Animals and treatments
C57BL/6 female mice of 4 months of age were supplied by Charles River Laboratories (Calco, Italy). Animals were allowed to food and water access ad libitum and kept in temperature-controlled facilities on a 12-h light and dark cycle. Animals were housed in the animal care facility of the Department of Pharmacological and Biomolecular Sciences at the University of Milan.
Animal investigation has been conducted in accordance with the ethical standards and according to the Declaration of Helsinki and according to the Guide for the Care and Use of Laboratory Animals, as adopted and promulgated by the US National Institute of Health, and in accordance with the European Guidelines for Animal Care and Use of Experimental Animals.
Female mice identified in metaestrous phase were selected for our analyses; the phase of the reproductive cycle in female mice was assessed by blind analysis of vaginal smears mounted on glass microscope slides and stained with May-Grünwald-Giemsa method (MGG Quick Stain Kit; Bio-Optica, Milan, Italy) according to the manufacturer’s protocol.
Mice were injected i.p. with 100 μl of 0.9% NaCl containing 5 μg of mouse recombinant IL-4 (Peprotech, London, UK). 17β-estradiol (E2; Sigma-Aldrich Corp., Milan, Italy) was administered by a 100-μL subcutaneous (s.c.) injection of 5 μg/kg E2 dissolved in corn oil by stirring in the dark and at room temperature o/n.
For in vivo BrdU labeling experiments, mice were injected i.p. with 30 μl of a 10 mg/ml solution of BrdU (Sigma-Aldrich) dissolved in 0.9% NaCl. Animals were sacrificed 2 h after BrdU injection (n = 6).
Two-day-old newborn rats (CD rats, Charles River) were supplied by Charles River Laboratories for the preparation of microglia and astrocytes primary cultures.
Intracerebroventricular (i.c.v.) injections were performed as previously described . Briefly, mice were anesthetized with a s.c. injection of a ketamine/xylazine solution (78 and 6 mg/kg, respectively) and positioned on a specific stand for surgical operation. Injections in the third cerebral ventricle were performed according to specific stereotaxic coordinates (bregma, − 0.25 mm; lateral, 1 mm; depth, 2.25 mm). IL-4 was injected at the concentration of 5 μg in 3.0 μl of 0.9% NaCl for expression studies on purified adult microglia or 250 ng in 2.5 μl 0.9% NaCl for all other experiments; CSF-1 (Peprotech) was injected at the concentration of 1 μg in 3 μl of 0.9% NaCl. Animals injected with the same volume of vehicle alone (0.9% NaCl) were used as controls. Injections were made using a 26S–gauge Hamilton syringe at a rate of 0.1 μl/3 s, and the needle was kept in place for additional 30 s and then removed slowly. The skin incision was closed with a suture and animals were allowed to recover for 24 h before sacrifice by a lethal ketamine/xylazine solution (150 and 12 mg/kg, respectively). Whole brains were removed and immediately processed for microglia isolation; brain areas (frontal cortex, hippocampus, and striatum of left hemisphere, ipsilateral to the injection site) were collected, immediately frozen on dry ice, and stored at − 80 °C until processed for RNA preparation. Whole brains were fixed in 4% formalin solution and stored until processed for immunohistochemistry.
Isolation of peritoneal macrophages
After 24 h of IL-4 or vehicle i.p. injections, peritoneal cells were isolated by peritoneal lavage and incubated with anti-CD11b antibody-loaded MicroBeads (Miltenyi Biotec, Bologna, Italy), as previously described . Briefly, 5 ml of pre-chilled 0.9% NaCl were injected into the peritoneal cavity using a 21 G needle; cell suspension was recovered and centrifuged; following incubation with ammonium-chloride-potassium (ACK) solution (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM EDTA; pH 7.3) for 5 min at 4 °C, cells were either plated and treated as described in the primary cell culture section or incubated with cd11b beads for macrophage purification. For cd11b+ cell isolation, after washing with PBS + 0.5% BSA, cells were resuspended in 90 μL PBS + 0.5% BSA and incubated with 10 μL CD11b MicroBeads for 15 min at 4 °C. After washing, cells were resuspended in 500 uL PBS + 0.5% BSA and applied to MS Miltenyi columns (Miltenyi Biotec) for the magnetic separation procedure. After three washing steps, CD11b-positive cells were eluted from the columns and counted. CD11b-positive cells obtained from each animal were divided into three aliquots; two aliquots of 2.5 × 105 cells each were used to detect BrdU and Ki67 by flow cytometry, the remaining aliquot was stored in TRIzol reagent (Invitrogen-Thermo Fisher Scientific, Milan, Italy) and used for gene expression studies.
