Animals
200–250 g male Sprague-Dawley (SD) rats were used in this study. For this study, we used the following experimental groups: (1) Several SD rats were sham group.(2) Experimental group performed bilateral common carotid artery ligation (BCCAO) .(3) Several SD rats were intraperitoneally injected with clemastine (1 mg/kg) after BCCAO. The rats were then allowed to raise under normoxic conditions for 0.5, 1, 3, 7, 14, 21, 28 days (d) before being sacrificed. Animal handling and experiments were approved by Institutional Animal Care and Use Committee, Shang hai, China.
Primary Culture of Astrocytes
In brief, cerebral hemispheres were separated from 1-day-old postnatal SD rats (Model Animal Research Center, Xin Hua Hospital, Shanghai Jiao Tong University School of Medicine, License No. SYXK (Shanghai) 2018-0038), the meninges and superficial vessels were carefully removed. The cerebral cortex was harvested and digested with 10 ml 0.125% trypsin containing 600 U DNase (Sigma, St. Louis, MO, USA, Cat. No. D4527) for 15 minutes in 37 °C thermostatic water bath. After this, 10 ml of Dulbecco’s modified Eagle’s medium-F12 nutrient mixture (DMEM-F12) (Invitrogen Life Technologies Corporation, Carlsbad, CA, USA; Cat. No. 31330-038) containing 10% fetal bovine serum (FBS) (Invitrogen Life Technologies Corporation; Cat. No. 10099-141) was added. The tissue was then triturated several times with a 5 ml pipette. The un-dissociated tissue clumps were allowed to settle for one to two minutes. Subsequently, the supernatant was collected and passed through a 70 µm cell strainer to remove the remaining small clumps of tissue; the cell suspension was then centrifuged at 1,500 rpm for five minutes. The supernatant was discarded and the cell pellet was resuspended in 10 ml of DMEM-F12 supplemented with 10% FBS. The resuspended cells were seeded into poly-L-lysine-coated 75 cm2 flasks at a density of 250,000 cells/ml and cultured at 37 °C in humidified 5% CO2/95% air. The medium was changed after 24 h and then replaced every three to four days. After 10 days, the bottom of the flask showed a confluency of cells with mixed glia, including mainly microglia/oligodendrocytes. The mixed cells were cultured for 10 days at 37℃ and 5% CO2 before shaking at 180 revolutions per minute (rpm) and 37℃ for 1 h to remove microglia cells and oligodendrocytes. After incubation in a humidified atmosphere of 95% air and 5% CO2 at 37 °C for 24 h, the cells were subjected to different treatments. The astrocytes cultures with above 90% purity were used in this study.
Treatment of Astrocytes Culture
The purified astrocytes were cultured for 1 d at 5% CO2 and 95% air at 37℃, the cells were subjected to different treatments. To study whether clemastine would affect astrocytes release of inflammatory mediators and autophagy, astrocytes were treated with (Clemastine group) or without (oxygen glucose deprivation, OGD group) 20 ng/ml clemastine for 1 h at 3% oxygen, 5% CO2 and 92% nitrogen at 37 °C. Control group with equal volume of PBS, Clemastine group were treated with 20 ng/ml clemastine.
Primary Culture of OPCs
The mixed culture is the same as the astrocytes. The mixed cells were cultured for 10 days at 37 °C and 5% CO2 before shaking at 180 revolutions per minute (rpm) and 37 °C for 1 h to remove microglia cells. The medium was then replaced with fresh DMEM/FBS, and the cultured cells were again shaken at 250 rpm and 37 °C for 20 h to harvest OPCs, followed by incubating on a 10 cm Petri dish for 60 minutes (min) at 37 °C to remove contaminating astrocytes and microglia. Purified OPCs were plated on Poly-D-lysine (PDL) or laminin-coated coverslips and cultured in oligodendrocyte precursor cell medium (OPCM) (Sciencell Research Labortories, USA, No.1601) at 5% CO2 and 95% air at 37 °C. The OPCs cultures with above 90% purity were used in this study.
Treatment of OPCs Culture
To examine the effects of IL-1β on the differentiated OPCs. The cells were subdivided into the Control group (0.01M PBS); 30 ng/mL IL-1β group; 30 ng/mL IL-1β + 20 ng/mL IL-1 receptor antagonist (IL-1Ra) group; 20 ng/mL IL-1Ra group. The OPCs were cultured in oligodendrocyte precursor cell differentiation culture medium (OPCDM) (Sciencell Research Labortories, USA, No.1631) for 7 d at 5% CO2 and 95% air at 37 °C.
Co-culture experiment with astrocytes and OPCs
The purified astrocytes were plated on tissue culture inserts for 6-well plates (0.4 µm, Millipore, Cat. No. MCHT06H48) at a density of 1 × 106 cells. The astrocytes were incubated for 1 h in the presence or absence of clemastine (20 ng/mL) or 3-MA (a specific autophagy inhibitor, Topscience Co., Ltd, 5 mM). In order to minimize OPCs damage due to pretreatment of OGD challenge, we have chosen to treat the target drugs (clemastine and 3-MA) before OGD challenge. Each tissue culture insert was placed on the OPCs in 6-well plates. Co-culture system were treated for 1 h at 3% oxygen, 5% CO2 and 92% nitrogen at 37 °C, then, cultured in OPCDM at 5% CO2 and 95% air at 37 °C.
