Primary Central Nervous System (CNS) Cultures with Mixed Neural Cell Types to Study Correlative Effects of High Glucocorticoids on Astrocytes, Oligodendrocytes, and Myelination Markers

Abstract

Dissociated glial and neuronal precursors from the spinal cord or cerebral cortex of late rat embryos are cultured on top of glass coverslips to ascertain the in vitro effects of high glucocorticoid levels on the process of myelination and on astrocyte markers during postnatal development and determine the dependence of those effects on glucocorticoid receptor activation. The study includes the immunohistochemical processing of culture-carrying coverslips with antibodies to astrocytic, neuronal, oligodendroglial, and myelin proteins to determine changes in those markers after glucocorticoid treatment. In addition, we describe oligodendrocyte–astrocyte cultures from neonatal embryos to determine the effects of high glucocorticoids on the morphology of oligodendrocytes in the absence of developing neurons.

1. Introduction

Stress responses, and their major physiological hallmark, elevated glucocorticoid levels, have been demonstrated to cause morphological and molecular changes in neurons and glial cells. There is also evidence for the regulation of myelin formation or repair by glucocorticoids, while other research shows that the expression of some functionally and structurally relevant astrocytic proteins is influenced by stress or altered glucocorticoid concentrations [1]. During brain development, glucocorticoids further modulate myelin formation and glia differentiation. Here, we describe a method to obtain primary cultures from the spinal cord or cerebral cortex of rats that results in a mixture of astrocytes, neurons, and oligodendrocytes, where myelination of axons by oligodendrocytes and astrocyte differentiation occur following a time course similar to the in vivo myelination process in rodents [2]. The cultures are amenable to determine the effects of glucocorticoid levels and glucocorticoid receptor activation on the myelination process, the concomitant expression of major astrocytic markers such as glial fibrillary acidic protein (GFAP) or connexin 43, and the morphology of oligodendrocytes co-cultured with astrocytes.

2. Materials

2.1. Animals

1. Pregnant Sprague–Dawley or Wistar rats and 1-day-old postnatal offspring were obtained commercially and maintained and euthanized according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Mississippi Medical Center in accordance to the directives of the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals and the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals.

2.2. Dissecting Instruments and Cell Culture Vessels

1. Large scissors and forceps.

2. Small dissecting scissors, 3/4 in. blade.

3. Dissecting scissors: 1 1/2 in. blade.

4. Forceps, straight and curved.

5. Curved microdissecting forceps.

6. Straight microdissecting forceps.

7. Surgical scalpel and blades.

8. A dissecting microscope.

9. Glass coverslips (18 mm in diameter).

10. 12-well cell culture plates.

11. Petri dishes (35 and 90 mm in diameter).

12. Laminar flow hood.

13. CO₂ cell culture incubator.

2.3. Solutions and Cell Culture Media

1. Mixed N2 medium and neurobasal medium (NBM) with B27 supplement (1:1 ratio) is used for the first 2 weeks of culture. Later, insulin-free N2 is used, and its ratio to NBM-B27 is adjusted accordingly (see primary culture section). N2 medium is made in the laboratory, while NBM and B27 are obtained commercially. DMEM/F12.

2. HBSS (with Ca²⁺/Mg²⁺).

3. HBSS (without Ca²⁺/Mg²⁺).

4. Trypsin-EDTA solution.

5. NBM with B27 supplement.

6. N2 medium composition: Dilute stock solutions into DMEM/F12 (with glutamine, without NaHCO₃) to make N2 medium. To make 500 mL N2, add 10 mg N-acetyl-l-cysteine, 2.5 mL transferrin (10 mg/mL), 0.25 mL insulin (10 mg/mL), 0.5 mL sodium selenite (5.2 μg/mL), 0.18 mL hydrocortisone (50 μg/mL), 0.5 mL putrescine (16.1 mg/mL), 0.156 mL progesterone (20 μg/mL), 0.5 mL biotin (10 μg/mL), 5 mL penicillin-streptomycin (100×), 6.6 mL BSA (7.5%), 1.2 g NaHCO₃, and 0.9 g HEPES to 450 mL DMEM/F12. Gently stir with a pipet to mix, and add DMEM/F12 to bring the final volume to 500 mL. Sterilize the resulting medium using a 0.22 μm filter system. Store in 50 mL aliquots at 4 °C.

7. Insulin-free N2: N2 medium without insulin.

8. Soybean trypsin inhibitor solution (SBTI) (see Note 4): Add 25 mg soybean trypsin inhibitor, 2 mg DNase I, and 2 mL BSA (7.5%) to 48 mL DMEM/F12. Mix completely. Pass through a sterile 0.22 μm filter system. Store aliquots at −20 °C.