Isolation of microglia from adult brains
After i.c.v. treatments, microglia cells were sorted from adult brains (n = 4), as previously described . Briefly, whole brains were dissected and washed in Hank’s balanced salt solution (HBSS; Life Technologies-Thermo Fisher Scientific); after removing the meninges, enzymatic cell dissociation was performed using Neural Tissue Dissociation Kit P (Miltenyi Biotec), with some protocol modifications: after enzymatic digestion with papain, samples were dissociated mechanically, homogenized, and filtered through a 40-μm cell strainer. After extensive washes in HBSS, myelin was removed by suspending samples in 10 ml of cold 0.9 M sucrose solution and centrifuging the dissociated brain cells at 850g and 4 °C for 10 min without braking. Floating myelin and the supernatant were discarded, and cells were processed for microglia magnetic sorting by incubating with CD11b MicroBeads (diluted 1:10 in PBS + 0.5% BSA; Miltenyi Biotec) for 15 min at 4 °C; after washing, cells were suspended in 500 μl of PBS + 0.5% BSA and applied to a magnetic column to purify CD11b+ cells. Immediately after isolation, cells were stored in TRIzol reagent (Invitrogen-Thermo Fisher Scientific) for gene expression.
Primary cell cultures
For in vitro assay, peritoneal cells were incubated with ACK solution, as described above, counted, and seeded at the concentration of 1 × 106 cells/ml in RPMI + GlutaMax (Gibco™-Thermo Fisher Scientific) supplemented with 10% endotoxin-free FBS, 1% penicillin/streptomycin, and 1% Na pyruvate (RPMI + 10% FBS). After 45 min and several washes in PBS, medium was replaced with RPMI + 10% FBS for IL-4 and CSF-1 treatment and in RPMI w/o phenol red supplemented with 10% dextran-coated charcoal (DCC)-FBS (RPMI + 10% DCC) for E2 treatment. After 3 h, cells were treated for 16 h with vehicle or 20 ng/ml of recombinant murine IL-4 or 20 ng/ml of recombinant murine CSF-1. For estrogen treatment, cells were treated on the next day for 3 h with vehicle (0.01% ethanol (EtOH)) or E2 105 M.
Astrocytes and microglia cell cultures
Primary cultures of glial cells were prepared from 2-day-old newborn rats as previously described . After meninges removal, brains were mechanically dissociated and digested in a solution of 2.5% trypsin (Sigma-Aldrich) and 1% DNAse (Sigma-Aldrich), filtered through a 100-μm cell strainer, and seeded at the confluence of 5 × 106 in a 75-cm2 flask in minimum essential Eagle’s medium (MEM) supplemented with 10% FBS, 0.6% glucose, 1% penicillin and streptomycin, and 1% L-glutammine (MEM + 10% FBS). Glial cells were grown at 37 °C under a humidified 5% CO2 and 95% air atmosphere, and medium was replaced every 3 days. After 10 days, microglia were obtained by shaking the confluent monolayer of mixed glial cells at 260 rpm for 2 h and seeded in 12-well plates at the confluence of 5 × 105 cells/well. The medium was changed with MEM + 15% FBS or MEM + 5% FBS 30 min after microglia plating in order to remove contaminating cells. In order to purify astrocytes, enriched astroglia cultures following microglia separation were incubated with 5 mM L-leucine methyl ester (Sigma-Aldrich) to eliminate contaminating microglia cells and seeded in six-well plates at the confluence of 5 × 105 cells/well in MEM + 15% FBS or MEM + 5% FBS.
Astrocytes and microglia were treated for 16 h with 20 ng/ml of recombinant rat IL-4, 20 ng/ml of recombinant rat CSF-1 or vehicle. For in vitro proliferation assay, cells were treated with 10 μM BrdU for 2 h before cell processing for flow cytometry analysis.
Flow cytometry analysis
For Ki67 staining, cells were fixed in 4% paraformaldehyde for 15 min, extensively washed with 125 mM glycine in PBS and permeabilized o/n in PBS containing 0.5% Triton X-100 and 1% BSA, at 4 °C. Cells were incubated with rabbit anti-mouse Ki67 antibody conjugated with eFluor660 (Affymetrix eBioscience, Milan, Italy) diluted 1:100 in incubation solution (PBS containing 0.5% Triton X-100 and 0.05% BSA) at room temperature for 1 h. After extensive washes in PBS, cells were analyzed with a flow cytometry system (NovoCyte® 3000 flow cytometer, ACEA Biosciences, San Diego, CA) and analyzed with NovoExpress® Software (ACEA Biosciences).