Treatment of Co-culture
To explore whether clemastine would affect OPCs differentiation via autophagy, The co-culture cells were subdivided into Control group, OGD group, OGD + clemastine, OGD + clemastine + 3-MA group.
Western Blot
Proteins were extracted from the corpus callosum or from primary cell culture using a protein extraction kit (Pierce Biotechnology Inc, IL) according to the manufacturer’s protocol. Protein concentrations were determined by the Bradford method using bovine serum albumin (BSA) (Sigma-Aldrich, St Louis, MO) as a standard. Samples of supernatants containing 50 µg of protein were heated to 95 °C for 10 min and were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis in 10% gel in a Mini-Protein 3 apparatus (Bio-Rad Laboratories, Hercules, CA). Protein bands were electroblotted onto 0.45 lm polyvinylindene difluoride membranes (BioRad) at 1.5 mA/cm2 of membrane for 1 h in Towbin buffer, pH 8.3, to which 20% (volume/volume (v/v)) methanol had been added. After transfer, the membranes were blocked with QuickBlock™ Blocking Buffer (Cat. P0231, Beyotime, China) for 1 h, then incubated with the primary antibodies according to the manufacturer’s recommendations. The primary antibodies used were as follows: IL-1β (Rabbit polyclonal IgG 1:1,000), Histamine H1 Receptor (HH1R) (Rabbit polyclonal IgG 1:1,000), 2’,3’-cyclicnucleotide 3’-phosphodiesterase (CNPase) (Rabbit polyclonal IgG 1:1,000), Olig2 (Rabbit polyclonal IgG 1:1,000), Nod-like receptor protein 3 (NLRP3) (Rabbit polyclonal IgG 1:1,000) [all from Bioworld Technology, Inc. Cat. No. BS6067, BS2733, BS3461, BS90984, BS90948], PLP (rabbit polyclonal IgG 1:1000) (Sigma; Cat. No. SAB1410977), Nkx2.2 (mouse monoclonal IgG 1:1,000) (Santa Cruz; Cat. No. sc-398951), p62 (Rabbit polyclonal IgG 1:1,000), Beclin-1 (Rabbit polyclonal IgG 1:1,000), LC3B (Rabbit polyclonal IgG 1:1,000), VCAM-1 (Rabbit polyclonal IgG 1:1,000), β-actin (Rabbit polyclonal IgG 1:1,000) [all from Cell Signaling Technology, Cat. No. #23214, #3495, #3868, #14694, #8457]. After three washes with TBST, the membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology; Cat. No. 7074 (anti-rabbit IgG), 7076 (anti-mouse IgG) ) for 1 h. The immunoblots were developed using the enhanced chemiluminescence detection system (Pierce Biotechnology Inc, Rockford, IL). Blots were stripped with stripping buffer (Cat. P0025N, Beyotime, China) and hybridized with total kinases or β-actin. The signal intensity of these proteins levels relative to control was measured with Quantity One Software, version 4.4.1 (BioRad Laboratories).
Double Immunofluorescence
The Cat. No. of antibody is the same as that of western blot and it is listed separately if there are differences. Immunofluorescence intensity is expressed by mean fluorescence intensity via Image J. The sections from control and BCCAO rats (in each group at 1, 3, 7, 14 and 21 days) were divided into four groups. The sections in group I from control and BCCAO rats at 1, 3 and 7days were then incubated with antibodies directed against anti-IL-1β (Rabbit polyclonal IgG 1:500) or anti-Histamine H1 Receptor (Rabbit polyclonal IgG 1:200) or NLRP3 (Rabbit polyclonal IgG 1:200) and GFAP (Mouse monoclonal IgG 1:100) (Bioworld Technology, Cat. No. MB9017). The sections in group II from control and BCCAO rats at 14 and 21 days were incubated with Olig2 (Rabbit polyclonal IgG 1:200) and NG2 (mouse monoclonal IgG 1:100) .
The sections in group Ⅲ from control and BCCAO rats at 1 day were incubated with LC3B (Rabbit polyclonal IgG 1:1,000). The sections in group Ⅳfrom control and BCCAO rats at 21 day were incubated with CNPase (mouse monoclonal IgG 1:1,000) (Chemicon International; Cat.No. NE1020) or PLP (rabbit polyclonal IgG 1:1000). The incubation for all groups was carried out at 4 °C overnight. On the following day, the sections were washed and incubated with a secondary antibody: Alexa Fluor 555 goat anti-rabbit IgG (1:100, Life, Cat. No. A21428) or Alexa Fluor 555 goat anti-mouse IgG (1:100, Life, Cat. No. A21424) or FITC goat anti-mouse IgG (Bioworld Technology, Inc. Cat. No. BS50950) at room temperature for 1 h. The ratio of NG2/Olig2-positive oligodendrocytes was calculated and averaged. The ratio of NG2/Olig2-positive oligodendrocytes in the corpus callosum was calculated by counting four randomly selected microscopic fields in sections obtained from each rat at 40 × objective by a blinded observer.