9. Plating medium: 100 mL horse serum, 40 mL 1× HBSS (containing Ca²⁺/Mg²⁺), and 60 mL DMEM/F12.

10. Poly-L-lysine (PLL) solution (for coating coverslips): Make a 0.1% PLL stock solution, sterilize with a 0.22 μm filter system, and store at 4 °C (~6 months). Dilute this stock solution 50× with ddH20 to make the working solution.

11. Antibody blocking solution: 10% normal goat serum and 0.2% Triton in PBS.

12. Antibody diluting solution: 10% normal goat serum and 0.1% Triton in PBS.

2.4. Drugs

1. Corticosterone.

2. Dexamethasone.

3. Mifepristone.

3. Methods

3.1. Primary Mixed-Cell Type Myelinating Cultures

1. The cultures are established on the top of PLL-coated glass coverslips housed within 12-well culture plates. Coverslips with an 18 mm diameter can be prepared according to our previous methods [2] or can be obtained from various commercial suppliers. Immediately before adding dissociated central nervous system (CNS) cells, individual coated coverslips are carefully placed at the bottom of 22 mm diameter wells of 12-well culture plates.

2. Embryos to prepare CNS cell cultures are obtained from timed-pregnant Sprague–Dawley or Wistar rats at gestation day 16 (embryonic day 16 or E16). Ten to 12 embryos can be obtained normally from a single pregnant rat, resulting in a sufficient number of cells to establish cultures in at least 36 coverslips housed in the corresponding wells of three 12-well culture plates.

3. Embryos are obtained from rats anesthetized with an overdose of isoflurane and immediately euthanized by pneumothorax. Immediately upon euthanasia, the uterus is removed and transferred to a Petri dish containing ice-cold HBSS (with Ca²⁺ and Mg²⁺).

4. Under the hood, embryos are separated from their placentas, extracted, and transferred to another dish containing the same HBSS medium. Then, in each embryo, the cranium and/or the spine are separated from each other. The spinal cord is collected by cutting longitudinally the vertebrae along the spine using fine-tip scissors and carefully removing the split bone without searing the spinal cord. If cerebral cortex tissue is targeted, the cranium is split cutting along its dorsal aspect without damaging the underlying brain. The cortical mantle is then separated, and the frontal lobes (anterior to the optic chiasm) are dissected out. Transfer the blocks of cortex or spinal cord into a dish with fresh HBSS medium.

5. Mince either the spinal cords or the frontal cortex blocks with fine-tip scissors or a surgical blade attached to a scalpel, into smaller pieces, and transfer the fragments to 15 mL conical tubes with 1 mL of 0.1% Trypsin-EDTA solution while gently agitating. Following incubation at 37 °C for 15 min in a water bath, add 2 mL of the SBTI solution, and gently agitate the mixtures until tissue clamps separate.

6. Centrifuge the tube at 300 g for 5 min. Remove the supernatant, and add 2 mL of plating medium to resuspend the remaining tissue.

7. Triturate the tissue by slowly pipetting through a 1 mL fine-tip pipette about 15 times. Most tissue blocks will dissociate into a suspension of single cells. Continue pipetting if chunks of tissue are still conspicuous.

8. Filter the cell suspension through a cell strainer (40 μm) to exclude tissue debris, which will remain on the strainer. To increase yields of dissociated cells, wash the strainer with 1 mL of plating medium.

9. Mix 100 μL of cell suspension with 100 μL of trypan blue to assess the percentage of viable cells in a hemocytometer. A successful cell dissociation process should result in at least 80% of viable cells (trypan blue negative).

10. Adjust cell density to 4 × 10⁵ cells/mL by diluting with the plate medium. Add about 250 μL of the suspension to each coverslip placed at the bottom of the culture plate well. Carefully spread the cell suspension on the top of coverslip without spilling over into the well (see Note 1).

11. Carefully bring the culture plates to the CO₂ incubator (avoid cells spilling out of the coverslips), and wait for 2 h to allow for strong attachment of the cells to the coverslip surface.

12. In the hood, add 750 μL pre-warmed N2/NBM-B27 medium (mixture of N2 and NBM-B27 at 1:1) with growth factors (NGF 50 ng/mL and NT3 10 ng/mL, used only for the first 10 days of culture to support neuronal development) to each culture coverslip, slowly delivering the medium at the wall of the culture well to avoid disturbing the recently seeded cells (see Note 2).

