Animal groups and drug administration
Both Mice C57BL/6J and thy1-YFP-16 transgenic mice (average weight of 27 ± 2 g) were randomly divided into six groups n= 20, denoted as Normal, TQ, TP, Stroke, TQ+ Stroke, and TP+ Stroke. The stroke model was conducted only on the last 3 groups. The Normal group was injected with a mixture of saline solution and DMSO 1% (Thermofisher, 85,190) intraperitoneally at a dose of 0.2 mL/20 g of body weight. TQ and TQ+ Stroke groups were injected intraperitoneally (I.P) with 5mg/kg body weight daily for 5 days before implementing the model and 7 days after [11-13]. TQ (Sigma, 274666) was dissolved in DMSO at a concentration of 10 mg/mL and then diluted with normal saline to attain a final concentration of 0.5 mg/mL. TP and TP+ Stroke groups were injected intraperitoneally with 0.5 mg/kg, 24 h before establishing the model, and 7 days after [14-16]. TP (Med Chem Express, USA#39836) was dissolved in DMSO at a concentration of 5 mg/mL and then diluted by normal saline to a final concentration of 0.05 mg/mL. Further information about the establishment of the YFP mice (Supplementary data section 1 & Fig. II).
Inducing focal cerebral ischemia
Various stroke models were used to produce moderate-to-severe levels of ischemia . Rose Bengal (RB) photoinduced thrombus was used to create core ischemia which locally reduces the blood flow, and this strategy is an alternative path to middle cerebral artery occlusion . The RB photoinduced thrombus model simulates the human stroke where the blood flow drops to 10% and neuronal cell death takes place in the ischemic core [18-19]. As well as, due to the difficulties to keep the animal within a stereotaxic frame to access carotid arteries and the complications associated with collateral circulation, RB photoinduced model is found to be a good choice.10 mg/mL RB dye (Sigma-Aldrich, #R3877) solution dissolved in Phosphate-buffered saline (PBS) was injected intraperitoneally at a dose of 100 mg/kg. 5 min later, a light-emitting diode cold light source (OPLENIC, MIL- HAL 3001) was fixed above the target area which covers the right forelimb motor cortex (1.75 mm lateral to Bregma, +0.5 mm left hemisphere) of the mice brain. To induce focal cerebral ischemia, the target area was illuminated for 15 min then the wound was sutured.
The animals were monitored daily up to the 7th day of treatment for signing moribundity and mortality if any. The body weight of animals was recorded daily before and after treatment.
The movement coordination and anti-fatigue ability of mice were determined by analyzing the ratio of staying time on the rotarod . For more information, please see the supplementary data (section 2).
Cylinder test (spontaneous forelimb use asymmetry test)
To evaluate the spontaneous use of the forelimb, we investigated the cylinder test . For more information, please see the supplementary data (section 2).
This test was used to identify forelimb motor function . Briefly, we examined the number of pieces of sunflower seeds remaining after a fixed time as previously described . For more information, please see the supplementary data (section 2).
The total proteins of the cortex tissue were extracted with 1% PMSF (Phenylmethanesulfonyl fluoride, Beyotime ST505) in 1 mL of ice-cold RIPA buffer, with added protease inhibitor cocktail EDTA-free and phosphatase inhibitors. After homogenizing and centrifuging at 12000 rpm for 20 min at 4 °C, supernatant proteins were preserved at −80 °C. Sample concentrations were determined with BCA kits (KeyGEN, Nanjing, China). Further information about the antibodies and methodologies are provided in the supplementary data (Section 3).
Mice were anesthetized as previously described in the literature , transcardially perfused with 50 mL (±) of 0.9% saline to flush the vascular blood, and then perfused with 4% paraformaldehyde in 0.01 M phosphate-buffered saline (PBS, pH 7.4). The sectioning and staining processes are addressed in the supplementary data (Section 4).
