Animals

C57BL/6 mice were provided by the Experimental Animal Center of Xi’an Jiaotong University. Mice were housed in a condition of 22 ± 1 °C air temperature, humidity of 50 ± 1% and 12/12 h light/dark cycle. The inclusion criteria for middle cerebral artery occlusion (MCAO) were male, aged from 8 to10 weeks and weighed from 22 to 25 g. Mice used for primary astrocytes separation were postnatal 1 day old. A total of 436 mice including 5 neonatal mice were used in the experiment. The experimental design and protocols were all complied to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23), which were also approved by the Animal Care and Use Committee of Xi’an Jiaotong University. A total of 431 mice were subjected to MCAO surgery or sham operation, and 143 were excluded because of failure of MCAO surgery or death after MCAO.

Mouse cerebral ischemia and reperfusion injury

We only used male mice in this study because estrogen has a significant impact on central nervous system. Before surgery, mice were fed freely with food and tap water. As described previously [7], the right middle cerebral artery was transient occluded in mice. Briefly, mice received MCAO under isoflurane anesthesia with inhaled concentration of 1.5–2%. After anesthesia, a silicon-coated suture (RWD Life Science) was placed through the right external carotid artery and advanced into the internal carotid artery to block the blood flow into middle cerebral artery. The suture was removed to allow reperfusion after 60 min ischemia. The rectal temperature of mice was monitored and maintained at 37 ± 0.5 °C by using a heating pad. The blood flow of the ischemic area was monitored by a laser Doppler system and the probe was placed on the defined spot of the skull (2 mm caudal and 4 mm lateral to the bregma). An 80% decrease and 70% recovery of the local cerebral blood flow was regarded as a successful MCAO surgery. Sham operation was performed only without insertion of the suture.

Drug treatment and adeno-associated virus injection

GTA from Sigma-Aldrich (90240, Germany) was orally delivered on day 2 and 4 after MCAO. The dosage was 4 g/kg body weight of mice according to a previous report [31]. C75, a specific inhibitor of FASN from MedChemExpress (HY-12364, China) and prepared in normal saline with the solubilizers of 5% DMSO, 40% PEG300 and 5% Tween-80. The concentration of C75 was 1 mg/ml. The C75 was administrated intraperitoneally with a dosage 10 mg/kg body weight 72 h after MCAO as we used before [7]. Vehicle control contained the corresponding solubilizers in normal saline. Mouse recombinant IL-33 (rIL-33) from Biolegend (580502, CA, USA) was prepared in normal saline to a concentration of 0.2 mg/ml and administrated into the lateral cerebral ventricle 72 h after MCAO as we used before [7]. Equal volume of normal saline was used as vehicle control of rIL-33. The astrocytes-specific adeno-associated virus (AAV) 2/9 encoding for FASN-shRNA-EGFP or scrambled RNA (scRNA)-EGFP with GFAP promoter was constructed by BrainVTA Co., Ltd. (Wuhan, China). The shRNA sequence was: 5’-CCGGCGTCTATACCACTGCTTACTACTCGAGTAGTAAGCAGTGGTATAGACGTTTTTG-3’. The virus injection was stereotactically guided into the peri-infarcted area 4 weeks before MCAO surgery. The amount of AAV was 200 nl (titer 2 × 1012 vg/ml) per coordinate. And the stereotactic parameters were AP 0.3 mm, ML 2.0 mm, DV 1.8 mm from bregma for cortex, and AP 0.3 mm, ML 2.0 mm, DV 3.00 mm from bregma for striatum. The knockdown efficiency of the shRNA was confirmed as we reported [7].

Neurological function assessmentNeurological defects evaluation

A neurobehavior assessment based on Garcia was performed on day 7 after MCAO [32]. The observer was unaware of the experimental grouping. The whole system consists of 6 tests with multiple evaluations of sensory and motor function based on spontaneous activity, symmetrical movements of the upper and lower limbs, forepaw outstretching, climbing, body proprioception and response to vibrissal touch.

The forelimb grip strength test

The long-term forepaw grip strength was assessed after MCAO. Briefly, the maximum grip strength was gauged using a strength meter (BIOSEB, BIO-GS3, France). A T-shaped metal rod was connected to the meter. The mouse was gently suspended by the tail. Then, the forepaws of mice were symmetrically allowed to grip both transverse tips of the T-shaped rod. The mouse body was maintained in a horizontally position and then pulled away from the rod until it let go. The maximum strength would be automatically recorded. Three repetitions were conducted for each mouse and the average maximum strength was recorded.

The adhesive removal test

The adhesive removal test is reliable in evaluating sensorimotor function of mouse after cerebral ischemia [33]. This test was performed by attaching two small adhesive tape strips (0.3 cm × 0.4 cm) onto the bare part of both forepaws. The contact time (mouse began to react to the presence of the adhesive tapes: shaking paws or bringing paws to mouth) and removal time for each tape were recorded. The asymmetry magnitude of each parameter was also compared.

