A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Summary

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

Abstract

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.

To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.

To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Introduction

According to data from the World Health Organization, about 5.5 million people die every year from ischemic stroke¹. This condition is characterized by the interruption of blood flow to a certain brain region, resulting in a reversible or irreversible loss in the supply of oxygen and nutrients to the tissue, which alters tissue function and leads to mitochondrial dysfunction, calcium dysregulation, glutamate excitotoxicity, inflammation and cell loss^2,3.

Apart from vascular cells, neuronal and glial cells are involved in the pathophysiology of the ischemic stroke⁴. In particular, astrocytes are essential to the maintenance of neurons and recently were shown to play a critical role in the response to the ischemic lesion⁵. This type of glial cell performs functions of structural support, defence against oxidative stress, synthesis of neurotransmitters, stabilization of cell-cell communication, among others⁶. Along with neurons, astrocytes play a direct role in synaptic transmission, regulating the release of molecules such as adenosine triphosphate, gamma-aminobutyric acid and glutamate⁷. Part of the injury induced by ischemia is caused by the excessive release of glutamate and its accumulation at the synaptic cleft, leading to the overactivation of N-methyl-D-aspartate receptors, activating downstream signalling cascades, ultimately resulting in excitotoxicity⁸. Since astrocytes are able to remove glutamate from the synaptic cleft and convert it into glutamine, they are crucial in defending against excitotoxicity, thereby exerting a neuroprotective effect on ischemia. These cells also play a role in ischemia-induced neuroinflammation. After the ischemic insult, activated astrocytes undergo morphologic changes (hypertrophy), proliferate, and show an increase in glial fibrillary acidic protein (GFAP) expression. They can become reactive (astrogliosis), releasing pro-inflammatory cytokines such as tumour necrosis factor-α, interleukin-1α and interleukin-1β, and producing free radicals, including nitric oxide and superoxide, which in turn can induce neuronal death^9,10. In contrast, reactive astrocytes may also play a neuroprotective effect, since they release anti-inflammatory cytokines, such as transforming growth factor-β, that is upregulated after stroke¹¹. Moreover, they can generate a glial scar, which can limit tissue regeneration by inhibiting axonal sprouting; however, this glial scar can isolate the injury site from viable tissue, thus preventing a cascading wave of uncontrolled tissue damage^12,13.

Thus, it is imperative to establish models that allow studying neuron-glia interactions under an ischemic injury in order to find therapeutic strategies that limit or reverse the effects of ischemic injury. Compared to other models used to study ischemic injury, namely in vivo models^14,15,16, organotypic cultures^17,18,19 and acute brain slices^20,21,22, primary cell cultures are less complex, which makes possible the study of individual contributions of each cell type in the pathophysiology of ischemic stroke and how each cell type responds to possible therapeutic targets. Typically, in order to study the interactions between neuron-enriched cultures and astrocyte-enriched cultures, neurons and glial cells of postnatal origin are used^23,24, or postnatal glial cells and embryonic neurons^25,26. Herein is proposed a simple approach to establish neuron- or astrocyte-enriched cultures and neuron-glia cultures from the same tissue. These primary cells are obtained from rat embryonic cortex, a region frequently affected by stroke^27,28. Moreover, the dissociation of the tissue is performed only by a mechanical procedure. Therefore, this protocol allows isolating cells in the same stage of development, in a fast and inexpensive way, and with high performance and reproducibility.

The crosstalk between astrocytes and neurons can be explored using a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes seeded in multiwell plates. Small paraffin spheres can be used to ensure the separation of the two cell cultures. This approach allows independent manipulation of each cell type before they are brought into contact. For example, it is possible to silence a specific gene in astrocytes and see how it can influence the neuronal vulnerability or protection against ischemic-induced damage. An established method to induce ischemic-like conditions in vitro is oxygen and glucose deprivation (OGD)³, which consists in replacing the regular atmosphere (95% air and 5% CO₂) by an anoxic atmosphere (95% N₂ and 5% CO₂), associated with the omission of glucose.

