Name: monitoring astrocyte reactivity and proliferation in vitro under ischemic-like conditions
Uploaded: 2017-10-21T13:00:00.000+00:00
Duration: 15 min 8 s
Description: Watch this Scientific Journal Video about Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions at JoVE.com

The authors want to thank Paola L&#243;pez Pieraldi for the technical assistance. A.H.M. is grateful for the grants 8G12MD007600 and U54-NS083924 that supported this publication. We thank NIH-NIMHD-G12-MD007583 grant for the facility support. D.E.R.A. is grateful for the fellowship provided by NIHNIGMS-R25GM110513.We are grateful for the use of the Common Instrumentation Area and the aid of Dr. Priscila Sanabria for the use of the Optical Imaging Facility of the RCMI program by grant G12MD007583. In addition, we want to thank Jose Padilla for his outstanding role in filming and editing the visual protocol.

Postnatal rats (Sprague Dawley) 1-4 days old are used to isolate cortices. The method of euthanasia is decapitation, as approved by NIH guidelines.
1. Preparation of Instruments and Materials for Surgery
<ol>
	<li>Sterilize instruments in an autoclave (temperature: 121 &#186;C, pressure: 15 psi, time: 30 mins) using a steel box or an instant sealing sterilization pouch. See materials in Table of Materials.</li>
</ol>
2. Complete DMEM Preparation
<ol>
	<li>In a one liter beaker containing 700 mL autoclaved water, add Dulbecco&#39;s Modified Eagle&#39;s Medium powder at room temperature.</li>
	<li>Add 3.7 g/L sodium bicarbonate, while the solution is stirred with a magnetic stirrer.</li>
	<li>Adjust the pH to 7.4, with autoclaved water bring volume up to 1 L, then filter. For culture purposes, supplement the desired volume of media with 10% FBS and 1% Penicillin/Streptomycin, and warm at 37 &#730;C. See materials in Table of Materials. 
	Note: The pH was adjusted to 7.4 via inserting the pH meter electrode into the media. The media must be stirring, then slowly add 1M NaOH using a dropper.</li>
</ol>
3. Primary Astrocyte Culture
Note: After seeding, cell media must be changed every three days and cells can be grown up to confluency (11-13 days). On the third day, tap the flask several times to lift microglial cells and oligodendrocyte progenitor cells off the culture, remove all the &#39;old&#39; media, wash twice using 10 mL of PBS, aspirate the PBS, then add fresh new media. See materials in Table of Materials.
<ol>
	<li>Dissection of newborn rat brain
	<ol>
		<li>Perform the dissection in a tissue culture hood. Obtain 1-4 days old newborn rat pups (2 rat brains are used per 75 cm2 flask).</li>
		<li>Prepare three 60 mm petri dishes and pipette 5 mL of complete DMEM on each. 
		Note: They can be divided as follows: #1 for each rat brain, #2 for all the cortices of the brains, and #3 for all the peeled cortices (without meninges).</li>
		<li>Grasp the pup and spray with 70% ethanol. Decapitate it and discard the body in a small biohazard waste container.</li>
		<li>Using the micro-dissecting tweezers, hold the head and cut the skin on top of the head to expose the cranium.</li>
		<li>With another pair of scissors, make a &#34;T&#34; incision starting from the back of the skull towards the nose. Use a pair of forceps to gently remove the skull.</li>
		<li>Remove the exposed brain with a pair of tweezers and place the brain in one of the petri dishes previously filled with medium.</li>
		<li>Use another set of sterile instruments for the subsequent steps.</li>
		<li>Use micro-dissecting forceps to gently place one brain on the inverted lid of a 60 mm petri dish. When the petri dish is placed on sterile gauze, it is possible to view the brain clearly and it is easier to dissect.</li>
		<li>Steady the brain with micro-dissecting forceps and separate the cerebral hemisphere by gently teasing along the midline fissure with the sharp end of the micro-dissecting forceps.</li>
		<li>Deflect and peel the cortices, leaving behind the white matter, and transfer them to a petri dish, previously labeled #2. Repeat the procedure for all the brains, combining all the dissected cortices in the petri dish labeled #2.</li>
		<li>Take out the hippocampus from underneath each cortex and discard it.</li>
		<li>Use the micro-dissecting tweezers and a&#160;60 mm petri to gently peel the meninges from the individual cortical lobes, and place them into the petri dish labeled #3.</li>
	</ol>
	</li>
	<li>Tissue dissociation: homogenizer blender method
	<ol>
		<li>Pour tissue/medium suspension (peeled cortices of the brain) into the sterile blender bag and add enough medium to bring the total volume in the bag to 5 mL.</li>
		<li>Place the bag into the homogenizer blender, leaving approximately 2 cm of the bag visible above the closed door. The cell dissociation is done during 2.5 min at high speed. 
		Note: Alternatively, this can be done by closing the bag and gently pounding it with a beaker in the hood. Make sure to use a cloth in between the bag and beaker to avoid friction.</li>
		<li>Pour the cell suspension into a number 60&#160;mesh sieve and then pour the filtered flow through onto the number 100 sieve, allowing it to filter by gravity.</li>
		<li>Using 15 mL tubes, centrifuge the cell suspension at 200 x g in a clinical centrifuge (preferably swing bucket rotor) for 5 min.</li>
		<li>Pour off the supernatant in a beaker, then using a serological pipette, re-suspend and dissociate the pellet of cells in a maximum of 5 mL of media.</li>
	</ol>
	</li>
	<li>Cell counting
	<ol>
		<li>With a micropipette, collect 100 &#181;L of cell suspension in a 1.5 mL microcentrifuge tube and add 100 &#181;L of trypan blue.</li>
		<li>Insert 10 &#181;L of the collected suspension in the hemocytometer and count all the cells that are viable in the four corners of the square.</li>
		<li>The formulae for calculating the density of the cells before preparing the flasks are: 
		<img alt="Equation 1" src="/files/ftp_upload/55108/55108eq1.jpg" /> 
		<img alt="Equation 2" src="/files/ftp_upload/55108/55108eq2.jpg" /></li>
		<li>Add at least 500,000 cells per 25 cm2 flask. Pipette up to 5 mL of DMEM into each flask. The mixture should not reach the neck of the flask. Place them in a 37 &#176;C incubator at 5% CO2, for 3 days, then change the media.</li>
	</ol>
	</li>
	<li>Astrocyte purification techniques: media change, mechanical, and chemical strategies 
	Note: The metabolic rate of astrocytes is high compared to other cell types, therefore, in nutritionally deprived conditions, these cells show low survival rates after a few days. Unlike astrocytes, microglia are not affected by nutritional deprivation and proliferate in such environments18. To maintain a reduced number of microglial cells, the authors change the astroglial cell culture media every 3 days. This constant change in cell media will also decrease the microglial population that can grow on top of the astrocytic cell monolayer in the flask19.
	<ol>
		<li>Use a Pasteur pipette to aspirate the media with non-adherent microglial cells from each of the flasks. Wash any debris left behind using sterile-filtered PBS to each of the flasks in a dropwise manner (5 mL/flask).</li>
		<li>Add the sterile-filtered DMEM (with antibiotic and fetal bovine serum) dropwise to each flask (5 mL/flask). Gently agitate in a circular motion to cover the entire surface.</li>
		<li>Place cells in a 37 &#176;C incubator at 5% CO2 for 8-10 days until the flasks reach confluency (&#62;95% plate coverage). 
		Note: Several protocols have shown that when astrocytes reach confluency , mechanical methods, such as shaking the culture from 2 to 24 h, leads to the detachment of cells that lie on top of these astrocytes, mainly microglia and precursor cells19,20.</li>
		<li>Non-astrocytic cells are reduced in culture by performing orbital shaking at 180 rpm for 30 min. After removing cells in supernatant, fresh media is pipetted into the flask (~15 mL).</li>
		<li>To eliminate the oligodendrocyte progenitor cells, increase shaking speed to 200 rpm for 6 h21,22,23 , aspirate supernatant, and pipet fresh media. 
		Note: To decrease the presence of microglia in the cultures, two chemical methods can be used sequentially (not simultaneously) in the primary cell culture methodology: Arabinose cytosine (Ara-C) and L-leucine methyl ester (LME)24.</li>
		<li>Immediately when cells reach confluency (100%), 3-4 days after the mechanical removal of microglia, these cells can be treated with 8 &#181;M of the antimitotic compound Arabinose cytosine (Ara-C) in DMEM for 5 days (change to fresh Ara-C in DMEM daily). 
		Note: The time point to add Ara-C is critical since this compound is an antimitotic drug, that reduces the number of rapidly-dividing cells such as microglia19,25 and fibroblasts26. In contrast to microglia, when confluent, astrocytes stop proliferating via contact inhibition27.</li>
