Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

The following protocol is intended to show the isolation of a primary astrocytes from brains of pup rats to perform oxygen glucose deprivation or OGD. Exposure of purified astrocytes, to oxygen glucose deprived environment is a module that allows us to test the reaction to some of the conditions found in ischemic strokes. Methodical changes, after exposure to oxygen glucose deprivation can be observed and measured using selective astrocyte markers such as GFAP.

In summary, these isolated cells exposed to ischemic-like conditions can be used to test new part ways activated in hypoxic conditions and other drugs that protect against ischemic stroke. In this part of the protocol astrocytes will be isolated from pup rat cortices. The purpose of this procedure is to obtain a primary astrocytic cell culture to perform oxygen glucose deprivation experiments.

The materials for this procedure are the following, Autoclave all materials before using them in this procedure. And expose hood to at least 20 minutes of UV lights before starting the protocol. To begin this procedure, pipet three to five milliliters of complete DME media in each plate.

It will be divided as following, one for each mouse brain, two for all of the cortices, and three will be for all of the cortices without the meninges. Decapitate a pup after disinfecting with 70%ethanol. Make an incision starting from the back of the head towards the nose.

Remove skull with curved forcep and gently scrape out the cerebellum exposing the cortices. Remove the exposed brains with a pair of tweezers and place the brain in the first petri dish with media. Repeat these steps for all of the other pups.

Separate the cerebral hemispheres by gently dividing along the midline fissure with the sharp end of the micro dissecting forceps. Peel the cortices by removing the white matter and hippocampus using the tweezers with a pinching movement, leaving them behind. Transfer the peel cortices to the petri dish labeled number two.

Repeat the procedure for all the brains combining all the dissected cortices in the petri dish. Gently shake the dish to equally distribute the media between all the cortices. Use the dissection microscope to identify the meninges lining the peeled cortices.

Meninges are a very thin transparent layer that can be identified by searching for the small red capillaries surrounding the tissue. Carefully inspect and remove the meninges lining the cortices and gently peel them off from the individual cortical lobes using fine tweezers on both sides of each cortex. Place the clean cortices into a new petri dish labeled number three.

For tissue dissociation using the stomach or blender method, pour the clean, peeled cortices and the media from the petri dish into a sterile blender bag. And add enough media to bring the total volume of the bag to five milliliters. Place the bag into the stomacher 80 leaving approximately two centimeters of the bag visible above the closed door.

The cell dissociation in this instrument is done after 2.5 minutes at high speed. Alternatively, tissue dissociation can be made by closing the sterile bag and gently pounding the tissue with a beaker in the hood until tissue particles have become a fine suspension. Make sure to use a cloth in between bag and beaker to avoid friction and breaking the bag.

Pour the cell suspension into the center of sleeve with a mesh number 60. Pour the filtered cells into the middle of a mesh number 100 allowing it to filter by gravity. Pour the filtered cell suspension into a conical 15 milliliter tube.

Center fuge the cell suspension at 200 times G in a center fuge preferably with a swing bucket router for five minutes. Remove the supernatant and discard in a beaker. Add complete fresh media to the cell palette.

Using a serological pipet resuspend and dissociate the palette of cells in a maximum of five milliliter of medium. Add the desired amount of cell suspension on a flask and complete with media. Add the resuspended palette into the flask with medium.

Distribute cells evenly throughout the flask and place them in an incubator at 37 degrees Celsius with 5%carbon dioxide. Remember to change the media every three days. The following methodology is intended to deprive cells of oxygen and glucose in a controlled environment.

The purpose of this model is to study the response of astrocytes in an ischemic-like stroke condition. The materials used for this procedure are, Purge the glucose-free medium by inserting a serological pipet connected to the gas system. Bubble the solution with the gas mixture for 10 minutes at a flow of 10 to 15 litters per minute.

After the 10 minutes are done, adjust the pH of the OGD medium to 7.4. Filter the purged glucose-free medium using the 50 milliliter tube filter system. It is important to always make fresh OGD medium for every experiment.

This will ensure that the medium is purged of oxygen. Remove astrocyte media and wash with cellular PBS two times to remove glucose from the cells. Add the OGD medium to the wells.

Place the multi well dish with the lid open inside the hypoxia chamber. Connect the gas mixture to the entrance valve and leave the exit valve open. Flush the chamber with the gas mixture at 10 to 15 litter per minute, for five to seven minutes.

Stop the gas flow and first close the clamp on the exit valve. Then, close the clamp on the gas entrance valve tubing. Place the hypoxic chamber into the cell culture incubator at 37 degrees Celsius for the desired time in OGD.

After incubation time is over, aspirate the OGD media and carefully add complete DMEM to the cells for the normoxic condition. Return the cells to the culture incubator for zero 24 and 48 hours of normoxic conditions. This protocol describes the isolation of astrocytes from neonatal rat cortices.

After validating the purity of the primary cultures OGD experiments were performed. In this ischemic-like model, glucose and oxygen deprivation induced a more phological change that was monitored by the immunofluorescence using astrocytic markers such as GFAP or proliferation markers such as PCNA. One of the advantages of this model is that is allows us to study a specific cell type response.

Although the limitation in this technique is the lack of cell to cell interaction due to the isolation of astrocytes. One of the possible ways to overcome this limitation, is to expose astrocytes to condition media from other cells types or a co-culture. In summary, the oxygen glucose deprivation method in an isolated cell system, allows us to directly test the effects of different neural protective or neurotoxic molecules.

Also, it is a valid tool to study astrocyte biology. Finally, this model sets a strong basis to study developmental drugs in experimental conditions using in vivo models.