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

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue and results in neuronal loss. Numerous evidences suggest that the interaction between glia and urinal cells exert the pressure effects after an ischemic event. In order to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment.

Here we present a simple approach to isolate astrocytes and neurons from rat embryonic cortex, and that by using specific cultural medium allows the establishment of neuron or astrocyte-enriched cultures or neo-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 cover slips are maintained in contact with a monolayer of astrocytes plated in multi-well plates. The two cultures are maintained a part by small paraffin spheres.

This approach allows independent manipulation and the application of specific treatment 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.

Start with rat embryonic cortex insulation. The embryos were obtained from a family of rats at 15 days of gestation and are placed in a sterile Falcon tube containing PBS. Still inside the yolk sachet embryos are placed inside a petri dish containing cold PBS.

With the help of scissors and tweezers, the yolk sac is broken, and the embryo is removed and placed in another petri dish containing cold PBS over an ice pack. It is necessary to be very careful when breaking the yolk sac not to damage the embryo. Position the embryo under a dissecting microscope, gently fix the embryo using a twizzer.

The initial incision should be parallel to the cortex. Be careful not to decapitate the animal. The scalp and are carefully removed not to damage the cortical brain tissue.

This next incision separates the cortex. Remove the blood vessels present in the tissue. Finally, using a pipette, transfer the cortical tissue to a Falcon tube with PBS.

The single cell suspension is obtained through mechanical digestion of the cortical brain tissue using tips with decreasing diameter. Make sure that the tissue is well homogenized After the digestion, centrifuge the material at 400 Gs for three minutes. Discard the supernatant, and resuspend the sediment in culture medium, previously warmed to 37 degrees.

Calculate the total number of cells present in the suspension using a Neubauer chamber and preparate the dilution for the adequate cell density. Finally, seed the cells in the multi-well and incubate at 37 degrees. In order to prepare the material for the co-culture system, heat the paraffin in a heating block until it becomes liquid.

Then, with the help of a stereo glass Pasteur pipette prepare small spheres over the cover slips that were previously placed in a multi-well, and coated with Poly-D-Lysine. 24 hours before the two cultures are brought in contact, change the culture medium of neurons and astrocytes to supplement it NBM with or without heat inactivated FBS. When the two cultures are ready to use, transfer the neuron, seated in the cover slips with paraffin spheres to wells containing astrocytes using a tweezers previously immersed in 70%ethanol.

After placing both cell types in contact, wait eight to 12 hours, and then start the different stimuli and procedures. The oxygen and glucose deprivation is performed in a culture with seven days of growth. Remove the culture medium, and wash the cells two times with HBSS medium without glucose supplementation.

Be sure that all the medium containing glucose is washed. Seal the Hypoxia Chamber and add the gas mix containing 95%nitrogen and 5%carbon dioxide for four minute with a flow of 20 liters for minutes in order to replace the oxygen present inside the chamber. Then, stop the flow and place the Hypoxia Chamber in an incubator at 37 degrees.

After the period of oxygen and glucose deprivation, replace the medium, HBSS without glucose supplementation with the appropriate culture medium for the remaining procedures and incubate the cells at 37 degrees. In order to characterize the type of cortical culture, we performed immunohistochemistry in the three types of cortical cultures to assess the number of cells that expressed GFPA or MAP2, which are markers widely used for astrocytic and neuronal cells respectively. These analysis revealed that when this protocol, we were able to obtain a pure enriched culture of astrocytes with about 97%of the cells expressing GFAP.

Regarding the neuron-enriched culture, we verified about 78%of the cells expressing MAP2. However we identify about 18%of GFAP and MAP2 negative cells. For the neuron-glia cortical culture, It was observed that about 49%of the cells are MAP2 positive.

31%are GFAP positive. And 20%they're not liable for GFAP nor MAP2. Seven days after the cortical culture establishment, the neuron-glia culture and the neuron-enriched culture were subjected to an OGD procedure for 4 and 6 hours.

After this procedure, cells were labeled with MAP2 and then the number of MAP2 positive cells were quantified using fluorescence microscopy. In neuron-glia culture, it was observed a decrease of about 30%in the number of the neurons, when the culture was submitted to an OGD period of 4 hours. After an OGD period of 6 hours, the extension of the lesion increases, reaching about 60%of neuronal loss.

Regarding the enriched culture of neurons exposed to OGD, it was observed about 41%and 64%decrease in the number of MAP2 cells. Moreover, it was observed that in the neuron-enriched culture, there was a slight increase in the injury extension when compared to the neuron-glia culture also exposed to OGD during 4 hours. In conclusion, here represent an in vitro model to study ischemic stroke established in a simple, fast and expensive and reproducible way.

Additionally, the method described also allows to implement neurons and astrocyte-enriched primary cultures, but also a neuron-glia culture. Thus, providing a great in-vitro model for modeling several brain diseases with a higher level of complexity, than immortalized cell lines and pure neuronal or glial cultures.