		<li>Allow 2 days for cells to recover from Ara-C treatment.</li>
		<li>To further reduce the microglial contamination, after cells reach confluency, use 50 mM of L-leucine methyl ester (LME) in DMEM fixed at pH 7.4, for 1 h at 37 &#730;C. 
		Note: LME crosses the membrane of cells and organelles, and after being hydrolyzed, the L-leucine produced accumulates in the lysosomes of these cells. The accumulation of this amino acid leads to an osmotic swelling and rupture of lysosomes28,29. LME is highly effective at eliminating microglia, compared to other cells20,30.</li>
	</ol>
	</li>
</ol>
4. Cultivating Astrocytes in 6-Well Plates
<ol>
	<li>Coating coverslips with poly-D-lysine
	<ol>
		<li>Put one coverslip in each 35 mm petri dish, then add 100 &#181;L of the diluted Poly-D-Lysine solution to each coverslip and wait 1 h.</li>
		<li>Remove the Poly-D-Lysine and wash twice with 2 mL sterile water.</li>
		<li>Remove water and wait for coverslips to dry inside the hood. Store the coverslips at -20 &#176;C for up to two weeks. 
		Note: Freeze the diluted Poly-D-Lysine solution; it can be stored for up to two weeks.</li>
	</ol>
	</li>
	<li>Trypsinization
	<ol>
		<li>Using a Pasteur pipette, aspirate the medium from the flasks. Add 5 mL/flask of sterile-filtered PBS to gently rinse the cells.</li>
		<li>Aspirate off the PBS, and then add 5 mL/flask of sterile-filtered 0.25% Trypsin and 0.5 mM EDTA solution. Place the flask in the 37 &#730;C, 5% CO2 incubator for 10 min. 
		Note: While the cells are incubating, warm 5 mL/flask of fresh complete DMEM at 37 &#176;C.</li>
		<li>After 10 min of incubation, agitate to make sure all the cells are lifted off the flask. Quickly add the 5 mL of warm complete DMEM to each flask, to neutralize the trypsin.</li>
		<li>Gently pipette the cells up and down several times to break up any &#34;clumps&#34;. Transfer suspended cells from two flasks to a 15 mL conical centrifuge tube.</li>
		<li>Centrifuge for 5 min at 200 x g to collect the cell pellet. Re-suspend the pellet in 500 &#181;L of medium and proceed to place cells in the flasks. 
		Note: Examples of analyses to be made with cells: 1) Reverse transcription polymerase chain reaction (RT-PCR) to analyze gene expression; 2) Western blot to evaluate protein expression; 3) Flow cytometry analysis to quantify the number of neural progenitor cells and proteins of interest; 4) Immunofluorescence to analyze protein expression and localization.</li>
	</ol>
	</li>
	<li>Seeding astrocytes
	<ol>
		<li>After treating the primary astrocytes with trypsin (refer to 4.2), collect 100 &#956;L of cell suspension in a 1.5 mL microcentrifuge tube and add 100 &#956;L of 0.5% trypan blue.</li>
		<li>Add 10 &#956;L of the collected suspension in trypan blue to the hemocytometer and count all the cells that are viable.</li>
		<li>Use the formulae in step 3.3 to calculate the density of the cells before preparing the flasks.</li>
		<li>Prepare the multi-well dishes by adding one sterile, poly-D-Lysine coated coverslip to each well. Pipette the 50,000 cells and fill the rest of the volume in each multi-well dish with fresh medium (approximately 2 mL).</li>
		<li>Place the multi-well dishes into the 37 &#730;C incubator at 5% CO2 and, after 5-7 days, astrocytes may grow and cover the entire surface of the coverslips.</li>
	</ol>
	</li>
</ol>
5. OGD Protocol for Primary Astrocytes
<ol>
	<li>OGD medium preparation
	<ol>
		<li>Supplement Dulbecco&#39;s modified Eagle&#39;s medium-free glucose with 3.7 g/L sodium bicarbonate, and 1% Penicillin/Streptomycin. See materials in Table 4.</li>
		<li>Purge 20 mL of the glucose free medium by bubbling with 95% N2/5% CO2 for 10 min at a flow of 15 L/min (indicated with the flow meter) by inserting a serological pipette to the gas system in the medium. 
		Note: After purging the medium of oxygen, adjust the pH at 7.4 (refer to note on step 2.1).</li>
		<li>Filter the purged glucose-free medium using a 50 mL tube filter system. 
		Note: Make fresh OGD medium for every experiment, bubbling with 95% N2/5% CO2 before using, to ensure that cells are exposed to an environment purged of oxygen.</li>
	</ol>
	</li>
	<li>Experimental procedure
	<ol>
		<li>Inside the tissue culture hood, remove astrocyte media from previously prepared coverslips (refer to step 4.3.5) and wash with cellular PBS twice to remove any glucose on the cells.</li>
		<li>Add 2 mL of the OGD media to each well and place the multi-well dishes inside the hypoxia chamber with their lids off.</li>
		<li>Connect the gas mixture to the entrance valve and leave the exit valve open. Flush the chamber with the gas mixture of 95% N2/5% CO2, at 15 L/min for 5 min.</li>
		<li>Stop the gas flow, and first close the clamp on the exit valve, then close the clamp on the gas entrance valve tubing. 
		Note: Ensure there is no gas leakage by covering the seal of the chamber with soapy water. If the seal is secure, no bubbles should form.</li>
		<li>Place the entire chamber into the cell culture incubator at 37 &#176;C for 1 h or 6 h in OGD.</li>
		<li>After incubation time is over, aspirate the OGD media and carefully pipet 2 mL of complete DMEM to each 4.67&#160;cm2 well for the normoxic process 24 h.</li>
	</ol>
	</li>
</ol>
6. Protocol for Primary Astrocyte Immunofluorescence Preparation
Note: To assess astrocyte purity, different brain cell types such as neurons, microglia, and oligodendrocytes can be detected by immunofluorescence using different cell markers. Proliferation markers, PCNA, and propidium iodide (PI) can be used on primary astrocytes exposed to OGD followed by normoxic conditions. See materials in Table of Materials.
<ol>
	<li>Immunofluorescence preparation 
	Note: This is a general immunofluorescence protocol, minor modifications can be performed to achieve an optimal signal (e.g., longer incubation periods, incubation temperature). Each marker from Table 1 can be used according to the manufacturer&#39;s protocol.
	<ol>
		<li>To evaluate cell viability, cells can be incubated for 5 min at 37 &#176;C with PI (5 &#181;M in complete DMEM) and wash twice with 2 mL of PBS for 5 min. Cells that show PI staining are less viable. 
		Note: Use the cells treated in step 5.2.6.</li>
		<li>To fix cells after PI staining, add 2 mL of 4% formalin per 4.67&#160;cm2 well, for 20 min at 4 &#730;C. For samples labeled with PCNA, the preferred fixing solution is methanol for 10 min at 4 &#730;C. 
		Caution: Formalin and methanol are toxic. 
		Note: Always check specific antibody protocols from each company.</li>
		<li>Wash cells with 2 mL of PBS for 5 min and repeat this step 3 times.</li>
		<li>Incubate cells in blocking solution (0.5% non-ionic surfactant, 10% FBS in PBS) for 30 min at room temperature with gentle shaking.</li>
		<li>Wash the cells with 2 mL of PBS for 5 min, repeat wash 3 times.</li>
		<li>Incubate at 4 &#176;C, overnight (16 h), with primary antibody (PCNA, GFAP) in a solution of 1% FBS in PBS.</li>
		<li>Remove the primary antibody solution, wash the cells with 2 mL of PBS for 5 min, and repeat this step 3 times.</li>
		<li>Incubate with secondary antibody conjugated with fluorophore in a solution of 1% FBS in PBS, for 1 h, at room temperature.</li>
		<li>Wash the cells with 2 mL of PBS for 5 min, repeat this step 3 times. Add 1 &#181;g/mL of DAPI (4&#39;,6&#39;-diamidino-2-phenylindole) for 5 min to stain cell nuclei.</li>
		<li>Wash cells with 2 mL of PBS 5 min, repeat this step twice and keep in PBS.</li>
		<li>Prepare the coverslips on a microscope slide using mounting medium.</li>
	</ol>
	</li>
	<li>Confocal microscope
	<ol>
		<li>After setting up the microscope, place the coverslip side down on the microscope stage.</li>
		<li>Using the software, select a laser beam at 543nm for GFAP-Cy3 conjugated antibody.</li>
	</ol>
	</li>
</ol>
7. Cell Viability Assay
<ol>
	<li>Trypsinize and quantify astrocytes by following steps 4.3 and 4.4.</li>
	<li>Add 1x104 cells/well to 96-well plates and grow them to confluency.</li>
	<li>Using the methodology from step 5, expose cultured 96-well plates to either normoxic 1 h OGD or 6 h OGD.</li>
	<li>After OGD treatment, normoxic media is added to all cells for 24 h and viability is measured using MTT reagent assay (use as manufacturer&#39;s instructions indicate).</li>
	<li>From a 5 mg/mL MTT solution, add 10% of the total volume to each well and incubate for 3 h at 37 &#730;C with the MTT reagent.</li>
	<li>Remove media and resuspend crystals using 200 &#181;L dimethyl sulfoxide. Read the plate in a spectrophotometer at 570 nm. 
	Note: A decreased absorbance relative to control cells will correlate with less viability.</li>
</ol>