Assessment of cerebral infarct size

Staining with 2,3,5-triphenyltetrazolium chloride (TTC, MP210312610, MP Biomedicals) was performed to reveal the infarcted area on day 7 after MCAO. After euthanasia with overdose sodium pentobarbital (MP Biomedicals), mouse brains were quickly removed and cut into 1-mm coronal slices on ice. The slices were immersed in 2% TTC in saline at 37 °C for 20 min. After fixation in 4% paraformaldehyde for 24 h, the images of the stained slices were acquired using a digital camera. The healthy area pixels of contralateral (VC) and ipsilateral hemisphere (VL) were calculated by using an image analysis software. The relative infarct percentage (%I) was obtained using the following formula: % I = 100 × (VC – VL)/VC.

Evaluation of brain atrophy

Brain atrophy was assessed based on Nissl staining of cerebral slices 30 days after MCAO. The frozen sectioned mouse cerebral coronal slices (15-μm-thick) at 300-μm intervals approximately from 1.3 to − 1.7 mm to bregma were rinsed with PBS and stained in 1% methyl violet (C0117, Beyotime) for 10 min. Next, the slices were gently rinsed with water. Then, the slices were transferred into graded ethanol. After treatment with xylenes and mounted with neutral balata, the slices were scanned by SLIDEVIEW VS200 (Olympus, Japan). The relative atrophy percentage was according to the formula: (total VC area – total VL area) / total VC area × 100%.

Evaluation of BBB disruptionEvans blue extravasation

Evans blue was delivered into the blood circulation of mouse through tail vein injection 6 days after MCAO. Each mouse received 4 mg Evans blue in 200 μl normal saline. The mouse was euthanized and transcardially perfused with 20 ml saline 24 h after Evans blue injection. Mouse brains were then harvested and prepared into 1-mm coronal slices. The image of extravasated Evans blue in the cerebral slices was taken by a digital camera and the ischemic hemisphere was separated. The separated hemispheres were processed with lysis buffer. A standard curve of Evans blue concentration was plotted. And the concentration of Evans blue in each sample was calculated based on the standard curve through a fluorescence spectrophotometer.

Dextran extravasation

Dextran-Texas Red™ 10 000 MW (D1828, Invitrogen) was intravenously delivered into the blood circulation of mouse 7 days after MCAO. Each mouse received 0.5 mg dextran in 200 μl saline. The mice were fixed with 4% paraformaldehyde 120 s after dextran administration. Mouse brains were then collected and cut into 12-μm-thick slices. After immunofluorescence staining with rabbit anti-GFAP antibody (1:500, GTX108711, Genetex, CA, USA), the images of slices were acquired using a laser confocal fluorescence microscope (Olympus FluoView FV1200). The extravasated dextran was based on its fluorescence intensity by the ImageJ software (National Institutes of Health, USA).

Lipid droplets staining

Nile red staining was applied to reveal neutral lipid droplets on fixed brain slices or on primary astrocytes. Cerebral slices or cell coverslips were immunofluorescence stained with rabbit anti-GFAP antibody (1:500, GeneTex), and Alexa Fluor 647-conjugated donkey anti-rabbit secondary antibody (1:500, Jackson ImmunoResearch) was used as the secondary antibody. Then, the slices or coverslips were incubated with Nile red (1 μg/ml, HY-D0718, MedChemExpress) at room temperature for 15 min. After mounting with an antifade medium (VECTASHIELD, USA), the samples were subjected to confocal fluorescence microscope observation. LipidSpot™ 610 (1:1000, 70069T, Biotium) was also used to observe the lipid droplets in tissues or primary astrocytes. After staining of GFAP as above, the samples were stained with LipidSpot™ 610 at room temperature for 30 min. Then, the samples were mounted and observed.

Immunofluorescence staining

Mice were fixed by transcardial perfusion with 4% paraformaldehyde. Mouse brains were removed and subjected to dehydration in 30% sucrose. The brains were then frozen and cut into coronal slices with a thickness of 12-μm. The sections were stained as we previously reported [7]. Briefly, after permeabilization and blocking with donkey serum, the samples were immunoreacted with rabbit anti-FASN antibody (1:400, ab22759, Abcam, Cambridge, London, UK), chicken anti-GFAP antibody (1:800, GTX85454, GeneTex), rabbit anti-GFAP antibody (1:400, GeneTex), goat anti-IL-33 antibody (1:500, AF3626, R&D system, USA), at 4 °C overnight, and then Alexa Fluor 594-conjugated donkey anti-rabbit secondary antibody (1:500, Invitrogen), Alexa Fluor 488-conjugated donkey anti-chicken secondary antibody (1:500, Jackson ImmunoResearch), Alexa Fluor 647-conjugated donkey anti-rabbit secondary antibody (1:500, Jackson ImmunoResearch), Alexa Fluor 594-conjugated donkey anti-goat secondary antibody (1:500, Invitrogen), Alexa Fluor 594-conjugated donkey anti-mouse secondary antibody (1:500, Invitrogen) were used as the corresponding secondary antibodies. The samples were mounted with the antifade medium and observed under a laser confocal fluorescence microscopy (Olympus FluoView FV1200).

