Clinical and Translational Science Award program of NCATS UL1 TR0000427 and KL2 TR000428 (Cengiz P), UL1TR000427 to the UW ICTR from NIH/NCATS and funds from Waisman Center (Cengiz P), K08 NS088563-01A1 from NINDS (Cengiz P) and NIH P30 HD03352 (Waisman Center), NIH/NINDS 1K08NS078113 (Ferrazzano P). We would like to thank Albee Messing, PhD, for providing us the GFAP knockout mice.

NOTE: This study was conducted in accordance with the recommendation of the Guide for the Care and Use of Laboratory Animals of the National Institute of Health. The animal protocol was approved by the University of Wisconsin-Madison, Institutional Animal Care and Use Committee. Primary Astrocyte Culture protocol that is presented here is adopted from the protocols presented by Zhang Y19 et al. and Cengiz P20 et al. with some modifications.
1. Hippocampal Dissection and Astroc...

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None of the authors have competing&#160;or conflicting interests.

In order to study the sex differences in the properties and function of astrocytes under physiological and pathological conditions, preparation of sexed primary astrocytes in cell culture is an important tool to utilize. In the present study we report a highly efficient and a reproducible method to culture a highly enriched homogeneous population of sexed hippocampal astrocytes from newborn (P0-P2) C57Bl/6 (wild type) or K19F (GFAP null) mouse pups in-vitro. Establishing this methodology helps the investigators ...

Astrocytes are one of the most important key players in the central nervous system (CNS). Growing body of evidence indicates that the roles of astrocytes are more than providing&#160;neuronal support. In fact, the roles of astrocytes under physiological conditions can be very complex, such as guiding the migration of the developing axons1, regulating CNS blood flow2, maintaining the pH homeostasis of the synaptic interstitial fluid3, and participating in the blood brain barrier4 and synaptic transmission5. Under pathological conditions, astrocytes respond to injury with a process called reactive astrogliosis in which the morphology, number, location, topography (with respect to distance from insult) and function of the astrocytes may change in a heterogeneous way6,7. Astrogliosis seen following neonatal hypoxic ischemic encephalopathy maybe contributing to the morbidity and mortality of neonates8.
Recent clinical and experimental studies indicate that the severity of brain injury appears to be sex-dependent and that the male neonates are more susceptible to the effects of hypoxia/ischemia (HI)-related brain injury, resulting in more severe neurological outcomes as compared to females with comparable brain injuries9-11. Although the localization of the injury depends on the gestational age and the&#160;duration and the severity of the insult, hippocampus is one of the most commonly effected regions in the CNS after term neonatal HI,&#160;and increased hippocampal astrogliosis has been confirmed by up-regulation of the Glial Fibrillary Acidic Protein (GFAP) 3 d&#160;after the neonatal HI7,10,12,13. Sex differences in astrocyte function were shown in both neonates and adult rodents after cerebral ischemia14,15. In addition, male astrocytic susceptibility to in-vitro ischemia was shown by increased cell death compared to female cortical astrocytes in culture16.
Sex differences start in-utero and continue until death17. Over the last decade, the importance of including the sexes in experimental conditions in cell culture and in-vivo&#160;studies have been the emphasis of the Institute of Medicine and NIH to seek fundamental knowledge in the sex differences seen in physiological and pathological conditions17,18. Development of reliable methods to isolate and maintain populations of sexed hippocampal astrocytes is essential to understand the cellular basis of sex differences in the pathological consequences of neonatal HI. The present study was designed to provide the techniques to prepare enriched sex-specific hippocampal astrocyte cultures from newborn mice in order to assess the roles of GFAP-immunoreactive astrocytes following Oxygen/Glucose Deprivation (OGD) and reoxygenation (REOX),&#160;inducing&#160;HI in cell culture environment. This technique can be used to test any hypothesis pertaining to hippocampal astrocytes in neonatal males and females under normoxic and ischemic conditions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Astrocyte culture media</td>
		</tr>
		<tr>
			<td>DMEM, high glucose</td>
			<td>cellgro</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Gibco</td>
			<td>26050-070</td>
			<td>Final Concentration: 10%</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Cellgro&#160;</td>
			<td>30-002-CI</td>
			<td>Final Concentration: 1%</td>
		</tr>
		<tr>
			<td rowspan="2">L-Leucine methyl ester hydrochloride</td>
			<td rowspan="2">Aldrich</td>
			<td rowspan="2">L1002-25G</td>
			<td rowspan="2">Final Concentration: 5 mM</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td colspan="4">Solution for brain tissue digestion</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Life Technologies</td>
			<td>14170-088</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin</td>
			<td>cellgro</td>
			<td>25-050-CI</td>
			<td>Final Concentration: 0.25%</td>
		</tr>
		<tr>
			<td colspan="4">Other</td>
		</tr>
		<tr>
			<td>70% (vol/vol) ethanol</td>
			<td>Roth</td>
			<td>9065.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine 12mm round coverslips&#160;</td>
			<td>Corning</td>
			<td>354087</td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma</td>
			<td>W3500</td>
			<td>cell culture grade</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>cellgro</td>
			<td>21-040-CV</td>
			<td>cell culture grade</td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA&#160;</td>
			<td>Life Technologies</td>
			<td>25300-062</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m Sterile cell strainer&#160;</td>
			<td>Fisher scientific</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr>
			<td>3.5 cm petri dish</td>
			<td>BD Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml Falcon tube</td>
			<td>BD Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml Falcon tube</td>
			<td>BD Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps, fine&#160;</td>
			<td>Dumont</td>
			<td>2-1032; 2-1033</td>
			<td># 3c; # 5</td>
		</tr>
		<tr>
			<td>Forceps, flat tip</td>
			<td>KLS Martin</td>
			<td>12-120-11</td>
			<td></td>
		</tr>
		<tr>
			<td>13 cm surgical scissors</td>
			<td>Aesculap</td>
			<td>BC-140-R</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Nikon</td>
			<td>A1RSi&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5805000.017</td>
			<td>Centrifuge5804R</td>
		</tr>
		<tr>
			<td>Orbital Shaker</td>
			<td>Thermo Scientific</td>
			<td>SHKE 4450-1CE</td>
			<td>MaxQ 4450&#160;</td>
		</tr>
		<tr>
			<td>Anti-IBA1</td>
			<td>Wako</td>
			<td>019-19741</td>
			<td>Rabbit monoclonal</td>
		</tr>
		<tr>
			<td>Anti-MAP2</td>
			<td>Sigma</td>
			<td>M2320</td>
			<td>Mouse monoclonal</td>
		</tr>
		<tr>
			<td>Anti-HIF1alpha</td>
			<td>abcam</td>
			<td>ab179483</td>
			<td>rabbit monoclonal</td>
		</tr>
		<tr>
			<td>Anti-S100B</td>
			<td>Sigma</td>
			<td>HPA015768</td>
			<td>Rabbit polyclonal</td>
		</tr>
		<tr>
			<td>Anti-GFAP (cocktail)</td>
			<td>Biolegend</td>
			<td>837602</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASTAIN Elite ABC Kit (Rabbit IgG)</td>
			<td>Vector Labs</td>
			<td>PK-6101</td>
			<td>Contains 4 Reagents&#160;</td>
		</tr>
		<tr>
			<td>Goat Anti Rabbit Alexa-Fluor 488</td>
			<td>Invitrogen</td>
			<td>A11070</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti Mouse Alexa-Fluor 568</td>
			<td>Invitrogen</td>
			<td>A11004</td>
			<td></td>
		</tr>
	</tbody>
</table>