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D., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+separate+astroglial+and+oligodendroglial+cell+cultures+from+rat+cerebral+tissue.">Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.</a> J Cell Biol. 85 (3), 890-902 (1980).</li><li>Pont-Lezica, L., Colasse, S., Bessis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+microglia+from+primary+cellular+cultures.">Depletion of microglia from primary cellular cultures.</a> Methods Mol Biol. 1041, 55-61 (2013).</li><li>Svensson, M., Aldskogius, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+density+of+axotomized+hypoglossal+motorneurons+following+pharmacological+blockade+of+the+microglial+cell+proliferation.">Synaptic density of axotomized hypoglossal motorneurons following pharmacological blockade of the microglial cell proliferation.</a> Exp Neurol. 120 (1), 123-131 (1993).</li><li>Wong, V. K., Shapourifar-Tehrani, S., Kitada, S., Choo, P. H., Lee, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+rabbit+ocular+fibroblast+proliferation+by+5-fluorouracil+and+cytosine+arabinoside.">Inhibition of rabbit ocular fibroblast proliferation by 5-fluorouracil and cytosine arabinoside.</a> J Ocul Pharmacol. 7 (1), 27-39 (1991).</li><li>Nakatsuji, Y., Miller, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Density+dependent+modulation+of+cell+cycle+protein+expression+in+astrocytes.">Density dependent modulation of cell cycle protein expression in astrocytes.</a> J Neurosci Res. 66 (3), 487-496 (2001).</li><li>Reeves, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+amino+acids+by+lysosomes+incubated+with+amino+acid+methyl+esters.">Accumulation of amino acids by lysosomes incubated with amino acid methyl esters.</a> J Biol Chem. 254 (18), 8914-8921 (1979).</li><li>Thiele, D. L., Kurosaka, M., Lipsky, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotype+of+the+accessory+cell+necessary+for+mitogen-stimulated+T+and+B+cell+responses+in+human+peripheral+blood:+delineation+by+its+sensitivity+to+the+lysosomotropic+agent,+L-leucine+methyl+ester.">Phenotype of the accessory cell necessary for mitogen-stimulated T and B cell responses in human peripheral blood: delineation by its sensitivity to the lysosomotropic agent, L-leucine methyl ester.</a> J Immunol. 131 (5), 2282-2290 (1983).</li><li>Hamby, M. E., Uliasz, T. F., Hewett, S. J., Hewett, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+an+improved+procedure+for+the+removal+of+microglia+from+confluent+monolayers+of+primary+astrocytes.">Characterization of an improved procedure for the removal of microglia from confluent monolayers of primary astrocytes.</a> J Neurosci Methods. 150 (1), 128-137 (2006).</li><li>Corvalan, V., Cole, R., de Vellis, J., Hagiwara, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+modulation+of+calcium+channel+activity+in+cultured+rat+astrocytes.">Neuronal modulation of calcium channel activity in cultured rat astrocytes.</a> Proc Natl Acad Sci U S A. 87 (11), 4345-4348 (1990).</li><li>Butler, I. B., Schoonen, M. A., Rickard, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+dissolved+oxygen+from+water:+A+comparison+of+four+common+techniques.">Removal of dissolved oxygen from water: A comparison of four common techniques.</a> Talanta. 41 (2), 211-215 (1994).</li><li>Tasca, C. I., Dal-Cim, T., Cimarosti, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+oxygen-glucose+deprivation+to+study+ischemic+cell+death.">In vitro oxygen-glucose deprivation to study ischemic cell death.</a> Methods Mol Biol. 1254, 197-210 (2015).</li><li>Wu, D., Yotnda, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+testing+of+hypoxia+in+cell+culture.">Induction and testing of hypoxia in cell culture.</a> J Vis Exp. (54), (2011).</li><li>Rivera-Aponte, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperglycemia+reduces+functional+expression+of+astrocytic+Kir4.+1+channels+and+glial+glutamate+uptake.">Hyperglycemia reduces functional expression of astrocytic Kir4. 1 channels and glial glutamate uptake.</a> Neuroscience. 310, 216-223 (2015).</li><li>Berger, R., Garnier, Y., Pfeiffer, D., Jensen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharides+do+not+alter+metabolic+disturbances+in+hippocampal+slices+of+fetal+guinea+pigs+after+oxygen-glucose+deprivation.">Lipopolysaccharides do not alter metabolic disturbances in hippocampal slices of fetal guinea pigs after oxygen-glucose deprivation.</a> Pediatric research. 48 (4), 531-535 (2000).</li><li>Anderson, T. R., Jarvis, C. R., Biedermann, A. J., Molnar, C., Andrew, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+the+anoxic+depolarization+protects+without+functional+compromise+following+simulated+stroke+in+cortical+brain+slices.">Blocking the anoxic depolarization protects without functional compromise following simulated stroke in cortical brain slices.</a> Journal of neurophysiology. 93 (2), 963-979 (2005).</li><li>Jarvis, C. R., Anderson, T. R., Andrew, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anoxic+depolarization+mediates+acute+damage+independent+of+glutamate+in+neocortical+brain+slices.">Anoxic depolarization mediates acute damage independent of glutamate in neocortical brain slices.</a> Cerebral Cortex. 11 (3), 249-259 (2001).</li></ol>