For astrocytes cultured, the cells were treated with oxygen glucose deprivation (OGD) and OGD + clemastine for 1 h, respectively (for IL-1β or HH1R and NLRP3 detection), and then fixed in 4% paraformaldehyde for 30 min, blocked in 1% BSA for 30 min and incubated with primary antibodies overnight at 4 °C. The cells were divided into two groups. The cells in group I were carried out by using primary antibodies directed against anti-IL-1β (Rabbit polyclonal IgG 1:500) or anti-Histamine H1 Receptor (Rabbit polyclonal IgG 1:200) or NLRP3 (Rabbit polyclonal IgG 1:200) and GFAP (Mouse monoclonal IgG 1:100) for astrocytes cultured for 1 h.The cells in group II were carried out by using primary antibodies directed against anti-LC3B (Rabbit polyclonal IgG 1:200) and GFAP (Mouse monoclonal IgG 1:100) for astrocytes cultured for 1 h. The cells were then incubated with Alexa Fluor 555 goat-anti-rabbit secondary antibody (1:100, Life, Cat. No. A21428) and FITC goat anti-mouse IgG (Bioworld Technology, Inc. Cat. No. BS50950) for 1 h. Finally, the cells were counterstained with DAPI and examined under a fluorescence microscope (Olympus System Microscope Model BX53, Olympus Company Pte, Tokyo, Japan).
For cultured OPCs, the cells were treated with IL-1β or IL-1Ra and IL-1β + IL-1Ra, respectively, and the same volume of PBS as control for 7 d (for NG2 and PLP detection), then fixed in 4% paraformaldehyde for 30 min, blocked in 1% BSA for 30 min and incubated with primary antibodies overnight at 4℃. Immunofluorescence labeling was carried out by using primary antibodies directed against NG2 (Mouse monoclonal IgG 1:1,000) and PLP (Rabbit polyclonal IgG 1:1000) or VCAM1 (Rabbit polyclonal IgG 1:1000) and PLP (Mouse monoclonal IgG 1:1,000) (Sigma; Cat. No. SAB1404221), respectively. The cells were then incubated with Alexa Fluor 555 goat anti-rabbit IgG (1:100, Life, Cat. No. A21428) or Alexa Fluor 555 goat anti-mouse IgG (1:100, Life, Cat. No. A21424) or FITC goat anti-mouse IgG (Bioworld Technology, Inc. Cat. No. BS50950) for 1 h. Finally, the cells were counterstained with DAPI and examined under a fluorescence microscope (Olympus System Microscope Model BX53, Olympus Company Pte, Tokyo, Japan). Quantitative analysis of the ratio of NG2, and PLP positive cells was carried out through counting four randomly selected microscopic fields at 40 × objective by a blinded observer. The percentage of cells with positive expression for the respective antibodies was calculated and averaged. Each experiment was done in triplicate.
Electron Microscopy
The BCCAO rats, BCCAO + clemastine rats at 1, 28 days and their corresponding control rats were perfused with a mixed aldehyde fixative composed of 2% paraformaldehyde and 3% glutaraldehyde in 0.1M phosphate buffer, pH 7.2. After perfusion, the brain was removed and coronal slices (approximately 1 mm thick) were cut. Blocks of corpus callosum were trimmed from these slices.Vibratome sections (Model 3000TM, The Vibratome Company, St. Louis, MO) of 80–100 mm thickness were prepared from these blocks and rinsed overnight in 0.1M phosphate buffer. They were then post-fixed for 2 h in 1% osmium tetraoxide, dehydrated, and embedded in Araldite mixture. Ultrathin sections were cut and viewed in a Philips CM 120 electron microscope (FEI Company, Hillsboro, OR). Four non-overlapping regions of the medial corpusallosum from each animal were photographed at two different magnifications. The diameter of each axon as well as that of axon plus its associated myelin sheath was measured by using Image J software (SummaSketch III Summagraphics, Seattle, WA). The g-ratio was calculated per axon as axon diameter to total axonal fiber diameter, the transmission electron microscope was finally used to detect the autophagosomes in astrocytes.[it is equivalent to axon area/(axon + myelin sheath area)] by a researcher blind to control/BCCAO/clemastine injection group and compared among groups with one-way ANOVA (α = 0.05, two-tailed; n = 3 per group).
Statistical Analysis
All data were evaluated by the SPSS13.0 statistical software (IBM, Armonk, NY). Different statistical methods were applied according to different types of data. The distribution values were expressed as mean ± SD. Four-group univariate-factor measurement data were analyzed by one-way ANOVA if the data were homogeneity of variance; otherwise, they were analyzed by Welch ANOVA. Multiple comparisons were analyzed by the least significant difference (LSD) method if the data were homogeneity of variance; otherwise they were analyzed by Dunnett’s T3 method. The criterion for statistical significance was set at P < 0.05.