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 st....

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 µ....

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