The authors have nothing to disclose.

This protocol describes the isolation of astrocytes from rat cortices. In this method, it is critical to decrease contamination with other cellular types such as microglia, oligodendrocytes, and fibroblasts. To reduce the number of microglia, several steps can be taken: changing the media, orbital shaking, and chemical treatments. Once culture purity is confirmed by immunofluorescence using selective cellular markers or for the most prominent cell contaminants, experiments can be performed. For instance, antibody against ionized calcium binding adaptor molecule 1 (Iba-1) can be used to detect microglia. Neuronal death starts early in the culture and these cells are eliminated together with oligodendrocyte precursor cells (OPCs) after flask tapping and media change at day 331. Oligodendrocytic precursor cells can be identified using antibodies such as olig2 or NG2. GalC can be used to identify immature or mature oligodendrocytes, but at this culture stage these are unlikely to be found. Alternatively, non-GFAP positive cells can be quantified and contaminants can be estimated. This last methodology does not discern between the specific cells, but will be a faster and more economic methodology to determine the percentage of non-astrocytic cells in the culture.
Once an astrocyte enriched culture is produced, OGD experiments can be conducted to study astrocyte reactivity. Purging a solution with nitrogen by bubbling to substitute oxygen is a commonly used protocol32. In our methodology, we use the bubbling method to purge oxygen with nitrogen gas (95% N2, 5% CO2) into the glucose-free media at a flow rate of 15 L/min, for 10 min, for a total volume of 0.025 L. To confirm that the oxygen is purged, we measured the oxygen concentration remaining in the media by using an oxygen meter for liquids and air. The oxygen concentration remaining in the media after purging for 10 min decreased from 7.9 mg/L to 0.3 mg/L. To determine how long the chamber must be purged to replace oxygen, purging with N2 was performed using no less than the chamber manufacturer&#39;s suggestion (20 L/min, 2-5 min). After 5 min of purging at 15 L/min, the oxygen concentration remaining in the chamber was 0%. Other methodologies using similar chambers have purged at an 8-20 L/min flow rate, and times of 5-10 min to substitute air oxygen with nitrogen33,34.
Like other cellular models, the OGD has limitations and results should be interpreted carefully. The OGD&#160;can be harmful to neurons, however, astrocytes can withstand these conditions for up to 24 h35. Another limitation is that astrocytes are isolated in this system, lacking signaling from other cells. One way to overcome this limitation is to use conditioned media from other cells under the same ischemic-like conditions. Alternatively, cytokines or other factors can be added to the media after OGD36.
An alternative method to study the effects of energy deficiency in cells, is to add ouabain to the media. &#160;This compound mainly inhibits the sodium/potassium pump, which depletes the intracellular production of ATP, similar to the effects of OGD37,38. However, this method has the disadvantage that all its mechanisms are not fully understood and it induces off-target effects, which introduces variables which constitute an unrealistic ischemic stroke scenario.
In summary, the OGD method is an isolated cell system which allows us to directly test the effects of different astrocytic drug targets in vitro that could further contribute to neuroprotection. It also provides a tool to study basic astrocyte biology. This model can create a strong basis of evidence to justify experimental conditions for the development of drugs using an in vivo model of stroke.

