Animal models and treatments
The mfat-1 transgenic mice were obtained by prokaryotic microinjection as previous report [26], the coding region of C. elegans fat-1 cDNA was optimized to enhance the expression of fat-1 in mammalian cells. The mfat-1 heterozygotes of C57BL/6J and wild-type (WT) C57BL/6J mice were crossed to produce mfat-1 transgene mice and WT littermates. The genotypes of the mice were identified by polymerase chain reaction (PCR) amplification. Experimental mice were bred in Animal Core Facility of Nanjing Medical University. Feeding conditions: temperature: 22±2℃; relative humidity: 50-70%; 12-hour cycle lighting, free intake of food and water. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University, with protocol number of IACUC-1911024. Every effort was made to minimize animal suffering.
Acute hypoxic-ischemic brain damage (HIBD) model
The establishment of a perioperative acute hypoxic-ischemic stroke model is based on the Vannucci mode [32]. The offspring mice obtained by mating of mfat-1 transgenic mice and wild-type mice weighing 22±2g were selected for modeling grouping. After isoflurane ether anesthesia (2%), the left common carotid artery was exposed and ligated with 6-0 nylon wire, the above operations were performed under a stereo microscope (Nikon, Tokyo, Japan). After suture, hypoxia was performed in the hypoxia chamber (Tianchen XL-2CL, Nanjing, China) for 50 minutes. 92% nitrogen and 8% oxygen were pumped into the hypoxia chamber. Body temperature was maintained at 37.0 ± 0.5 ℃ throughout surgery. The sham group underwent the same anesthesia and exposure as the surgery group, ignoring ligation and hypoxia. This model has a mortality rate of about 10%.
Gas chromatography analysis of fatty acid profiles
Fatty acid composition of brain tissue was determined by capillary gas chromatography (Agilent 7890A, CA, USA). Briefly, the total lipids were extracted by the Fatty Acid Extraction kit (Sigma-Aldrich, Missouri, USA), and then dry under nitrogen for transesterification immediately. Fatty acids were methylated with boron trifluoride and methanol (heat at 70℃ for 1 hour). Add 1mL of hexane and 1mL of distilled water and then vortex and centrifuge at 500 x g for 5 minutes. Reconstitute the trans esterified lipids with hexane and transfer to a GC vial. Methylated fatty acids were then analyzed via gas chromatography flame ionization detection on gas chromatograph as previously described [33]. Peaks were identified by comparison with authenticated standards (Sigma-Aldrich, Missouri, USA). Fatty acid components were identified by comparison of retention times with those of authentic standards. The ratio of n-3/n-6 was calculated by the areas of peaks. Data are expressed as molar percent of fatty acids.
Longa score
Behavior deficit was taken by Longa's [34] 5-point scale at 24h after HIBD. A score of 0: no neurological deficit; a score of 1: failure to extend left forepaw fully; a score of 2: circling to the left; a score of 3: falling to the left; and a score of 4: did not walk spontaneously and had a depressed level of consciousness. Mice without symptoms of neurological impairment or dying after HIBD were rejected, and other rats were recruited.
Berderson score
Mice were held gently by the tail, suspended one meter above the floor, and observed for forelimb flexion. The evaluation standards of Bederson method were divided into 4 grades, normal grade 0: no observable deficit; moderate grade 1: forelimb flexion; severe grade 2: decreased resistance to lateral push (and forelimb flexion) without circling; grade 3: same behavior as grade 2, with circling [35].
Rotarod test
For the rotating test, an accelerating rotarod (Giliang DigBehv-010, Shanghai, China) was used as described previously [36, 37], which accelerated in speed of 20 rpm over a 5min period. Record the time for the mouse to fall for the first time and the number of drops (mice were reloaded on rotarod as soon as possible after falling) within 300s. mice were acclimatized to the rotarod for three trials, with an intertrial interval of 30 min.
2,3,5-Triphenyte-trazoliumchloride (TTC) staining
After anesthesia, fresh brain tissue was taken immediately, washed with pre-cooled buffer solution (1X PBS) and directly put into quick-frozen at -20 ℃ for 20-30 minutes. The brain was cut into 2-3mm tissue blocks with a lycra blade in the brain tank. Then put it into the pre-preheated TTC incubation solution (Sigma-Aldrich, Missouri, USA), placed in the oven at 37 °C and incubated in the dark for 15min. The reaction between TTC reagent and dehydrogenase in normal tissues is red, while the dehydrogenase activity in ischemic tissues is reduced and cannot be reacted, thus presenting a pale color. The Image Pro Plus software was used to analysis the infarct volume. Infarct volume (%) = (right ischemic pale area / left brain area + right brain area) × 100.