13. After returning to the incubator, refresh the medium every 4 days by removing about 2/3 of the old medium with the same amount of fresh medium.

14. Cultures are maintained to the desired length of time depending on the goals of the research. For example, first, myelin sheaths, as determined by co-localization of MBP with axons (visualized by MBP and pNF double-immunolabeling), appear after 10 days in vitro, but markers of differentiating astrocytes and oligodendrocyte appear earlier. By 28 days in culture, well-developed MBP immunoreactive myelin sheaths (co-labeled with pNF) can be easily detected under the fluorescence microscope.

3.2. Treatment of Myelinating Cultures with Corticosteroids and Mifepristone

1. Starting at day 12 in vitro (DIV), coverslips are divided into six groups (each group with at least three coverslips). The medium is refreshed every 4 days and contains either of the following compounds: 5 μM corticosterone (group 1), 50 μM corticosterone (group 2), 50 μM dexamethasone (group 3), 10 μM mifepristone (group 4), 50 μM corticosterone plus 10 μM mifepristone (group 5), or vehicle (DMSO 1%; group 6, control). Before use, all these additional compounds are previously dissolved in 100% DMSO and further diluted to a 1% DMSO to be used in cultures at the mentioned concentrations. The control group of coverslips only receives refresh medium containing the 1% DMSO vehicle. This schedule of refresh medium application is partly based on procedures described in Mulholland et al. [3].

2. After 28 DIV, the coverslips, while still at the bottom of the culture plate wells, are rinsed with PBS (carefully removing and replacing solutions down the wall of the well) and fixed for 10 min with a 4% paraformaldehyde PBS solution at room temperature.

3. After washing with PBS, coverslips containing the fixed cells are carefully removed from the bottom of the wells (see Note 3) and placed preferably on the top of tube caps with flat endings and a diameter smaller than the coverslip itself (the caps are previously glued to the bottom of a box by one of the endings, while the free ending supports the coverslip as a platform) as described in Pang et al. [2].

4. The coverslips are immediately subjected to the various steps of fluorescence immunohistochemical staining, carefully applying and decanting solutions as per the specific procedure described below.

3.3. Immuno-fluorescent Labeling of Cell Cultures

1. Coverslips with fixed cells are washed with PBS three times (5 min each time) while in the wells of culture plates and then carefully removed from the bottom of the well using curved-tip tweezers. Each coverslip is placed on the top of one of the tube caps glued to the inside of a slide box as described elsewhere [2].

2. Block nonspecific binding with antibody blocking solution for 30 min at room temperature. In this and the following steps with solutions containing Triton-X, it is necessary to pay attention to the concentration of Triton-X that best allows for detection of myelin proteins (see Note 4).

3. Apply primary antibodies (Table 1) diluted in the preincubation (blocking) solution. If performing simultaneous labeling of two or more different antigens, use antibodies raised in different host species diluted in antibody preincubation solution. A total of 150 μL of antibody solution is sufficient to cover one coverslip. Incubate for 2 h at room temperature.

4. Wash three times with PBS.

5. Apply secondary antibodies. Fluorescently tagged secondary antibodies that specifically bind the primary antibodies (based on the animal species of the primary antibody hosts) are then diluted in blocking solution and applied to the coverslips, typically at a 1:200 dilution for 30–60 min.

6. Decant secondary antibody solution, and wash three times with PBS; drain excessive PBS by touching the edge of the coverslip against a stack of water-absorbent tissue.

7. Let the coverslips air dry.

8. As soon as the coverslips dry, apply a drop of mounting medium on a clean glass slide, and mount the coverslip on the slide being mindful that the fixed culture faces the drop of mounting medium on the slide. If visualizing cell nuclei is required, use a mounting medium containing DAPI.

9. Once mounted, the immunofluorescently labeled cells in the coverslip cultures can be best viewed at a laser confocal microscope. At the microscope, the ability of using different filter sets to detect fluorescent light of different wavelengths will allow to visualize different labels derived from secondary antibodies with different fluorophores. Since microscopes allow taking micrographs from light emitted at a specific wavelength (or channel), pictures of each channel provide an opportunity to determine/quantify the numbers or morphology of cells (or their parts) containing a specific molecular marker. In addition, the ability to merge channels imaged from a specific location allows to establish with higher confidence the identity of particular structures and quantify them (see Figs. 1 and 2 and Note 5).