Stroke is defined as &#34;an acute neurological dysfunction of vascular origin with either sudden or rapid development of symptoms and signs, corresponding to the involvement of focal areas in the brain&#34;1,2. There are two types of stroke: hemorrhagic and ischemic. When vascular dysfunction is caused either by an aneurysm or an arteriovenous malfunction, accompanied by weakening with posterior rupture of an artery,&#160;this is termed hemorrhagic stroke3 which, in most cases, leads to death. When a thrombus or an embolus obstructs blood flow, causing a temporary deprivation of oxygen and glucose to a brain region, it is called ischemic stroke4. Failure to nourish cells around the affected area or ischemic core leads to a homeostatic and metabolic imbalance, energetic dysfunction, neuronal death, and inflammation5, which can induce a life-long disability for patients6.
Ischemic stroke is a multifactorial injury involving several types of cells that react and exert their effects at different time points. Many interactions create a difficult environment to study the behavior of individual cells. So, how do we study the contribution of a specific cell type under such a complex environment? An accepted in vitro model of ischemia consists of exposing cells to oxygen and glucose deprivation (OGD), for a certain period, followed by the restoration of cells to a normoxic environment. This system simulates an ischemic stroke followed by blood reperfusion. In this method, cells or tissues are exposed to a glucose-free media in an environment purged of oxygen, using a specialized hypoxic chamber. The OGD incubation time can vary from a few minutes up to 24 h, depending on the hypothesis that wants to be tested. Studies have shown that depending on the times of OGD and normoxic environment, specific phenotypes of stroke (i.e., acute or subchronic) can be achieved. Primary isolated astrocytes, exposed to OGD with posterior restoration to normoxic conditions, is a well-studied cellular model to mimic stroke in vitro7. Using OGD is possible to reveal the independent molecular mechanisms of isolated cells under a stroke-like environment.
As our knowledge of astrocyte biology increases, it has become evident that they are crucial for maintaining synapses and sustaining neural repair, development, and plasticity8. Under normal conditions, astrocytes release and respond to cytokines, chemokines, growth factors and gliotransmitters, keeping metabolic balance and homeostasis within synapses5,9. In acute neuroinflammation, such as ischemic stroke, these cells can become reactive, show a long-term overexpression of Glial Fibrillary Acidic Protein (GFAP), and show hypertrophy in their morphology5,10,11,12. As the ischemic infarct develops, the homeostasis provided by astrocytes becomes affected, regarding normal glutamate uptake, energy metabolism, exchange of active molecules, and antioxidant activity13.
Reactivated astrocytes proliferate around the infarct tissue while leukocytes migrate towards the lesioned area14. Astrocytic proliferation can be measured using markers such as proliferating cell nuclear antigen (PCNA), Ki67, and bromodeoxyuridine (BrdU)15. This proliferative response is generated in a time-dependent manner and it helps forming the glial scar, an array of irreversibly reactive astrocytes along the parenchyma of the damaged site after an injury9. One of the initial functions of this scar is to limit the immune cell extravasation from this area. However, studies have shown that the scar becomes a physical impediment for axons to extend, as they release molecules inhibiting axonal growth, and create a physical barrier preventing axons from extending around the injured area16. Nevertheless, there is scientific evidence showing that after a spinal cord injury, completely preventing glial scar formation can impair the regeneration of axons17. Thus, the context in which the specific astrocytic response is measured, must be considered upon the framework of the injury studied.
The presented methodology can be applied to study the individualized function of astrocytes after oxygen glucose deprivation and it can be modified depending on the questions that the investigator wants to answer. For example, besides the morphological change and the markers expressed at different OGD times, the supernatants from astrocytes exposed to OGD can be further analyzed to identify soluble factors released by these cells, or used as a conditioned media to assess its effect in other brain cells. This approach enables studies on astrocyte reactivity that could lead to the elucidation of the factors that govern and modulate their response in an ischemic-stroke scenario.