Imaging and Dendritic spine analysis
The bright-field images were captured using an Olympus BX61 microscope and fluorescence images were captured using an Olympus BX53 manual microscope (Olympus Instruments, Yokohama, Japan). Confocal images were captured using an Olympus FV1200 microscope (Olympus, Yokohama, Japan) with an 80 m working distance 63×/1.4-numerical aperture oil-immersion objective (see the supplementary data-section 5)
2,3,5-Triphenyl tetrazolium chloride (TTC) and Histological staining
TTC staining was performed on the 7th day of the experiment . TTC staining process and calculation of infarct volume can be found in the supplementary data (Section 6). The most frequently routine histological stains HE and Nissl were employed to assess the temporal and spatial patterns of postischemic morphological alterations . Sectioning and staining processes are addressed in the supplementary data (Section 7).
Real-time PCR was conducted to detect RNA expression levels of ATG5, c-FOS, c-MYC, iNOS and 5-HT in the cortex. Real-time PCR specific primers for mouse β-actin (internal control) were designed using Primer Express software. The Trizol (Invitrogen) extraction reagent was used to extract cortex RNA. All procedures were carried out according to the manufacturer's instructions. The concentration and purity of RNA samples were determined by Thermo Nanodrop 2000. One microgram of RNA from each sample was reverse transcribed into cDNA, according to the instructions of the DBI-2220 Bestar qPCR RT Kit. 2044 Bestar SYBR Green qPCR master mix. The reaction program for the PCR was as follows: 50 °C for 2 min; 95 °C for 10 min; 40 cycles of 95 °C for 5 s, 55 °C for 30 s, and 72 °C for the 30s. All mRNA expression levels were detected by RT-PCR. Results were analyzed using the Biorad CFX manager program, version 3.0. Each experiment was done three times.
The primers used were as follows:
β-actin Forward: 5′- CTGTCCCTGTATGCCTCTG -3′.
β-actin Reverse: 5′- ATGTCACGCACGATTTCC -3′.
ATG5 Forward: 5′- AACTGAAAGAGAAGCAGAACCA -3′.
ATG5 Reverse: 5′-TGTCTCATAACCTTCTGAAAGTGC -3′.
iNOS Forward: 5′-CACCTTGGAGTTCACCCAGT-3′
iNOS Reverse: 5′-ACCACTCGTACTTGGGATGC-3′
c-FOS Forward: 5′-GGGGACAGCCTTTCCTA-CTA-3′
c-FOS Reverse: 5′-CTGTCACCGTGGGGATAAAG-3′
c-MYC Forward: 5′-TCGCTGCTGTCCTCCGAGTCC-3′
c-MYC Reverse: 5′-GGTTTGCCTCTTCTCCACAGAC-3′
5-HT Forward: 5′-CCATCAGCAAGGACCACGGCTA-3′
5-HT Reverse: 5′-CCCGTAGAGAACCAGCATGAGCAA-3′
Cell line Groups and Establishment of OGD/R model
In vitro immunofluorescence staining
Cells on the coverslips were fixed in a 4% paraformaldehyde for 1 hr. at room. The fixed cells were permeabilized with 0.2% Triton X-100 in 0.01 M PBS for 15 min. After blocking with 5% BSA in 0.01 M PBST for 2 hr., the fixed cells were incubated with the primary antibody at 4°C overnight (Supplementary data-section 10).
FACS analysis by using Annexin V/PI staining:
Cells were washed twice with cold PBS and then resuspend in 1X Binding Buffer at a concentration of 1 x 10^6 cells/ml. We transferred 100μl of the solution (1 x 10^5 cells) to a 5 ml culture tube, then 5μl of FITC Annexin V and 5μl PI were added. cells were gently vortex and incubated for 15 min at RT (25°C) in the dark. Finally, we added 400μl of 1X Binding Buffer to each tube. Samples were analyzed by the CytoFLEX LX flow cytometer machine from Beckman within 1 h. and data calculation by CytExpert software and significant values obtained by SPSS 20.0.
Data were analyzed by one-way ANOVA using SPSS 20.0, followed by post hoc Fisher's least significant difference (LSD) tests. The P values less than 0.05 were considered statistically significant. Histograms were generated in GraphPad Prism 5. All data are expressed as mean ± SEM. Gray values of western blot results were calculated using Image Lab. Immunofluorescence and histological results were analyzed by Pro Image Plus. ⁎P < .05, ⁎⁎P < .01, ⁎⁎⁎P < .001 was determined as statistically significant.