Enriched endogenous omega-3 polyunsaturated fatty acids protects cortical neurons from experimental ischemic injury
Zhe Shi 1, Huixia Ren 1, Chuanming Luo 1, Xiaoli Yao 2, Peng Li 1, Chengwei He 1, Jing-X Kang 3, Jian-Bo Wan 1, Ti-Fei Yuan 4, *, Huanxing Su 1, *
1State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China
2Department of Neurology, National Key Clinical Department and Key Discipline of Neurology, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, China
3Laboratory for Lipid Medicine and Technology, the Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
4School of Psychology, Nanjing Normal University, Nanjing, 210097 China
*Corresponding author: Huanxing Su, State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China. Tel: (853) 8397 8518; Fax: (853) 2884 1358 Email: ; Ti-Fei Yuan,
Supplementary Material and methods
Animals
Experimental mice were obtained by mating male fat-1 mice (C57BL/6 background obtained from Dr. Jing X. Kang, Harvard Medical School, MA, USA) with female C57BL/6 wild type (WT) mice. The fat-1 phenotypes of each animal were characterized using isolated genomic DNA and fatty acid composition analysis from mouse tails. Mice were fed a modified diet containing 10% corn oil (TROPHIC Animal Feed High-tech Co., Ltd, China), with a fatty acid profile high in n-6 (mainly linoleic acid) and low in n-3 PUFAs (~0.1% of the total fat supplied), until the desired age for primary neuron cultures (E16-18) and focal cortex infarction surgery (8-10 weeks).
All animal procedures were performed in compliance with guidelines approved by the Ethics Committee for Animal Experiments of the University of Macau.
Primary cortical neuron cultures and Oxygen-glucose deprivation/reperfusion (OGD/R)
Cortical cultures were obtained from E16.5 WT or fat-1 embryos. The presence of the fat-1 gene was confirmed by genotyping on each embryo. Cerebral cortices were removed, and stripped of meninges. Tissues were digested in 0.05% trypsin, and triturated. Cells were seeded in 6 or 24-well plates pre-treated with poly-L-lysine and laminin. Cultures were maintained in Neurobasal medium containing 2% B27 supplement and 0.5mM GlutaMAXTM-I (Life Technologies). Cultures were kept at 37 °C, 100% humidity and in a 95% air/5% CO2 atmosphere. Unless indicated, experiments were performed after 7 days in vitro (DIV).
For OGD/R, cultures were placed in a hypoxia chamber containing an atmosphere of <0.2% O2, 5% CO2, 95% N2, >90% humidity, and 37°C. Within the chamber, the medium was removed and replaced with oxygen/glucose-free balanced salt solution (BSS, in mmol/l: 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 1 mM NaH2PO4.2H2O, 262 mM NaHCO3, 1.8 mM CaCl2, pH 7.2, <0.1% O2), which was previously saturated with 95% N2/5% CO2 at 37°C. Still within the chamber, cells were washed twice with oxygen/glucose-free BSS. Cultures were taken out of the chamber after 4h and transferred to the regular cell culture incubator. Sham-treated cultures were always handled in parallel and received similar wash steps as OGD/R-treated cultures with the difference in that BSS contains 4.5 g/l glucose and regular oxygen.