<table><tbody><tr><td>&lt;strong&gt;Instruments for Surgery - Step 1&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Operating scissor 5.5&amp;rdquo;</td><td>Roboz Company</td><td>RS-6812</td><td>Tools used to decapitate the rats.</td></tr><tr><td>Curved forceps 7&amp;rdquo;&amp;nbsp;</td><td>Roboz Company</td><td>RS-5271</td><td>Holds the skin of the rat while the skull is removed.</td></tr><tr><td>Micro-dissecting scissors 4&amp;rdquo;&amp;nbsp;</td><td>Roboz Company</td><td>RS-5882</td><td>Cuts both the skin and skull of the rat.</td></tr><tr><td>Micro-dissecting forceps 4&amp;rdquo; angled, fine sharp&amp;nbsp;</td><td>Roboz Company</td><td>RS-5095</td><td>Holds the skin of the rat while the skull is removed.</td></tr><tr><td>Micro-dissecting forceps 4&amp;rdquo; slightly curved 0.8</td><td>Roboz Company</td><td>RS-5135</td><td>Tool used to separate cortices.</td></tr><tr><td>Micro-dissecting tweezers</td><td>Roboz Company</td><td>RS-4972</td><td>Peels brain meninges.</td></tr><tr><td>Dissection microscope</td><td>Olympus</td><td>SZX16</td><td>Important for removing the meninge from the cortices.</td></tr><tr><td>&lt;strong&gt;DMEM Preparation - step 2&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium (DMEM)</td><td>GibCo. Company</td><td>11995-065</td><td>Supports the growth of cells.</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich Company</td><td>S7277</td><td>Supplement for the cell culture media.</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>GibCo. Company</td><td>10437-010</td><td>Serum-supplement for the cell culture.</td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>GibCo. Company</td><td>15140-148</td><td>Inhibits the growth of bacterias in the cell culture.</td></tr><tr><td>Filter System 1L with 0.22um pore</td><td>Corning</td><td>431098</td><td></td></tr><tr><td>&lt;strong&gt;Astrocyte culture - step 3&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Serological pipets 5mL</td><td>VWR</td><td>89130-896</td><td>To pipette DMEM to containers with cells.</td></tr><tr><td>Serological pipets 10mL</td><td>VWR</td><td>89130-898</td><td>To pipette DMEM to containers with cells.</td></tr><tr><td>Serological pipets 25mL</td><td>VWR</td><td>89130-900</td><td>To pipette DMEM to containers with cells.</td></tr><tr><td>Centrifuge conical tube 15mL</td><td>Santa Cruz Biotechnology</td><td>sc-200250</td><td></td></tr><tr><td>Safe-lock tube 1.5mL</td><td>Eppendorf</td><td>022363204</td><td></td></tr><tr><td>Barrier Tips 200 uL</td><td>Santa Cruz Biotechnology</td><td>sc-201725</td><td></td></tr><tr><td>Barrier Tips 1 mL</td><td>Santa Cruz Biotechnology</td><td>sc-201727</td><td></td></tr><tr><td>Biohazard Orange Bag 14 x 19&quot;</td><td>VWR</td><td>14220-048</td><td></td></tr><tr><td>60mm petri dishes</td><td>Falcon</td><td>351007</td><td></td></tr><tr><td>Sterile gauze pads</td><td>Honeywell Safety</td><td>89133-086</td><td></td></tr><tr><td>Stomacher 80 Biomaster</td><td>Sewar Lab System</td><td>030010019</td><td>Triturate the brain tissue.</td></tr><tr><td>Stomacher 80 Blender Sterile Bags</td><td>Sewar Lab System</td><td>BA6040</td><td>Sterile bag for the stomacher cell homogenizer.</td></tr><tr><td>Beaker 400mL</td><td>Pyrex</td><td>1000</td><td></td></tr><tr><td>Sterile cell dissociation sieve, mesh #60&amp;nbsp;</td><td>Sigma-Aldrich Company</td><td>S1020</td><td>To obtain a uniform single cell suspension.</td></tr><tr><td>Sterile cell dissociation sieve, mesh #100</td><td>Sigma-Aldrich Company</td><td>S3895</td><td>To obtain a uniform single cell suspension.</td></tr><tr><td>Invert phase microscope</td><td>Nikon</td><td>Eclypse Ti-S</td><td>Verify cells for contamination or abnormal cell growth.</td></tr><tr><td>75cm2 sterile flasks</td><td>Falcon</td><td>353136</td><td></td></tr><tr><td>Multi-well plate</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>Micro cover glasses (coverslips), 18mm, round</td><td>VWR</td><td>48380-046</td><td></td></tr><tr><td>Bright-Line hemacytometer</td><td>Sigma-Aldrich Company</td><td>Z359629</td><td></td></tr><tr><td>Pasteur pipettes</td><td>Fisher Scientific</td><td>13-678-20D</td><td></td></tr><tr><td>Ethyl alcohol&amp;nbsp;</td><td>Sigma-Aldrich Company</td><td>E7023</td><td></td></tr><tr><td>L-leucine methyl ester hydrochloride 98% (LME)</td><td>Sigma-Aldrich Company</td><td>L1002</td><td>Promotes the elimination of microglia cells in the primary cortical astrocyte cultutre.</td></tr><tr><td>Cytosine &amp;beta;-D-arabinofuranoside (Ara-C)</td><td>Sigma-Aldrich Company</td><td>C1768</td><td></td></tr><tr><td>Poly-D-Lysine Hydrobromide, mol wt 70,000-150,000</td><td>Sigma-Aldrich Company</td><td>P0899</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>GibCo. Company</td><td>15400-054</td><td></td></tr><tr><td>Trypan Blue</td><td>Sigma-Aldrich Company</td><td>T8154</td><td></td></tr><tr><td>Phosphate buffer saline (PBS) tablets</td><td>Calbiochem</td><td>524650</td><td></td></tr><tr><td>Sterile Water</td><td>Sigma-Aldrich Company</td><td>W3500</td><td></td></tr><tr><td>&lt;strong&gt;&amp;nbsp;OGD Medium Preparation - step 5&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Centrifuge conical tube 50 mL</td><td>VWR</td><td>89039-658</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium-free glucose</td><td>Sigma-Aldrich Company</td><td>D5030</td><td>Supports the growth of cells.</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich Company</td><td>S7277</td><td>Supplement for the cell culture media.</td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>GibCo. Company</td><td>15140-148</td><td>Inhibits the growth of bacterias in the cell culture.</td></tr><tr><td>200mM&amp;nbsp; L-glutamine&amp;nbsp;</td><td>GibCo. Company</td><td>25030-081</td><td>Amino acid that supplements the growth of cells.</td></tr><tr><td>Phospahet buffer saline (PBS) tablets</td><td>Calbiochem</td><td>524650</td><td></td></tr><tr><td>Filter System 50mL with 0.22um pore</td><td>Corning</td><td>430320</td><td></td></tr><tr><td>Centrifuge conical tube 50 mL</td><td>VWR</td><td>89039-658</td><td></td></tr><tr><td>Single Flow Meter&amp;nbsp;</td><td>Billups-Rothenberg</td><td>SMF3001</td><td>Measure gas flow in oxygen purge.</td></tr><tr><td>Hypoxia Incubator Chamber&amp;nbsp;</td><td>StemCell</td><td>27310</td><td>Generates a hypoxic environment for the cell culture.</td></tr><tr><td>Traceable Dissolved Oxygen Meter</td><td>VWR</td><td>21800-022</td><td></td></tr><tr><td>95% N2/ 5% CO2 Gas Mixture</td><td>Linde</td><td></td><td>Purges the environment of oxygen.</td></tr><tr><td>&lt;strong&gt;primary astrocyte immunofluorescence - step 6&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Phosphate buffer saline (PBS) tablets</td><td>Calbiochem</td><td>524650</td><td></td></tr><tr><td>Formaline Solution Neutral Buffer 10%</td><td>Sigma-Aldrich</td><td>HT501128</td><td>Solution used to fix cells.</td></tr><tr><td>Methanol&amp;nbsp;</td><td>Fisher</td><td>A4544</td><td>Solution used to fix cells.</td></tr><tr><td>Non-ionic surfactant (Triton X-100)</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>GibCo. Company</td><td>10437-010</td><td>Serum-supplement for the cell culture.</td></tr><tr><td>Anti-NeuN</td><td>Cell Signaling</td><td>24307</td><td>Detects mature neurons, serves to validate the astrocytic culture.</td></tr><tr><td>Anti-PCNA</td><td>Cell Signaling</td><td>2586</td><td>Detects proliferating cells.</td></tr><tr><td>Propidium Iodide (PI)</td><td>Sigma-Aldrich Company</td><td>P4170</td><td>Apoptosis staining.</td></tr><tr><td>Anti-Olig1</td><td>Abcam</td><td>AB68105</td><td>Detects mature oligodendrocytes.</td></tr><tr><td>Anti-Iba1+</td><td>Wako</td><td>016-20001</td><td>Detects microglial cells.</td></tr><tr><td>Anti-GFAP Conjugated with Cy3&amp;nbsp;</td><td>Sigma-Aldrich Company</td><td>C9205</td><td>Detects reactive astrocytes in the treated cells.</td></tr><tr><td>Alexa Fluor 488</td><td>Molecular Probe Life Technology</td><td>A1101</td><td>Anti-Mouse Secondary Antibody.</td></tr><tr><td>Alexa Fluor 555</td><td>Molecular Probe Life Technology</td><td>A21428</td><td>Anti-Rabbit Secondary Antibody.</td></tr><tr><td>4&amp;rsquo;,6&amp;rsquo;-diamidino-2-phenylindole (DAPI)</td><td>Sigma-Aldrich Company</td><td>D9542</td><td>Nuclear staining.</td></tr><tr><td>Confocal microscope</td><td>Olympus</td><td></td><td></td></tr></tbody></table>