The method described is suitable for studying the interactions between neurons and astrocytes in the context of ischemic stroke, in a simple, fast, reproducible and inexpensive way.

Protocol

All animals used were bred at the CICS-UBI Health Science Research Centre in accordance with the national ethical requirements for animal research and with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Directive 2010/63/EU).

1. Rat embryo cortex primary cell culture

1. Culture medium preparation
   1. Prepare the Neurobasal Medium (NBM) by adding the following supplements: 2% B27, 0.5 mM glutamine, 25 µM glutamate and 120 µg/mL gentamicin. Homogenize, adjust the pH to 7.2 and sterilize the medium with a vacuum filtration step, using a 0.22 µm filter. For the neuron-glia cell culture, supplement the NBM cell culture medium with 10% of Heat-Inactivated Fetal Bovine Serum (HI-FBS).
   2. Prepare the Minimum Essential Medium Eagle (MEM) medium with the following supplements: sodium hydrogen carbonate 2.2 g/L, insulin from bovine pancreas 5 mg/L, D-glucose anhydrous 3.4 g/L, penicillin (12 U/mL) /streptomycin (12 µg/mL) and 10% HI-FBS. Homogenize, adjust the pH to 7.2 and sterilize the medium with a filtration step.
2. Preparation of materials and equipment
   1. Puncture the terminal part of a 1 mL plastic micropipette tip from side to side, using a needle with a specific diameter (0.5 mm for S; 0.6 mm for M; 0.8 mm for L) and seal the opening of the tip using a flame.
   2. Sterilize all the glassware. Throughout the dissection keep the tools used (e.g., scissors, tweezers, scalpel) immersed in 70% ethanol. Spray the materials with 70% ethanol before entering them in the laminar flow chamber.
   3. Set the water bath temperature to 37 °C and place the cell culture medium in the water bath before starting the procedure.
      NOTE: For immunocytochemistry assays, poly-D-lysine coating should be performed in multiwell plates containing coverslips.
3. Rat embryonic cortex culture
   1. Remove the rat embryos from a female Wistar rat with 15-16 days of gestation (the end of the mating, which should last 24 h, is considered the 1^st day of the embryonic development). For that purpose, anaesthetize the female with ketamine (87.5 mg/kg) and xylazine (12 mg/kg) and remove the embryos. Then euthanize the female rat by cervical dislocation, following standard protocol.
   2. Place the embryos in a 50 mL sterile tube, add phosphate buffer saline (PBS) until it covers the embryos and quickly take them to the culture room.
   3. Still inside the yolk sac, place the embryos in a Petri dish containing 25 mL of cold PBS. With the help of scissors and tweezers, break the yolk sac, remove the embryo and transfer it to another Petri dish containing also cold PBS. The PBS in the Petri dish should be enough to cover the entire embryo.
      NOTE: Be careful when opening the yolk sac to avoid damaging the embryo. In 1.3.3 and 1.3.4 we used 90 mm diameter Petri dishes placed on top of ice packs covered with absorbent paper to keep the PBS at low temperature.
   4. For dissection of the embryo, transfer it to another Petri dish containing 30 mL of cold PBS. Place the embryo under a dissecting microscope and immobilize it using a tweezer. Make the initial incision parallel to the cortex, going from the ocular cavity to the end of the muzzle and be careful not to decapitate the animal.
   5. Carefully remove the scalp and the meninges using tweezers, in order not to damage the cortical brain tissue. Make the next incision to separate the cortex. Transfer the cortical tissue to a 15 mL tube containing 5 mL of PBS using a Pasteur pipette.
   6. Perform the mechanical digestion of the cortical brain tissue using the 1 mL plastic tips prepared in 1.2.1. Triturate 10 times with a regular pipette and repeat the process using pipettes with progressively smaller holes (L, M and S), until the chunks have fallen apart.
   7. After the mechanical digestion, centrifuge the suspension at 400 x g for 3 min. Discard the supernatant and resuspend the sediment with the appropriate cell culture medium previously warmed at 37 °C.
   8. Determine the total number of cells present in the cell suspension (cell density) using a Neubauer chamber, make the appropriate dilutions and plate the cells. The initial cell density was defined based on a previous study⁵.
      1. For a neuron-enriched culture, use 0.21 x 10⁶ cells/cm² as the initial cell density and maintain the cells in NBM culture medium without HI-FBS.
      2. For the neuron-glia cultures use 0.14 x 10⁶ cells/cm² as the initial cell density and maintain the cells in NBM culture medium supplemented with 10% HI-FBS.
      3. For an astrocyte-enriched culture, use 0.26 x 10⁶ cells/cm² as the initial cell density and maintain the cells in MEM supplemented as previously indicated.
      4. Place the cells in an incubator set at 37 °C, 95% O₂ and 5% CO₂.
         NOTE: For longer culture periods, an anti-mitotic such as 27 μM 5-fluoro-2′-deoxyuridine with 68 μM uridine should be added to suppress cell growth.