Focal cortical infarction (FCI)
Eight to ten weeks old male fat-1 mice (n=10) and their WT littermates (n=10) were subjected to FCI following previous studies reporting that small cortical infarcts could be generated by damaging the endothelium of targeted pial or penetrating arterioles using focused femtosecond laser pulses with minor modifications (Nishimura et al., 2006; Schaffer et al., 2006). Mice were deeply anesthetized with isoflurane 5% and maintained with 2% isoflurane in a mixture of 20% oxygen and 80% air. A cranial window of 3.5 mm×3.5 mm size was thinned on the left region over the distal main branch of middle cerebral artery between bregma and lambda with a dental burr. For in vivo two-photon imaging, the mouse was fixed with a custom-fabricated metal frame by holding the head with a cyanoacrylate and dental cement, and then fixed on the stage of Leica DM6000 CFS. The blood serum was labeled by intravenously injecting 0.2ml of 2% (wt/vol) solution of 2-MDa fluorescein-dextran (FD2000S; Sigma) in saline. To conduct focal cortical infarction in the somatosensory cortex, we used a 5× magnification air objective to obtain images of target arteriole as well as leptomeningeal anastomoses and penetrating arterioles, and then outline the area supplied by target arteriole with Camera (Leica DFC 365 FX CCD). Next, we modified the area supplied by the distal branch of the middle cerebral artery to an approximated 2.0×2.0 mm area containing its leptomeningeal anastomoses if possible. Subsequently, we performed a clot of the target arteriole and its connected vessels induced by focusing femtosecond-duration laser pulses. After surgery, all mice were allowed to recover for 24h in a recovery cage under a heat lamp and had free access to drinking water and eating food.
Fatty acid analysis
To examine whether the expression of the fat-1 gene altered the PUFA composition in the primary cultured cortical neurons of the fat-1 and WT groups, fatty acid analysis were processed by using gas chromatography-mass spectrometry (GC-MS), as described previously 1. Briefly, cell samples were ground to powder under liquid nitrogen and subjected to fatty acid methylation by 14% boron trifluoride–methanol reagent at 100°C for 1h. Fatty acid methyl esters were analyzed by an Agilent GC-MS system (Agilent Technologies, Palo Alto, CA) consisting of an Agilent 6890 gas chromatography and an Agilent 5973 mass spectrometer. Fatty acids were identified in forms of their methyl esters by three means: (i) searching potential compounds from NIST MS Search 2.0 database, (ii) comparing retention time with those of reference compounds (Nu-Chek Prep, Elysian, MN) eluted under the identical chromatographic condition, (iii) comparing their mass spectra plots with those of authentic standards. Quantification was performed by normalizing individual peak area as the percentage of total fatty acids.
Immunocytochemistry and histopathology analysis
Immunocytochemistry was conducted on 7 DIV. Neurons were washed with cold phosphate-buffered saline (PBS) (pH 7.4), followed by 4% paraformaldehyde (PFA) for 30min. After washing with PBS containing 0.1% Tween, cells were incubated with 5% goat serum for 1h, and then incubated with primary antibodies against, Tuj-1 (neurons, 1:500) and GFAP (astrocytes, 1:500) at 4°C overnight. After PBS washing, they were incubated with secondary antibody, counterstained with 0.1μg/mL DAPI, for 15min at room temperature. Immunostaining was analyzed using a fluorescence microscope (Leica DM6000 B) interfaced with a digital camera and an image analysis system.
Histopathology was performed after completion of behavioral tests. Mice were sacrificed after by deep anesthetization with isoflurane 5% and transcardial perfusion with cold heparinized saline, followed by perfusion of 4% paraformaldehyde. Brains were removed and post-fixed overnight in paraformaldehyde, before being dehydrated and embedded in paraffin. Brains were cut into 10μm thick sections in the coronal plane and stained with hematoxylin and eosin (HE). Brain sections including the somatosensory cortex territory were used for quantification of infarct area. Infarct volume was calculated by multiplying infarct areas with the distance between sections.
Terminal transferase dUTP nick end labeling (TUNEL) assay
To identify apoptotic neurons, TUNEL assays were performed using an in situ cell death detection kit (Roche, No.11 684 795 9101). After washed three times by ice-cold PBS, the cell samples were fixed with a freshly prepared fixation solution for 1h and incubated in permeabilization solution for 2min on ice. Then, 50μl TUNEL reaction mixture was added on each sample. Slides were incubated in a humidified atmosphere for 60min at 37°C in the dark, followed by counterstaining with DAPI. The number of TUNEL-positive cells will be counted in 10 randomized fields per well under a fluorescence microscope. Results were the average ± SEM of data from 5 experiments unless stated otherwise in the legends.