10. The intensity of fluorescent signal registered in the eventual micrograph taken at the microscope depends on the nature of the label and the amount of label bound to cells. It also depends on the intensity of the laser beam directed to the tissue and the gain at the photomultiplier detector capturing the light reflected from the tissue. Thus, to ensure that the intensity and tissue extent of label in images from a particular experiment genuinely represent the actual presence of labeled structures, the laser intensity and detector gain for each wavelength channel must be uniform for all pictures taken from coverslips in the experiment.

11. Micrographs taken at the experiment then can be analyzed with software packages for image analysis, such as NIH-supported ImageJ. These applications allow to quantify intensity of labeling, number of labeled structures, and geometric parameters of labeled structures such as volume or area fraction occupied by them.

Table 1.

List of antibodies used to label specific glial and neuronal proteins in the cultures

Directed against	Provider	Catalog no.	Dilution	Host
Myelin basic protein (MBP)	Millipore	#AB9348	1:200	Chicken
Phosphorylated neurofilament (pNF)	BioLegend	#801602	1:1000	Mouse
Connexin 43 (Cx43)	BD Biosciences	#610062	1:300	Mouse
2′,3′-Cyclic nucleotide 3′-phosphohydrolase (CNPase)	Millipore	#MAB1580	1:1000	Mouse
Glial fibrillary acidic protein (GFAP)	Millipore	#AB5804	1:1000	Rabbit

Fig. 1.

Micrographs taken at a laser confocal microscope from a mixed culture from the cerebral cortex after 28 days in vitro. The cultures originated from a rat embryo collected at embryonic day 16. The immunofluorescent signals (green and red) correspond to antibodies to myelin basic protein (MBP, green) and axon marker phosphorylated neurofilament (pNF, red). Note the colocalization of MBP immunosignal along pNF-immunoreactive axon bundles (arrows) indicating myelinated axonal segments. DAPI is a fluorescent agent that binds to chromatin in the cell nucleus. The merged panels are overlays of MBP, pNF, and DAPI micrographs. The four panels to the left correspond to the same optic field from a control culture (CONT) viewed with different fluorescence filters to detect MBP (a), pNF (b), and DAPI (c), with those three channels merged in (d). The two panels to the right are merged pictures from cultures treated either with corticosterone (e, CORT) or corticosterone+mifepristone (f, CORT+MFPS). The 50 micrometer scale bar shown in A applies to all 6 micrographs

Fig. 2.

Micrographs at a laser confocal microscope from three different primary mixed cultures from the cerebral cortex after 28 days in vitro. One of the cultures was treated only with vehicle (left panel), another with 50 μM corticosterone (CORT, middle panel), and the third one with 50 μM CORT and 10 μM mifepristone (CORT+MFSP, right panel). The culture originated from a rat embryo collected at embryonic day 16. The immunofluorescent signals correspond to antibodies to astrocyte marker GFAP (red) and Connexin 43 (Cx43, green). Note that CORT causes a drastically lower amount of Cx43 immunoreactive granules (middle panel) that is largely prevented by glucocorticoid receptor antagonist mifepristone (CORT+MFPS). DAPI (blue) is a fluorescent agent that binds to chromatin in the cell nucleus

3.4. Mixed Oligodendrocyte–Astrocyte Cultures to Study Oligodendrocytes’ Response to High Corticosteroids

1. Mixed oligodendrocyte–astrocyte cultures allow to identify oligodendrocytes differentiated in cultures in the absence of neurons (Fig. 3). The cultures are originated from the cerebral cortex of postnatal day 1 rats using procedures similar to those described previously [4, 5], while immunofluorescence labeling is performed as described above. Specific details to prepare the cultures from the neonatal frontal cortex follow here.

2. The frontal cortices are separated from the brain and transferred to L15 medium. The meninges are separated and discarded, and the frontal cortical lobes are excised out, collected, minced, digested with trypsin-EDTA, and centrifuged as above to obtain a suspension of cells. Dissociated cells are cultured in DMEM with10% FBS in 75 mL flasks.

3. Cells are kept in culture until they reach confluency (about 10 days).

4. Then, culture flasks are shaken at 200 rpm at 37 °C overnight. The shaking results in mostly astrocyte and oligodendrocyte precursors remaining attached to the flask bottom, while other cell types go into suspension in the supernatant and can be removed.

5. The supernatant is then replaced with fresh medium, and the cells in the flask are cultured for at least 1 week. Cells in this culture are then dissociated with trypsin and plated on poly-L-lysine-coated glass coverslips to an approximate density of 1.5 × 10⁵ cells per coverslip.

6. The mixture of cells on coverslips is maintained for 10 days and then treated once with either CORT5, CORT50, DEX50, or vehicle as described above and cultured for 4 days before fixing them with 4% PFA for immunofluorescence processing.