For BrdU staining, ex vivo peritoneal cells or in vitro primary cells, detached by 0.25% Trypsin-EDTA (Life Technology) for astrocytes or Accutase (Merck-Millipore, Vimodrone (MI), Italy) for microglia, were fixed and permeabilized in 70% EtOH for 30 min at 4 °C and DNA was denaturated with 2 N HCl/0.5% Triton X-100 and incubated 30 min at room temperature. Cells were washed with 0.1 M sodium tetraborate (pH 8.5) and incubated with rat anti BrdU antibody (AbD Serotec—Bio-Rad, Segrate, Italy) diluted 1:100 in incubation solution (PBS containing 0.05% Tween-20 and 1% BSA). After washes in PBS + 1% BSA, cells were incubated with Alexa647-conjugated goat anti-rat secondary antibody (1:200 in incubation solution; Molecular Probes, Monza, Italy) for 1 h at room temperature. After extensively washing with PBS, ex vivo peritoneal macrophages were resuspended in PI solution (H2O containing 10% NP40, 1 mg/ml RNase A and 5 μg/ml PI stock; Sigma-Aldrich), instead primary cells were resuspended in PBS. Samples were analyzed using NovoCyte® 3000 flow cytometer and analyzed with NovoExpress® Software (ACEA Biosciences). Animals with no pulse of BrdU and in vitro samples without BrdU treatment were used for gating strategy to evaluate non-specific signals. Doublets were removed based on FL2 scatter width (FL2-W)/FL2 scatter area (FL2-A).
RNA preparation and expression analyses
Following 24 h of vehicle or IL-4 (250 ng) i.c.v. treatment, brain area (frontal cortex, striatum, and hippocampus) were first homogenized using steel beads and tissue Lyser (Qiagen, Milan, Italy) at 28 Hz, for three cycles of 20 s followed by 30 s, on ice and in RLT buffer. Total RNA from tissue or cells was purified using RNeasy minikit protocol (Qiagen), according to the manufacturer’s instructions, including a step with deoxyribonuclease incubation. For real-time PCR, 1 μg RNA (500 ng for striatum and primary cultures, 100 ng for isolated microglia, 300 ng for ex vivo peritoneal macrophages) was used for cDNA preparation using 8 U/μl of Moloney murine leukemia virus reverse transcriptase (Promega, Milan, Italy) in a final volume of 25 μl; the reaction was performed at 37 °C for 1 h, and the enzyme inactivated at 75 °C for 5 min. Control reactions without the addition of the reverse transcription enzyme were performed (data not shown). A 1:4 cDNA dilution was amplified using GoTaq®qPCR Master Mix technology (Promega) according to the manufacturer’s protocol. The PCR was carried out in triplicate on a 96-well plate using QuantStudio® 3 real-time PCR system (Applied Biosystems-Thermo Fisher Scientific) with the following thermal profile: 2 min at 95 °C; 40 cycles, 15 s at 95 °C, 1 min at 60 °C. Primer sequences are reported in Additional file 1: Table S1. Data were analyzed using the 2−ΔΔCt method.
Brains were trimmed using a brain matrix (Adult Mouse Brain Slicer Matrix BSMAS005-1, Zivic Instruments, Pittsburgh, PA, USA), and sections were routinely processed, paraffin embedded, and sectioned in 4-μm serial sections. After heat-induced epitope retrieval, performed in Dewax and HIER Buffer H (TA-100-DHBH, Thermofisher Scientific, Waltham, MA, USA) for 40 min at 94 °C, sections were incubated with a 10% normal goat serum for non-specific binding blocking. Sections were immunostained with rabbit polyclonal anti-Ki67 antibody (Clone SP6, Thermo Fisher Scientific, Waltham, MA, USA), incubated with biotinylated goat anti-rabbit secondary antibodies (VC-BA-1000-MM15, Vector Laboratories, Peterborough, UK) and labeled by the avidin-biotin-peroxidase procedure with a commercial immunoperoxidase kit (VECTASTAIN® Elite ABC-Peroxidase Kit Standard, VC-PK-6100-KI01, Vector Laboratories). The immunoreaction was visualized with DAB (Peroxidase DAB Substrate Kit, VC-SK-4100-KI01, Vector Laboratories) substrate, and sections were counterstained with Mayer’s hematoxylin (C0302, Diapath, Italy). Digital image analysis was performed by scoring the number of Ki67-positive cells in three ×400 microscopic fields in the parenchyma of vehicle, IL-4 and CSF-1-treated mice (n = 3); monocyte-like cells were excluded from the analysis. For double immune-fluorescence analysis, after heat-induced antigen retrieval in Dewax and HIER BufferH pH 9 (Thermofisher Scientific) and anti-ki67 antibody (RM-9106, Thermofisher Scientific) incubation, goat anti-rabbit green fluorescent antibody (Alexa Fluor 488, Thermo Fisher Scientific) was used; after washing with PBS, sections were re-incubated with anti-GFAP (Z0334, from DAKO Agilent, Santa Clara, CA, USA) or anti-Iba1 (Wako Chemicals USA, Richmond, VA) for 1 h at room temperature followed by a goat anti-rabbit red fluorescent secondary antibody (Alexa Fluor 555, Thermo Fisher scientific). Sections were mounted on coverslips with ProLong Gold Antifade mountant with DAPI (P36941, Thermofisher Scientific).
Unless otherwise stated, all values are expressed as mean ± standard error of the mean (SEM) of n observations. The results were analyzed by the Student unpaired two-tailed t test using GraphPad Prism 5 software, after a normality test (Kolmogorov-Smirnov) . A value of p < 0.05 was considered significant.