Western blot analysis

Mouse brains were harvested 3 or 7 days after MCAO. The tissues corresponding to the peri-infarcted areas were separated on ice. The sample proteins were collected by sonic lysis in RIPA lysis buffer (Beyotime, China) on ice. A BCA kit from Beyotime was used to determine the protein content of each sample. The denature condition was 100 °C for 2 min. Proteins samples (40 μg per lane) were electrophoresis separated by a 10% sodium dodecyl sulfate–polyacrylamide gel. The separated proteins were transferred to a polyvinylidene difluoride membrane. The membranes were then blocked with 10% skim milk at room temperature for 1 h. For immunoblotting of the membrane, the following primary antibodies were used at 4 °C overnight: rabbit anti-FASN antibody (1:1000, ab22759, Abcam), rabbit anti-ZO-1 antibody (1:1000, 21773-1-AP, Proteintech, China), rabbit anti-Occludin antibody (1:1000, 27260-1-AP, Proteintech), rabbit anti-Claudin 5 antibody (1:1000, AF5216, Affinity Biosciences, China), goat anti-IL-33 antibody (1:2000, AF3626, R&D system), rabbit anti-β-actin (1:3000, NC011, Zhuangzhibio, China). The following secondary antibodies were used to reveal the protein bands on the membrane: horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5000, EK020, Zhuangzhibio) and horseradish peroxidase-conjugated donkey anti-goat secondary antibody (1:5000, EK030, Zhuangzhibio). The incubating condition was room temperature for 2 h. The images of immunoblotted membranes were taken by the ChemiDoc MP imaging system (Bio-Rad, CA, USA).

Primary astrocyte culture and oxygen–glucose deprivation

Primary astrocytes from cortex were separated from the neonatal C57BL/6 pups of 1-day old according to a previous study with slight modification [34]. Purified primary astrocytes were cultured in DMEM (Thermo Fisher Scientific) with 10% fetal bovine serum (Thermo Fisher Scientific). For oxygen–glucose deprivation (OGD), the cell culture medium was changed into glucose-free DMEM (Thermo Fisher Scientific). After that, cell plates were placed into a hermetic chamber (Billups-Rothenberg, USA) flushed and suppled with an anaerobic gas mixture of 5% CO2 and 95% N2 at 37 ºC. After 120 min of OGD, astrocytes were returned to normal culture conditions with complete culture medium and supplied with 5% CO2 in the air. GTA and C75 were added 24 h after OGD. The concentration was 2 mM for GTA and 10 μM for C75 [35, 36]. After 48 h, astrocytes were fixed with 4% paraformaldehyde for 15 min at room temperature or collected for protein extraction.

Liquid chromatography-tandem mass spectrometry (LC–MS/MS)

On day 3 after MCAO, mouse was anesthetized and transcardially perfused with 20 ml saline. Then, mouse brain was removed and the peri-infarct area was quickly dissected on ice. The sample was then snap frozen in liquid nitrogen and stored at − 80 ºC until analysis. The sample was homogenized in tenfold-volume water. Then tenfold-volume methanol (containing 5 μg/ml of internal standards) and tenfold-volume chloroform was mixed. The mixture was processed by 10 min ultra-sonication. After centrifugation at 3000g for 10 min, 32 μl chloroform layer was evaporated to dryness. The residues were mixed with 20 μl HoBt (in DMSO), 40 μl cholamine (in DMSO with 200 mM TEA) and 20 μl HATU (in DMSO) and incubated at room temperature for 5 min. A 120 μl acetonitrile was added and followed by centrifuging at 14,000g for 15 min at 4 °C prior to LC–MS/MS analysis.

Target fatty acids were quantified by an Agilent 1290 Infinity II UHPLC system coupled to a 6470A Triple Quadrupole mass spectrometry (Santa Clara, CA, United States). Samples were injected onto a Waters UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm). The mobile phase consisted of water (phase A) and acetonitrile (phase B), both with 0.1% formate. The chromatographic separation was conducted by a gradient elution program as follows: 0 min, 10% B; 4 min, 30% B; 8 min, 45% B; 11 min, 50% B; 14 min, 70% B; 15 min, 100% B; 18 min, 100% B; 18.1 min, 10% B; 20 min, 10% B. The column temperature was 40 °C. The temperatures of ESI + source drying gas was 300 °C and sheath gas was 350 °C. The flow rate of ESI + source drying gas and sheath gas were 5 and 11 L/min, respectively. The pressure of nebulizer was 45 psi, and capillary voltage was 3000 V. The dynamic multiple reaction monitoring (dMRM) was used to acquire data in optimized MRM transition (precursor—> product). The total scan time of per cycle was 500 ms. The raw data were processed by Agilent MassHunter Workstation Software (version B.08.00) and ChemStation (version E.02.02.1431).

Statistical analysis

The Prism 8 (Graphpad Software, San Diego, California, USA) was used for statistical analysis. Neurological scores based on Garcia were presented as median with interquartile range and tested by Kruskal–Wallis method. Other data were expressed as mean with standard deviation and tested by one-way ANOVA with post hoc Tukey method. Repeated data including grip strength and adhesive removal times were processed using two-way ANOVA, and multiple comparisons were corrected by Tukey method. A P value less than 0.05 was considered statistically significant.