Nissl staining and hematoxylin and eosin staining
After perfusion and fixation, drying, samples were dewaxed with xylene and hydrated with alcohol of class gradient concentration. Coronal brain sections (4um) were used to observe the change of the gross morphology by Hematoxylin-Eosin staining and Nissl staining, including brain hemisphere swelling, subcortical petechial hemorrhage, tissue necrosis, nerve cell loss, and inflammatory cell infiltration. The damaged neurons appear vacuolated and pale blue. Normal cells have relatively large, plump nidellae with a darker bluish tint.
Immunohistochemical analysis
Immunofluorescence staining was carried out to detect GPR120 expression in mice after HIBD. Paraffin-embedded formaldehyde fixed specimens were cut into 4um thick slices, dewaxed with xylene, and rehydrated with a series of graded alcohols. Microwave high temperature antigen repair in citric acid buffer (3% trisodium citrate and 0.3% sodium citrate). Brain serial coronal sections were washed with PBS before fixed with 4% paraformaldehyde at room temperature for 30 min. Subsequently, they were incubated with a blocking solution (5% FBS) for 30 min at 37 °C. Then, they were incubated with anti-GPR120 antibody (Santa Cruz Biotechnology, CA, USA) overnight at 4 °C. The antibodies used in this section also illustrated in supplementary Table S1. On the following day, they were washed with PBS and incubated with secondary antibodies anti-IgG Conjugates (Invitrogen, Carlsbad, CA, USA) for 1h and 4',6-diamidino-2-phenylindole (DAPI) for 1 min at room temperature in the dark. Images were obtained using a confocal microscope (CarlZeiss LSM710, Oberkochen, German).
Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay
TUNEL staining was performed according to the manufacturer’s instructions (KeyGEN BioTECH, Nanjing, China) for quantification of neuronal cell death [38].Paraffin sections (4μm) were dried for 1 h at 70 °C, and deparaffinized in xylene and graded ethanol solutions. Images were obtained using a confocal microscope (CarlZeiss LSM710, Oberkochen, German). The TUNEL-positive cells were counted in the cortex, hippocampus and striatum in three separate fields for each animal by an observer who was blind to the experimental condition. Data was expressed as ratio of TUNEL-positive neurons (%).
Enzyme-linked immunosorbent assay (ELISA)
The concentrations of IL-1β, IL-6, and TNF-α in ischemic penumbra in brain of each group were quantified using an ELISA kit (Elabscience Biotechnology, Wuhan, China) according to the manufacturer’s instruction. Absorbance at 450nm was recorded and the concentration of the target protein was calculated according to the standard curve and normalized against the protein of the samples. Result was expressed as pg/mg protein.
RNA-seq analysis
Total RNA extraction
Total RNA was extracted from the tissues using Trizol (Invitrogen, Carlsbad, CA, USA) according to manual instruction. Ischemic side brain tissues were ground into powder by liquid nitrogen, followed by being homogenized and rested horizontally. The mix was centrifuged, then the supernatant was transferred into a new EP tube with chloroform/isoamyl alcohol (24:1). The mix was shacked vigorously for 15s, and then centrifuged, the upper aqueous phase where RNA remained was transferred into a new tube with equal volume of supernatant of isopropyl alcohol, then centrifuged at 4°C. After deserting the supernatant, the RNA pellet was washed twice with 75% ethanol, then the mix was centrifuged at 4°C to collect residual ethanol, followed by the pellet air dry in the biosafety cabinet. Finally, DEPC-treated water was added to dissolve the RNA. Subsequently, total RNA was qualified and quantified using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA).
mRNA Library Construction
Oligo(dT)-attached magnetic beads were used to purified mRNA. Purified mRNA was fragmented into small pieces with fragment buffer at appropriate temperature. Then First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. afterwards, A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. The cDNA fragments obtained from previous step were amplified by PCR, and products were purified by Ampure XP Beads, then dissolved in EB solution. The product was validated on the Agilent Technologies 2100 bioanalyzer for quality control. The double stranded PCR products from previous step were heated denatured and circularized by the splint oligo sequence to get the final library. The single strand circle DNA (ssCir DNA) was formatted as the final library. The final library was amplified with phi29 to make DNA nanoball (DNB) which had more than 300 copies of one molecular, DNBs were loaded into the patterned nanoarray and single end 50 bases reads were generated on BGIseq500 platform (BGI-Shenzhen, China).
