Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrocytes are one of the most important key players in the central nervous system (CNS). Here, we are reporting a practical method of sexed hippocampal astrocyte culture protocol in order to study the mechanisms underlying the astrocyte function in male and female neonate pups after in-vitro ischemia.

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The overall goal of this technique is to prepare enriched sex-specific hippocampal astrocyte cultures from newborn mice as a tool to study the sex-specific roles of astrocytes following oxygen and glucose deprivation and reoxygenation in order to mimic in vivo hypoxic ischemia in a cell culture environment. This method can help answer key mechanistic and translational questions pertaining to the sex-specific role of hippocampal astrocytes following hypoxic ischemia in developing male and female brains. Dissect the brain from the head of a newborn mouse pup by using small scissors to create a posterior to anterior midline incision through the skin to expose the cranium.

Cut the cranium midline carefully from the neck to the nose. Use sterile flat tip forceps to peel away the skull. Remove the brainstem with the help of curved forceps and place the remaining brain into a dissecting dish.

Using curved forceps, flip the brain so that the ventral surface is facing up and then separate the hemispheres with a sharp, sterile surgical blade. With flat curved forceps, peel back the cerebral hemispheres and carefully remove the bulging midbrain and thalamic tissue to reveal the hippocampus, a small seahorse shaped structure in the medial temporal lobe. Remove both the hippocampal lobes and carefully clean the meninges and fibroblasts from the surface of the lobes by pulling with fine forceps.

Transfer both hippocampal lobes from a single mouse into a second dish filled with approximately 5 milliliters of HBSS and place on ice. Mince the lobes approximately 4 times using a sharp sterile surgical blade. Use a 1 milliliter sterile pipette to transfer the HBSS and tissue into a 50 milliliter sterile conical tube and then centrifuge at 300 times G for 5 minutes.

After centrifugation, aspirate the supernatant. Add 3 milliliters of 0.25 percent trypsin, mix, and incubate the tissue at 37 degrees Celsius for 20 minutes with gentle shaking. Following the incubation, centrifuge the tube at 300 times G for 5 minutes.

After the spin, use a sterile glass pipette to carefully aspirate the supernatant and then add 10 milliliters of pre-warmed astrocyte plating medium. Triturate 20 to 30 times using a fire-polished glass pipette. Following another 5 minute centrifugation at 300 times G, aspirate the supernatant and add 2 milliliters of astrocyte plating media.

Pass the cell suspension through 70 micron mesh filter into new 50 milliliter conical tube. Add the entire cell suspension to a coated sterile T-25 culture flask containing 3 milliliters of astrocyte plating media. Incubate the flask at 37 degrees Celsius in a 5%carbon dioxide incubator.

The next day, observe the progression of astrocytic confluence under a light microscope, aspirate the media and replace with 2 milliliters of fresh astrocyte plating media. Around 11 days in vitro, harvest the confluent astrocytes by aspirating the media and then adding 2 milliliters of 0.25%trypsin. Gently rotate the flask a few times and then aspirate the trypsin using a sterile glass pipette.

Add another 2 milliliters of 0.25%trypsin and let the flask sit at room temperature for 4-5 minutes. After removing the trypsin, place the flask at 37 degrees Celsius in a 5%CO2 incubator for 10 minutes. Following the incubation, add 5 milliliters of astrocyte plating medium and detach the astrocytic layer by knocking the flask 3 to 4 times.

Gently triturate the cell suspension across the bottom of the flask to achieve complete detachment and then collect the astrocytes in a 15 milliliter conical tube. Centrifuge the tube at 300 times G for 5 minutes. Aspirate the supernatant and then add 1 milliliter of fresh astrocyte plating medium.

Add 10 microliters of the cell suspension to a hemocytometer and use an inverted phase contrast microscope with a 10x objective to count the cells in the large central gridded area. Dilute the cells to approximately 1 time 10 to 5th cells per 2 milliliters of astrocyte plating medium. Pipette 2 milliliters of the cell suspension onto a poly-d lysine coated glass coverslip in the well of a 24-well culture dish.

Incubate at 37 degrees Celsius in a 5%carbon dioxide incubator. To begin oxygen and glucose deprivation, aspirate the medium from the coverslip containing adherent astrocytes and rinse once with 1 milliliter of isotonic OGD solution. Add 0.2 milliliters of OGD solution to the well and place in a hypoxic incubator.

Gently mix with an orbital shaker set to 50 rpm for the first 30 minutes of hypoxia. For reoxygenation, replace the OGD solution with 2 milliliters of astrocyte plating medium and incubate in 5%carbon dioxide and atmospheric air at 37 degrees Celsius for 5 hours. This immunofluorescence image shows the purity of the primary mouse hippocampal astrocyte culture.

The astrocyte culture was immunostained to show the neuronal dendrite marker, MAP2, in red, and the microglia marker, IBA1, in green. Nuclei are stained with DAPI which appears blue. The arrow indicates microglia contamination.

This representative immunofluorescent image of cultured hippocampal astrocytes under normoxic conditions, shows GFAP immunoreactivity in red and HIF1-alpha labeling in green. Again, nuclei are stained with DAPI which appears blue. The arrow indicates low HIF1-alpha expression.

After 2 hours of oxygen and glucose deprivation and 5 hours of reoxygenation, HIF1-alpha expression was increased in cultured hippocampal astrocytes as indicated by the arrowhead indicating elevated HIF1-alpha nuclear expression. GFAP expression and S100b expression were upregulated in hippocampal astrocyte cultures from male mice following oxygen and glucose deprivation and rexoygenation. Upregulation of S100b and GFAP was also seen in astrocytes obtained from female mice.

Following this procedure, other methods like quantitative PCR, western blotting, siRNA, and immunohistochemical stainings can be performed in order to answer additional questions like sex-specific changes in certain gene and protein expressions following in vitro ischemia. After it's development, this technique can pave the way for researchers in the field of neuroscience to explore the effects and consequences of stroke in the hippocampus studying the astrocytes in culture.

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Journal (Neuroscience)

Journal (Neuroscience)

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

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