monitoring astrocyte reactivity and proliferation in vitro under ischemic-like conditions

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of oxygen and glucose, which causes energy failure and neuronal death. After an ischemic stroke insult, astrocytes become reactive and proliferate around the injury site as it develops. Under this scenario, it is difficult to study the specific contribution of astrocytes to the brain region exposed to ischemia. Therefore, this article introduces a methodology to study primary astrocyte reactivity and proliferation under an in vitro model of an ischemia-like environment, called oxygen glucose deprivation (OGD). Astrocytes were isolated from 1-4 day-old neonatal rats and the number of non-specific astrocytic cells was assessed using astrocyte selective marker Glial Fibrillary Acidic Protein (GFAP) and nuclear staining. The period in which astrocytes are subjected to the OGD condition can be customized, as well as the percentage of oxygen they are exposed to. This flexibility allows scientists to characterize the duration of the ischemic-like condition in different groups of cells in vitro. This article discusses the timeframes of OGD that induce astrocyte reactivity, hypertrophic morphology, and proliferation as measured by immunofluorescence using Proliferating Cell Nuclear Antigen (PCNA). Besides proliferation, astrocytes undergo energy and oxidative stress, and respond to OGD by releasing soluble factors into the cell medium. This medium can be collected and used to analyze the effects of molecules released by astrocytes in primary neuronal cultures without cell-to-cell interaction. In summary, this primary cell culture model can be efficiently used to understand the role of isolated astrocytes upon injury.

One of the main concerns of primary astrocytic culture is the presence of other cells such as neurons, oligodendrocytes, fibroblasts, and microglia. In Figure 1, isolated cells from rat cortices had media changes every 3 days and were either untreated or treated with added LME for 1 h. 24 h later, cells were immunostained for GFAP and counterstained with DAPI. Untreated cells showed an average of 39% non-GFAP positive cells, while LME-treated cells showed 8%. These results show how LME treatment and media change effectively decreased non-GFAP positive cells by 4.75 fold, producing an enriched-astrocyte culture. To determine if 1 h or 6 h OGD affects astrocyte viability after this methodology, Figure 2 shows an MTT assay of astrocyte cultures, 24 h after the insult. No significant difference in viability was found in any condition, showing that both times can be used to study astrocyte reactivity. Cell proliferation is one of the effects triggered by OGD in astrocytes. Proliferating cell nuclear antigen (PCNA) in Figure 3 increased from 10% in normoxic cells, to 30% after 1 h OGD, showing the expected in vivo astrocytic response15. Finally, astrocytes were exposed to 6 h of OGD followed by 24 h normoxic conditions. After this period of time, immunofluorescence against GFAP was performed. OGD-exposed cells were compared to astrocytes under normoxic conditions. Astrocytes without OGD show the traditional stellate morphology (Figure 4A) while cells that underwent OGD clearly present the characteristic hypertrophy of reactive astrocytes (Figure 4B). This hypertrophy can be visualized in the corresponding differential interference contrast (DIC) image of cortical rat astrocytes under normoxic and OGD conditions (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55108/55108fig1.jpg" /> 
Figure 1. Immunofluorescence to validate primary cortical rat astrocytes culture purity with GFAP and DAPI. (A) The left panel represents GFAP positive cells (red), the middle panel shows nuclei (DAPI, blue), and the right panel shows merged images of non-LME-treated (upper panel) and LME-treated (lower panel) cells. White arrows show non-GFAP-positive stained cells. (B) Quantification of non-GFAP positive nuclei in LME and non-LME treated cells. An unpaired t-test was performed (p&#60;0.0001) and the error bars represent the mean with SEM. Scale bars represent 50 microns and pictures were taken at 20x magnification. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55108/55108fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55108/55108fig2.jpg" /> 
Figure 2. Viability assay of astrocytes with 24 h exposure to OGD. Astrocytes were cultured in 96-well plates and exposed to either normoxic, 1 h OGD, or 6 h OGD conditions. After treatment, cells were added to normoxic media for 24 h and viability was measured using MTT assay. Absorbance at 570 nm shows the cell viability of OGD-exposed cells in culture when compared to cells in normoxic conditions. Data was analyzed with one-way ANOVA (p=0.9669) and presented with error bars that represent the mean with SEM. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55108/55108fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55108/55108fig3.jpg" /> 
Figure 3. Proliferation in rat astrocytes increases upon exposure to oxygen and glucose deprivation and can be detected using PCNA as a marker. (A) The left panel represents PCNA positive cells (green), the middle panel shows nuclei (DAPI, blue), and the right panel shows merged images of normoxic cells (upper panel) and 1 h OGD treated cells (lower panel). (B) Percent of PCNA positive cells in normoxic versus OGD treated cells. An unpaired-test was performed (p&#60;0.0001) and the error bars represent the mean with SEM. Scale bars represent 50 microns and pictures were taken at 20x magnification. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55108/55108fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55108/55108fig4.jpg" /> 
Figure 4. Expression of Glial Fibrillary Acidic Protein (GFAP) conjugated with Cy3 in primary rat cortical astrocyte culture. (A) Astrocytes in a normoxic environment show the characteristic stellate morphology. The intermediate filament protein, GFAP, fills the cell bodies and extends into the thin cytoplasmic processes. (B) After 6 h of OGD exposure astrocytes adopted a hypertrophic morphology. Scales represent 20 microns and pictures were taken at 40x magnification in a confocal microscope. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55108/55108fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55108/55108fig5.jpg" /> 
Figure 5. Cortical rat astrocytes under normoxic and oxygen-glucose deprivation. Immunofluorescence of astrocytes under normoxic conditions (upper left panel) or 6 h OGD (lower left panel), stained using a Cy3-conjugated antibody against GFAP. The corresponding differential interference contrast (DIC) image is shown on the right panel. Scale bar represents 20 microns and pictures were taken at 20x magnification. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55108/55108fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Purpose</td>
			<td>Antibody/Marker</td>
			<td>Description</td>
		</tr>
		<tr>
			<td rowspan="5">Astrocyte Culture Validation</td>
			<td>Iba-1</td>
			<td>Detects microglial cells</td>
		</tr>
		<tr>
			<td>NeuN</td>
			<td>Identifies mature neurons</td>
		</tr>
		<tr>
			<td>Olig1</td>
			<td>Detects mature oligodendrocytes</td>
		</tr>
		<tr>
			<td>Olig2, NG2</td>
			<td>Detects oligodendrocyte precursor cells</td>
		</tr>
		<tr>
			<td>GalC</td>
			<td>Identifies immature or mature oligodendrocytes</td>
		</tr>
		<tr>
			<td>Proliferation</td>
			<td>PCNA</td>
			<td>Binds p36 protein, expressed at high levels in proliferating cells</td>
		</tr>
		<tr>
			<td rowspan="2">Cell viability&#160;</td>
			<td>Propidium iodide (PI)</td>
			<td>Binds to the DNA, this marker indicates lack of cell viability</td>
		</tr>
		<tr>
			<td>MTT</td>
			<td>Measures mitochondrial functionality</td>
		</tr>
		<tr>
			<td>Reactivity</td>
			<td>GFAP</td>
			<td>Indicator of astrocyte reactivity&#160;</td>
		</tr>
	</tbody>
</table>
Table 1. List of reagents used to stain various types of cells and cellular processes