Bioinformatics analysis
Primary sequencing data produced by RNA-Seq (raw reads) were subjected to quality control (QC). The sequencing data was filtered with SOAPnuke (v1.5.2) by (1) Removing reads containing sequencing adapter; (2) Removing reads whose low-quality base ratio (base quality less than or equal to 5) is more than 20%; (3) Removing reads whose unknown base ('N' base) ratio is more than 5%, afterwards clean reads were obtained and stored in FASTQ format. The clean reads were mapped to the reference genome using HISAT2 (v2.0.4). Bowtie2 (v2.2.5) was applied to align the clean reads to the reference coding gene set, then expression level of gene was calculated by RSEM (v1.2.12) . The heatmap was drawn by pheatmap (v1.0.8) according to the gene expression in different samples. Essentially, differential expression analysis was performed using the DESeq2(v1.4.5) with Q value ≤ 0.05. To take insight to the change of phenotype, GO (http://www.geneontology.org/) and KEGG (https://www.kegg.jp/) enrichment analysis of annotated different expressed gene was performed by Phyper (https://en.wikipedia.org/wiki/Hypergeometric_distribution) based on Hypergeometric test. The significant levels of terms and pathways were corrected by Q value with a rigorous threshold (Q value ≤ 0.05) by Bonferroni.
Western blotting
Western blotting was performed according to the manufacturer’s specification. The ischemic side brain tissue samples were collected at 24h after HIBD. Proteins were extracted by homogenizing in RIPA buffer (Sigma-Aldrich, Missouri, USA) with phenylmethanesulfonyl fluoride (PMSF) and phosphatase inhibitor (Bimake, TX, USA) and further centrifuged at 12,000 rpm at 4 °C for 10 min. The concentrations were determined with BCA protein assay kit (Thermo Fisher Scientific, MA, USA). the samples were separated by 8–12% SDS–PAGE gel (Bio-Rad, CA, USA) and then transferred to polyvinylidene fluoride (PVDF) (Bio-Rad, CA, USA) membranes. Subsequently, the membranes were blocked in 5% BSA (Sigma-Aldrich, Missouri, USA) for 1 h at room temperature following incubation with primary antibodies overnight at 4 °C. Dilutions for primary and secondary antibodies were listed in supplementary Table S1. Membranes were washed three times in TBST and specific binding was visualized by ECL reaction (Bio-Rad, CA, USA). The density of bands was detected using an imaging densitometer, and the gray value of the bands was quantified using ImageJ Software (version 1.41).
Real-time PCR
Total RNA was extracted from the ischemic cerebral cortices at 24 h after HIBD using Trizol reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol. The quantity of total RNA was measured with a UV spectrophotometer (Thermo Fisher Scientific, MA, USA). Next, reverse transcription was performed using a cDNA synthesis kit HiScript II Q RT SuperMix (Vazyme Biotech, China). Quantitative PCR was performed with ChamQ SYBR qPCR Master Mix (Vazyme Biotech, China) at the following conditions (denaturing at 95 °C for 30s, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s) and detected by StepOnePlus Real-Time PCR Systems (Applied Biosystems; MA, USA). The expression of target genes was measured in triplicate and normalized to β-actin as an internal control. The −ΔΔCt values of each group were analyzed, and mRNA expression levels were normalized to 2−ΔΔCt. Primers are listed in Supplementary table S2.
Statistical analysis
GraphPad Prism 8.0.1 software (GraphPad Software Inc, La Jolla, CA) was used to analyze data and form the graphs in this work (including which tests were performed, exact P values, and sample sizes). Simply, one-way ANOVA with a test for linear trend, Tukey test was used as appropriate to analyze parametric statistics. The statistics of neural score longa score and Berderson score data use rank sum test in nonparametric test. At least three independent experiments were applied to collect effective data. Bias was avoided by making sure that assessor was blinded to collecting and analyzing data. P < 0.05 was considered significant. Average values represent the mean ± SD. *: P<0.05; **: P<0.01; ***: P<0.001.