Animals and reagents
Wild type C57BL/6j mice were acquired at Facultad de Ciencias Veterinarias, UBA. C57BL/6-Tg(CAG-EGFP)1Osb/J transgenic mice expressing the enhanced green fluorescent reporter under chicken beta actin gene promoter and cytomegalovirus enhancer (ACT::GFP) (RRID:MGI:2686900, Jackson, Bar Harbor, USA), was kindly provided by Dr. Campetella (IIBIO, CONICET). All mice strains were maintained in the animal care facility at IQUIFIB- UBA-CONICET, following institutional guidelines for animal care. Protocols were in accordance with guidelines from the Comité Institucional de Cuidado y Uso de Animales de Laboratorio (CICUAL)- at Facultad de Farmacia y Bioquímica- UBA and were approved by Res (D) Nº4538/2018 and Res (D) Nº 657/2020.
Cuprizone (Bis-cyclohexanone oxaldihydrazone), poly-L-lysine, 5-bromo-2´-deoxyuridine (BrdU), bisbenzimide a33258 (Höechst), propidium iodide (PI) and Triton X-100 were purchased from Sigma-Aldrich Argentina. DMEM-F12, Hanks balanced salt solution (HBSS) and B-27 Supplement were from Gibco Life Technologies. Plasticware was acquired from Elessar (Argentina), fetal calf serum (FCS) was from Cripion SRL (Argentina). Mowiol 4-88 was from Calbiochem. Paraformaldehyde (PFA), sucrose and other reagents were from Biopack. Epidermal growth factor (EGF) was purchased from Peprotech and basic fibroblast growth factor (bFGF) was a gift from Dr Baldi (IBYME, UBA-CONICET). H2DCFDA probe was a gift from Dr. Sandra Verstraeten (Universidad de Buenos Aires, CONICET). Primary antibodies are listed in Table 1. All secondary antibodies were purchased from Jackson ImmunoResearch.
Tissue processing and immunohistochemistry
Neonatal mice between 2-4 days old were euthanized by decapitation and brains were removed, washed in phosphate buffered saline (PBS) and fixed overnight in 4 % PFA-PBS. After rinsing in PBS, brains were sequentially set to soak in 15 % and 30 % sucrose solutions at 4 ºC and finally were frozen at -80 ºC until sectioned with a Leica CM1850 cryostat in 30 µm thick slices. Tissue sections were adhered to glass slides and processed for immunohistochemistry (IHC). After blocking with 5 % FCS, 0.1 % Triton X-100 in PBS solution, sections were incubated overnight with primary antibodies (Table 1) diluted in PBS with 1 % FCS and 0.02 % Triton X-100. Brain slices were then rinsed in PBS and incubated with the appropriate fluorescent secondary antibodies (1:2000) together with 5 µg/ml Höechst 33258 for nuclei staining. After washing in PBS, sections were air dried and mounted with Mowiol solution.
Neurosphere cultures and treatments
For all the experiments, 2-4 days old newborn mice were sacrificed by decapitation and SVZ was removed from brains. Cell suspensions were obtained by mechanical dissociation of SVZ tissue with a P1000 micropipette tip. Cells were washed in 5 ml DMEM-F12 and after 5 min centrifugation at 300 xg, supernatant was discarded. Cell pellets were resuspended and cultured in DMEM-F12 supplemented with 2 % B-27, 20 ng/ml bFGF and 20 ng/ml EGF (proliferative medium), for maintenance of undifferentiated and highly proliferative status. Growth factors were added to the culture medium every other day. In this proliferative condition, cells retained multipotency and undifferentiation, both when cultured as NS in suspension in T25 flasks or 24-well culture plates, as well as when grown as a monolayer on poly-L-lysine-coated coverslips. Differentiation of NSC/NPC into neural lineages was induced by changing culture medium to DMEM-F12 with 2 % B-27 Supplement without growth factors (differentiating medium).
CPZ solutions at different concentrations were prepared in 50 % v/v ethanol by serial dilutions from a 6 mg/ml CPZ stock freshly prepared in ethanol 50 % v/v. Identical volumes of each CPZ working solution, representing less than 5% of final volume, was added to the culture media to reach desired final CPZ concentrations, according to the previously used reference range (Pasquini et al., 2007). For CPZ0, we added the same volume of 50% v/v ethanol without CPZ to the medium. In every experiment we run in parallel a control condition containing culture media without any treatment (CTL).