Watch this Scientific Journal Video about Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions at JoVE.com

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

Ischemic stroke is a complex event in which the specific contribution of astrocytes to the affected brain region exposed to oxygen glucose deprivation (OGD) is difficult to study. This article introduces a methodology to obtain isolated astrocytes and study their reactivity and proliferation under OGD conditions.

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

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of ...

monitoring-astrocyte-reactivity-proliferation-vitro-under-ischemic

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.8K Views.  Universidad Central del Caribe. Ischemische beroerte is een complexe gebeurtenis waarin de specifieke bijdrage van astrocyten naar de hersenen van de getroffen regio blootgesteld aan zuurstof glucose ontbering (OGD) moeilijk te bestuderen. Dit artikel introduceert een methodologie voor het verkrijgen van geïsoleerde astrocyten en studeren hun reactiviteit en de proliferatie onder voorwaarden van OGD.

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

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Biology

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Bioengineering

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

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.

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.

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

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

Medicine

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, 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.

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.

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to use these cells for the purpose of studying the pathophysiology of amyotrophic lateral sclerosis (ALS) in the rat hSOD1G93A model. First, the protocol shows how to isolate and culture astrocytes and microglia from postnatal rat cortices, and then how to characterize and test these cultures for purity by immunocytochemistry using the glial fibrillary acidic protein (GFAP) marker of astrocytes and the ionized calcium-binding adaptor molecule 1 (Iba1) microglial marker. In the next stage, methods are described for dye-loading (calcium-sensitive Fluo 4-AM) of cultured cells and the recordings of Ca2+ changes in video imaging experiments on live cells.
The examples of video recordings consist of: (1) cases of Ca2+ imaging of cultured astrocytes acutely exposed to immunoglobulin G (IgG) isolated from ALS patients, showing a characteristic and specific response compared to the response to ATP as demonstrated in the same experiment. Examples also show a more pronounced transient rise in intracellular calcium concentration evoked by ALS IgG in hSOD1G93A astrocytes compared to non-transgenic controls; (2) Ca2+ imaging of cultured astrocytes during a depletion of calcium stores by thapsigargin (Thg), a non-competitive inhibitor of the endoplasmic reticulum Ca2+ ATPase, followed by store-operated calcium entry elicited by the addition of calcium in the recording solution, which demonstrates the difference between Ca2+ store operation in hSOD1G93A and in non-transgenic astrocytes; (3) Ca2+ imaging of the cultured microglia showing predominantly a lack of response to ALS IgG, whereas ATP application elicited a Ca2+ change. This paper also emphasizes possible caveats and cautions regarding critical cell density and purity of cultures, choosing the correct concentration of the Ca2+ dye and dye-loading techniques.

We present here a protocol on how to prepare primary cultures of glial cells, astrocytes, and microglia from rat cortices for time-lapse video imaging of intracellular Ca2+ for research on pathophysiology of amyotrophic lateral sclerosis in the hSOD1G93A rat model.

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to ...

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through multipotent intermediates. This technique not only holds great promises in the field of disease modeling, as it allows to convert, for example, fibroblasts for patients suffering neurodegenerative diseases into neurons, but also represents a promising alternative for cell-based replacement therapies. In this context, a major scientific breakthrough was the demonstration that differentiated non-neural cells within the central nervous system, such as astrocytes, could be converted into functional neurons in vitro. Since then, in vitro direct reprogramming of astrocytes into neurons has provided substantial insights into the molecular mechanisms underlying forced identity conversion and the hurdles that prevent efficient reprogramming. However, results from in vitro&#160;experiments performed in different labs are difficult to compare due to differences in the methods used to isolate, culture, and reprogram astrocytes. Here, we describe a detailed protocol to reliably isolate and culture astrocytes with high purity from different regions of the central nervous system of mice at postnatal ages via magnetic cell sorting. Furthermore, we provide protocols to reprogram cultured astrocytes into neurons via viral transduction or DNA transfection. This streamlined and standardized protocol can be used to investigate the molecular mechanisms underlying cell identity maintenance, the establishment of a new neuronal identity, as well as the generation of specific neuronal subtypes and their functional properties.

Here we describe a detailed protocol to generate highly enriched cultures of astrocytes derived from different regions of the central nervous system of postnatal mice and their direct conversion into functional neurons by the forced expression of transcription factors.

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through ...

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

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