In vitro intracellular ROS assessment
Intracellular ROS levels were measured by staining with CellROX® Green Reagent (Molecular Probes). CellROX® Green Reagent (5μM) was added to the medium for 30min at 37°C after the OGD/R treatment. After the culture was washed three times with PBS, they were fixed with 3.7% formaldehyde for 15min, and the cells were then examined under the fluorescence microscope. Fluorescence images were taken at the excitation/emission wavelengths of 488/520 nm. The levels of intracellular ROS were determined by the fluorescent intensity quantified with IOD values by Image-Pro Plus software.
Biochemical Assays of Hydrogen Peroxide and Glutathione peroxidase
Five cortical samples in each group were assessed in biochemical assays. Analyses were carried out using Hydrogen Peroxide Assay Kit (S0038, Beyotime Biotech) and Glutathione Peroxidase Detection Kit (S0056, Beyotime Biotech). For H2O2 detection, test tubes containing 50μL of supernatants and 100μL of test solutions were placed at room temperature for 20min and measured immediately with a spectrometer at a wavelength of 560nm. The concentration of H2O2 released was calculated from a standard concentration curve in triplicate experiments.
The detection of glutathione (GSH) content was conducted following the manual. After lysed with a lysis buffer, tissue extracts were mixed with test solutions. Then, the plate was measured at 340nm. Finally, the amount of GSH was vitality calculated according to the manual instruction.
Protein extraction and Western blot analysis
Cortical neurons in 6cm dishes were washed with ice-cold PBS for three times and lysed with a lysis buffer containing protease inhibitors (Beyotime Biotech) at 24h after OGD/R treatments. The protein concentration was determined using a BCA protein assay kit. Then, protein extracts were separated by electrophoresis on 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were sequentially incubated with primary antibodies and secondary antibodies, and enhanced chemiluminescence (ECL) solution and followed by autoradiography. The intensity of the blots was analyzed using Image Pro plus 6.0.
Behavioral analysis
The gait analysis of animals (five animals in each group) was performed at 2 weeks after FCI following the method described previously (Chen et al., 2014). In brief, the CatWalk XT system comes with a high-speed digital camera with a sample rate of 100 frames per second. The video camera transforms each scene (the area in front of the lens) into a digital image (an image composed of discrete pixels of digital brightness values). The Illuminated Footprint enables detection of intensity differences between animals’ paws. Prior to the FCI surgery, the animals were trained to traverse a glass walkway for three consecutive days. Fourteen days after FCI surgery, three complete trails across the walkway were recorded. If an animal failed to fluently complete a run, walked backwards, or reared during the run, the animal was given an additional re-run. Quantitative analysis of the data from the CatWalk XT system includes the following parameters: step sequence distribution, regularity index, print area, duration of swing and stance phases, and maximum contact maximum intensity (maximum intensity at the maximum contact of a paw).
The adhesive removal test was performed as described previously 2. Mice were habituated to the testing box for 1min. Then, two adhesive tape strips with equal pressure were gently applied on each paw of the animal so that they covered the hairless part of the forepaws. The animal’s behavior was observed for 2min. The time point that the mouse reacted to the presence of the adhesive tape strips (contact time) and attempted to remove the adhesives (removal time) was respectively recorded.
Data analysis
Statistical differences between two groups were determined by two-tailed Student's t test. Multiple group comparisons were made by one-way ANOVA and Tukey post hoc test. Data were presented as mean ± SEM. Significance levels were set to 0.05 for all comparisons.
1Wan, J. B. et al. Endogenously decreasing tissue n-6/n-3 fatty acid ratio reduces atherosclerotic lesions in apolipoprotein E-deficient mice by inhibiting systemic and vascular inflammation. Arterioscler Thromb Vasc Biol 30, 2487-2494, (2010).
2Bouet, V. et al. The adhesive removal test: a sensitive method to assess sensorimotor deficits in mice. Nat Protoc 4, 1560-1564, (2009).