NSC/NPC viability and proliferation assays
NS were grown in suspension in proliferative media (+bFGF/EGF) containing different CPZ doses by triplicate. After one week in culture, NS were fixed in 4 % PFA and photographed at low magnification for determination of total numbers of NS and NS diameter in each condition.
For quantitation of viable cells, intact or dissociated NS from ACT::GFP mice were seeded into poly-L-lysine-coated coverslips with proliferative or differentiating media and were grown with or without CPZ, depending on the experiment. Cells were fixed with 4 % PFA and after washing in PBS, they were stained with 5 µg/ml final Höechst 33258. GFP fluorescence associated to living cells, and Höechst staining of total nuclei were observed by fluorescence microscopy.
For proliferation assay, 24 hours before fixing with 4 % PFA, cells were incubated with 10 µM 5-Bromo-2’-deoxyuridine (BrdU) and then were analyzed by immunocytochemistry using anti BrdU antibody.
NPC migration and processes elongation in proliferating cultures
Whole NS were seeded by triplicate on poly-L-lysine coated coverslips and cultured for 48 hours in proliferative conditions (+bFGF/EGF) with CPZ treatments. During this period, numerous cell processes elongate from the NS surface and individual cells detached from the NS and migrate away from the NS border. Cells were washed in PBS twice, fixed in PFA 4 % solution for 20 min at room temperature and subjected to immunocytochemistry and Höechst staining. After culture images were taken, the outline of individual NS was manually demarcated to establish NS border. For each condition we selected NS which met the requirement of being isolated (without interaction with neighboring NS). For each NS, we determined the external edge/border of migratory cells to include all GFAP+ cell processes and Höechst-stained cell nuclei arising from the central NS to demarcate the complete migratory area. This area was subdivided in concentrical rings every 50 μm. We registered nuclei numbers inside each migratory ring. We also measured NS diameter and the maximal length of the external edge containing cells processes from individual NS.
Cell differentiation and cell death analysis
Cell suspensions from dissociated NS cultures were seeded onto poly-L-lysine-coated coverslips and were left one hour for adhesion. Cell differentiation was started by adding differentiating media (without bFGF/EGF) with CPZ treatments by triplicate. Cells were differentiated for one week and then they were washed twice in PBS, fixed in PFA 4% solution for 20 min at room temperature and finally rinsed in PBS. Cell differentiation was evaluated by immunocytochemistry for oligodendrocytes, astrocytes, neurons, or progenitors by detection of specific markers (see immunocytochemistry section). For cell death analysis, cells were incubated with propidium iodide 100 µg/ml before fixation for 30 min.
CPZ effects on mature oligodendrocytes, astrocytes, and neurons survival
Dissociated NS were seeded on coverslips and grown for six days in proliferative media. Then, media was switched to differentiating conditions (without mitogens) without any treatment and cultured for six days. Once differentiated, cells were exposed to CPZ treatments by triplicate for two additional days. Immunocytochemical analysis was performed to detect oligodendrocytes, astrocytes, neurons and oligodendroglial precursors. For each marker, one coverslip was removed before CPZ treatment was started, as a control for initial cell proportions as the pre-treatment condition (PRE).
PFA-fixed cells were blocked with 5% FCS– 0.1% Triton X-100 in PBS solution and incubated with primary antibodies listed in table 1. For BrdU immunodetection, antigen retrieval was performed after fixation by incubation with 2 N HCl for 20 min at 37 º C, followed by neutralization with 0.1 M Sodium Borate (pH 9) for 15 min at 37 º C and a blocking step overnight with 2 % FCS.
All antibodies were diluted in a 1% FCS, 0.02% Triton X-100 in PBS solution and incubated overnight at 4 ºC. After rinsing in PBS, cells were incubated with the appropriate secondary antibody (Jackson IR, 1:2000) together with Höechst 33258 for nuclei staining at 5 µg/ml. We performed red and green detection for two markers in cell cultures derived from wild type animals combined with blue nuclei Höechst staining. In some experiments in which NS were generated from ACT::GFP mice, green channel was reserved for GFP detection as a control for cell viability and only one additional marker was evaluated using a secondary antibody with red fluorophore by immunocytochemistry.