7. Oligodendrocytes and their processes are immunofluorescently identified using specific antibodies for MBP and CNPase. Under a laser scanning confocal microscope, micrographs of immunostained cells are obtained using the same laser intensity and fluorescence detector gain across samples as explained above.

8. To determine whether the extent and branching of processes and membrane expansions of cultured oligodendrocytes are affected by corticosterone or dexamethasone, the images of individual oligodendrocytes are analyzed using Sholl analysis as implemented in the Fiji version of the ImageJ image analysis software [6]. The analysis involves the overlaying of a series of concentric circles centered on the cell body, covering all visible fluorescently labeled cell processes. For the Sholl analysis, we have typically used circles with a radius difference between adjacent circles of 3 μm. The sampling consisted in all visible oligodendrocytes within at least 27 pictures (all under the 100× objective) per coverslip in 3 coverslips with cell culture.

Fig. 3.

Micrographs taken at a laser confocal microscope directed to three different oligodendrocyte–astrocyte cultures from the cerebral cortex. The cultures were originated from postnatal day 1 rats. Cultures were treated with vehicle only (left panel), 50 μM corticosterone (CORT 50, middle panel), and 50 μM of the corticosteroid analog dexamethasone (DEX 50). The immunofluorescent signal corresponds to antibodies to oligodendrocyte marker CNPase (green). Note that CORT and DEX cause significant shortening of cell processes from CNPase expressing oligodendrocytes as compared to the control cultures (left panel). DAPI (blue) is a fluorescent agent that binds to chromatin in the cell nucleus. The100 micrometer scale bar shown in the left panel applies to all 3 micrographs

4. Notes

1. When seeding the cell suspension on the coverslips at the bottom of the culture wells, particular attention must be paid to add the right volume of cell suspension (250 μL is sufficient), and do it while avoiding spilling the suspension out of the coverslip edge, because during 2 h, the cells will attach to any surface, and it is convenient that only the upper surface of the coverslip contains the attached culture cells when the additional culturing and immunohistochemistry steps are applied. The goal is to ensure a consistent starting cell density in all coverslips and preventing that cells attach to the lower surface of the coverslips or the sides of the well. This is less of a concern when the cells have already attached to the coverslip’s upper surface.

2. Throughout the cell culture procedures, while the slides carrying seeded cells are at the bottom of the wells, it is of the utmost importance that washes and delivery of solutions are made with the transfer pipettes tips applied to the walls of the well without touching or disturbing the cultured cells, always striving that the flow of liquids slowly and smoothly happens at the wall itself and not on top of the cell culture. If solutions are directly delivered to or aspired from the middle of the slide, turbulence and liquid eddies may disturb or damage the cultured cells.

3. When lifting the coverslips containing paraformaldehyde-fixed cultures from the bottom of the culture wells, maximum attention must be paid to only hold the slides by their edge with very fine-tip curved tweezers. Once transferred to the top of the plastic bottle cups, access to washing solutions or solutions with antibodies and/or fluorophores is straightforward, because after incubating with a solution, removal of it is easy by decantation. However, it is still important to add slowly solutions to the culture-containing side of the coverslips.

4. Binding of specific antibodies to MBP and other components of myelin can be impeded by the dense packing of the myelin membranes, so it is important for immunohistochemical demonstration of MBP to assess whether, in the particular preparation being processed, higher concentrations of Triton-X (up to 0.6% in PBS) and/or longer incubation times might be necessary, always being mindful that an excess of Triton-X may promote detachment.

5. As indicated, the extent of myelination in the mixed cultures was assessed at 28 DIV. At this stage, individual axons and some well-defined axon bundles that are labeled for both MAP and pNF are easily distinguishable, indicating the presence of myelinated axons. If MAP antibody is tagged using secondary antibodies carrying a fluorophore emitting green fluorescence and pNF is tagged to emit red fluorescence, then their coexistence in myelinated axons appears as a yellow fluorescence signal. Due to the simplicity, we chose to assess myelination by measuring the length of all yellow axon segments in a micrograph in relation either to the cell number or the total length of pNF (red) labeling within the micrograph. Later in culture period (beyond 28 DIV), many axons may become additionally myelinated and form a mesh in which length is more difficult to assess. In this case, the extent of co-labeled structures can be assessed with an image analysis system such as ImageJ [7] that can segment away the yellow portions of the micrographs, and the surface area of yellow labeled structures can be expressed in relation to the number of cells or the surface of pNF-labeled structures to calculate a myelination index.