein assay. Then, samples containing 80 µg of protein were separated by SDS-PAGE electrophoresis and electro transferred to PVDF membrane. Next, the membranes were blocked with TBST containing 5% skimmed milk and then incubated with primary antibodies of IL-6 (1:200,Abcam, ab9324, Mouse) overnight at 4 ℃, β-actin (1:1000, Abcam, USA) was used as a loading control. After washing three times in TBST for 5 min each, the membrane was incubated for 2 h at room temperature with a secondary antibody (HRP Goat Anti-Mouse IgG, 1:5000, Abbkine, A21010). Finally, the membrane was placed in a chemiluminescent reagent for 1min, and then exposed to X-ray film cassette. The immune blot was revealed with an ECL Western blot detection kit (Amersham Pharmacia Biotech, Buckinghamshire, England). Densitometry analysis was performed using Image J software.
2.15 Quantitative real-time polymerase chain reaction (QRT-PCR)
QRT-PCR was performed to detect the expression of IL-6 in cortical and cultured neurons in vitro. After tissue homogenate, 1ml Trizol reagent (Takara Bio Inc., Otsu, Japan) was added and total RNA was extracted according to standard specification. Then, 5 ul of the obtained RNA was reversely transcribed to cDNA with the RevertAid™ First Strand cDNA Synthesis Kit (Thermo, USA). QRT-PCR was performed to examine the messenger RNA (mRNA) level of IL-6. Afterwards, the QRT-PCR was performed. The primer sequences were listed as follows: IL-6: Forward, 5'-GAGGATACCACTCCCAACAGACC-3'; Reverse, 5'-AAGTGCAT CATCGTTGTTCATACA-3'. GADDH: Forward, 5'-TGACTTCAACA GCGACACCCA-3'; Reverse, 5'-CACCCTGTTGCTGTAGCCAAA-3'. BAX: Forward, 5'-TGGAGCTGCAGAGGATGATT5-3'; Reverse, 5'-CA GGGCCTTGAGCACCAGTT-3'. Casp3: Forward, 5'-TTCTTCAGAGG CGACTACT-3'; Reverse, 5'-TCCCACTGTCTGTCTCAAT-3'. AKT1: Forward, 5’-AGTCCCCACTCAACAACTTCT-3’, Reverse, 5’-GAAGG TGCGCTCAATGACTG-3’. STAT3: Forward, 5'-GTGCAGGATCTAG AACA GAA-3'; Reverse, 5'-GACTGGTTGTTTCCATTCAG-3'. ERK1: Forward, 5'-CAAGA ACAAGACCAACATGAAT-3'; Reverse, 5'-GGTA GGACACAAACTTGTAG-3'. JAK1: Forward, 5'-TTCACTGGAGTAT CTGTTTG-3'; Reverse, 5'-CAACTGCATCTTCTTCATCAT-3'. Subsequently, the reaction was carried out in a DNA thermal cycler (ABI 7300) based on the following standard protocol: a denaturation step of 95 ℃ for 3 min; 45 cycles of 95 ℃ for 15s and annealing temperature at 60 ℃ for 30 s. The threshold cycle of each sample was recorded, and data were analyzed by normalization to GAPDH values using the 2-△△Ct method.
2.16 Statistical analysis
All data in the experiment were presented as mean ± SD and were analyzed using SPSS 20.0. Comparisons between two groups were analyzed by Independent-Samples T-test. Non-parametric comparisons between three or more groups were made with one-way ANOVA test. P < 0.05 was considered statistically significant.