Determination of intracellular ROS
For oxidative stress determination, intracellular ROS levels were monitored by using (6)-carboxy-2´,7´-dichlorodihydrofluorescein diacetate probe (H2DCFDA) (Molecular Probes Inc., Cat #C-400), which is converted into a fluorescent compound (DCF) upon oxidation with ROS. Internalization of the probe into cells was confirmed by fluorescence microscopy. Briefly, NS were dissociated, and cells were attached to poly-L-lysine coated coverslips and cultured in proliferative conditions for six days. CPZ treatments were performed for 24 hours and then the medium was removed and replaced with HBSS containing 5 µM H2DCFDA probe. After 30 min of incubation at 37 ºC under 5 % of CO2, medium was discarded, and cells were rinsed twice with PBS and immediately photographed under a fluorescence microscope.
For quantitative analysis of DCF fluorescence, cells attached to 96-well plates were cultured in proliferative conditions for six days. At the end of this period, cells were treated with different CPZ doses for 3, 6, 9, 24, 48, or 72 hours. Medium from each well was removed and replaced with HBSS containing 5 µM H2DCFDA probe. After 30 min of incubation at 37 ºC under 5 % of CO2, medium was discarded, and cells were rinsed twice with PBS. Nuclei were dyed with 5 µM Höechst 33258, and fluorescence was monitored in a plate reader fluorometer (Flexstation 3, Molecular Devices) at 485 nm excitation and 535 nm emission wavelengths. The experiment included six replicates for each condition and was performed three times.
ROS determination by flow cytometry was performed both in proliferative and differentiating cultures. Wild type mice-derived NS were dissociated and seeded in poly-L-lysine-coated 24 well plates. For proliferative cultures, cells were grown in a proliferative medium for 6 days and then treated with CPZ for 24 hours. Culture medium was discarded, and cells were incubated with 5 µM H2DCFDA probe in HBSS for 30 min at 37 º C under 5 % of CO2. Cells were harvested and resuspended in PBS with 5 mM EDTA. Flow cytometry was performed using a Pas III flow cytometer (Partec, Germany). Background fluorescence intensity was determined in control cells in which incubation with the probe was omitted. Analysis of data was performed with FlowJo v10.0.8 software (FlowJo, Ashland, OR, USA) after excluding doublets and dead cells. For differentiating cultures, dissociated cells were cultured for 6 days in proliferative conditions and 6 additional days in differentiating medium. After treatment with CPZ for 24 hours, culture medium was discarded, and cells were incubated with 1 µM H2DCFDA probe in HBSS for 30 min at 37º C under 5% of CO2, and further processed for flow cytometry as for proliferative cultures.
Morphological analysis of oligodendrocytes
To examine morphological complexity of MBP+ mature oligodendrocytes, we compared high magnification images of cultures treated after differentiation with CPZ500 and CPZ0. Analysis was performed with ImageJ RRID:SCR_003070 https://imagej.net/. OL processes were outlined with NeuronJ plugin and Sholl analysis was employed to set a ring grid centered in the cell soma (starting radius: 10 µm; step size: 10 µm; end: 200 µm) as previously reported . The number of process crossings per ring for at least 15 MBP+ cells were determined from each condition. Mean total process crossings per ring was plotted against the distance from OL soma of the corresponding ring.
Images were captured with an Olympus BX50 fluorescence microscope (RRID:SCR_018838) and a DP73 digital camera (Olympus, RRID:SCR_017564) with the CellSens software (RRID:SCR_014551). Cell quantification, and image measurements were performed using Image-Pro Plus (RRID:SCR_007369, http://www.mediacy.com/imageproplus). Low magnification images for determination of NS size were acquired with a Nikon Elipse TE300 inverted microscope and for the total numbers of NS we used a Leica EZ4 stereomicroscope and a Kodak Easy Share Camera.
Statistical analysis was performed by using GraphPad Prism software (RRID:SCR_002798, http://www.graphpad.com/). Statistical tests are indicated in figure legends and significance is represented as ***p<0.001; **p<0.01; *p<0.05, bars without asterisks or indicated as ns correspond to non-significant differences.