2. Co-culture system

1. Preparation of materials
   1. Heat the paraffin at 150 °C in a heating block for approximately 7 min. Keep at 150 °C until finishing the procedure. Then, with the help of a 1 mm diameter sterile glass Pasteur pipette, add a small drop over sterilized and PDL-coated coverslips. The paraffin spheres are irregular, but they have approximately 2 mm diameter. The spheres will allow the two cultures to be separated by approximately 1.25 mm.
   2. As the paraffin is not sterile, place the multiwell with the paraffin spheres under ultraviolet radiation for 15 min.
   3. To establish the co-culture, transfer the coverslip with the paraffin spheres using a tweezer previously immersed in 70% ethanol for 15 min.
2. Co-culture
   1. When the two cultures are ready to use (i.e., after 7 days in culture under the conditions mentioned in step 1.3.8), transfer the neurons seeded in the coverslips with paraffin spheres to the wells containing the astrocytes.
   2. 24 h before the two cultures are brought in contact, change the culture medium of neurons and astrocytes to NBM supplemented, or not, with HI-FBS, depending on the purpose of the experiment.
   3. After placing both cell types in contact, wait 8-12 h before starting the different stimuli and procedures.
      NOTE: The schematic representation of the co-culture system is shown in Figure 1.

3. Oxygen and glucose deprivation

1. Culture Medium Preparation
   1. For the OGD experiments, use Hank's Balanced Salt Solution (HBSS). Prepare the HBSS medium with the following reagents: 1.26 mM CaCl₂, 5.36 mM KCl, 0.44 mM KH₂PO₄, 0.49 mM MgCl₂, 139.9 mM NaCl, 4.17 mM NaHCO₃, 3.38 mM Na₂HPO₄. Homogenize and adjust the pH to 7.2. Sterilize the medium by filtration.
      NOTE: If appropriate, supplement the HBSS solution with glucose (HBSSglu+), by adding 5.56 mM glucose. For cultures submitted to OGD do not supplement the HBSS medium with glucose (HBSSglu-).
2. OGD procedure
   1. 7 days after seeding the cells, remove the culture medium and wash two times with HBSSglu-. After washing add the HBSSglu- cell culture medium and place the multiwell in the hypoxia chamber.
   2. Seal the hypoxia chamber and add a gas mix containing 95% N₂/5% CO₂ for 4 min with a flow of 20 L/min to remove the oxygen present inside the chamber. After this, stop the flow and place the hypoxia chamber in an incubator at 37 °C for 4 h or 6 h, depending on the extent of the intended ischemia.
   3. After the period of OGD, replace the HBSSglu- medium with the appropriate culture medium for the remaining procedures.
      NOTE: The OGD experiment aims to simulate the in vitro conditions that the cells suffer during an ischemic event, so it is important to certify that all the media used previously is removed.

4. Immunocytochemistry assay

NOTE: Perform the immunocytochemistry assay as previously described⁵.