sex differences in mouse hippocampal astrocytes after in-vitro ischemia

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies indicate that the severity of brain injury appear to be sex dependent, and that the male neonates are more susceptible to the effects of HI-related brain injury, resulting in more severe neurological outcomes as compared to females with comparable brain injuries. The development of reliable methods to isolate and maintain highly enriched populations of sexed hippocampal astrocytes is essential to understand the cellular basis of sex differences in the pathological consequences of neonatal HI. In this study, we describe a method for creating sex specific hippocampal astrocyte cultures that are subjected to a model of in-vitro ischemia, oxygen-glucose deprivation, followed by reoxygenation. Subsequent reactive astrogliosis was examined by immunostaining for the Glial Fibrillary Acidic Protein (GFAP) and S100B. This method provides a useful tool to study the role of male and female hippocampal astrocytes following neonatal HI, separately.

Understanding the roles of sexed astrocyte functions under physiologic or pathophysiological conditions have been immensely elucidated by culturing these cells under in vitro conditions. The important aspect of performing sexed culturing is to determine the sex of the mouse pup prior to its use. We determined the sex of the mouse genetically by PCR and by visual assessment (Figure 2)16. The methodology of sex determination using PCR was adopted with mo...

Watch this Scientific Journal Video about Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia at JoVE.com

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.

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies ...

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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JoVE Journal

Neuroscience

13.2K visualizações.  University of Wisconsin. 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 fema...