1. Briefly, to characterize the different cortical cultures, incubate the cells overnight at 4 °C with rabbit anti-GFAP (1:2000) and mouse anti-microtubule-associated protein 2 (MAP2; 1:500); and then 1 h at room temperature with the following secondary antibodies: anti-rabbit conjugated to Alexa Fluor 546 and anti-mouse conjugated to Alexa Fluor 488, both at 1:1000 dilution.
2. Label the cell nuclei by incubation with 2 µM Hoechst 33342 for 10 min at room temperature.
3. Mount coverslips in fluorescence mounting medium and acquire images on an epifluorescence microscope with a 63x magnification.

5. Statistical analysis

1. Express data as percentage of the total number of cells or as a percentage of control and presented as the mean ± standard error of the mean (SEM) of at least 3 independent experiments performed in triplicate.
2. Perform statistical analysis with software (GraphPad Software Inc., San Diego, CA), using the unpaired Student’s t test. The results were considered significant when values of p < 0.05.

Results

To characterize the cultures, immunocytochemistry to assess the number of cells that expressed GFAP or MAP2, widely used markers of astrocytes and neurons (Figure 2), was performed in each type of cortical culture. This analysis revealed that astrocyte-enriched cultures presented 97% of the cells expressing GFAP (Figure 2A). Regarding the neuron-enriched culture 78% of the cells expressed MAP2, 4% of the cells expressed GFAP, and 18% of the cells were both GFAP ...

Discussion

The method here described consists of the astrocyte and neuron isolation from rat embryonic cortical tissue, allowing the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures. It was adapted from a previous study of our group⁵, where the cortical neuron–glia and neuron-enriched embryonic cultures isolation were described and the two cultures characterized. Using these cultures, Roque et al. found that astrocytes play a key role in responding to an ischemic damage a...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24 -well culture plates	Thermo Fischer Scientific	142475
95% N2/5% CO2 gas cylinder	ArLíquido
Anti-mouse conjugated to Alexa Fluor 488	Invitrogen	A11001	1/1000 dilution; incubation period - 1 h at room temperature
Anti-rabbit conjugated to Alexa Fluor 546	Invitrogen	A11010	1/1000 dilution; incubation period - 1 h at room temperature
B27 supplement (50x)	Gibco	17504-044
Dako Fluorescence Mounting Medium	Dako	S3023
D-glucose anhydrous	Fisher Scientific	G/0450/60	3.4 g/L
Epifluorescence microscope	Zeiss	AxioObserver Z1x	63x objective
Fetal Bovine Serum (FBS)	Biochrom	S0615	10%
Gentamicin	Sigma-Aldrich	G1272	120 µg/mL
Glutamate	Sigma-Aldrich	G8415	25µM
Glutamine	Sigma-Aldrich	G3126	0.5 mM
Hoechst 33342	Invitrogen	H1399	2 µM; incubation period - 10 min at room temperature
Hypoxia incubation chamber	Stemcell Technologies	27310	Chamber used for OGD induction
Insulin from bovine pancreas	Sigma-Aldrich	I5500	5 mg/L
Ketamine	Sigma-Aldrich	K-002	87.5 mg/Kg
Minimum Essential Medium Eagle medium	Sigma-Aldrich	M0268	warm up to 37 °C before use
Mouse Anti-MAP2	Santa Cruz Biotechnology	Sc-74421	1/500 dilution; incubation period overnight at 4 °C
Neurobasal medium	Gibco	21103-049	warm up to 37 °C before use
Paraffin pastilles for histology	Sigma-Aldrich	1.07164	Solidification point 56-58°C
Paraformaldehyde	Sigma -Aldrich	P6148	4% in PBS
Penicilin/Streptomycin	Biochrom	A 2213	penicillin (12U/mL) /streptomycin (12µg/mL)
Poly-D-lysine	Sigma-Aldrich	P1024
Rabbit Anti-GFAP	DAKO	Z0334	1/2000 dilution; incubation period overnight at 4 °C
Sodium hydrogen carbonate	Fisher Scientific	S/4240/60	2.2g/L
Xylazine	Sigma-Aldrich	X1126	12 mg/Kg