The authors acknowledge the funding support by Funda&#231;&#227;o para a Ci&#234;ncia e a Tecnologia through Projects UIDB/00709/2020, POCI-01-0145-FEDER-029311 and the fellowship SFRH/BD/135936/2018 to JP, by &#8216;&#8216;Programa Operacional do Centro, Centro 2020&#8221; through the project CENTRO-01-0145-FEDER-000013 and funding to the PPBI-Portuguese Platform of BioImaging through the Project POCI-01-0145-FEDER-022122.

All animals used were bred at the CICS-UBI Health Science Research Centre in accordance with the national ethical requirements for animal research and with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Directive 2010/63/EU).
1. Rat embryo cortex primary cell culture
<ol>
	<li>Culture medium preparation
	<ol>
		<li>Prepare the Neurobasal Medium (NBM) by adding the following supplements: 2% B27, 0.5 mM glutamine, 25 &#181;...

<ol>
	<li>Donkor, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stroke+in+the+21(st)+Century:+A+Snapshot+of+the+Burden,+Epidemiology,+and+Quality+of+Life.">Stroke in the 21(st) Century: A Snapshot of the Burden, Epidemiology, and Quality of Life.</a> Stroke Research and Treatment. 2018, 3238165 (2018).</li><li>Dirnagl, U., Iadecola, C., Moskowitz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathobiology+of+ischaemic+stroke:+an+integrated+view.">Pathobiology of ischaemic stroke: an integrated view.</a> Trends in Neurosciences. 22 (9), 391-397 (1999).</li><li>Woodruff, T. M., 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=Pathophysiology,+treatment,+and+animal+and+cellular+models+of+human+ischemic+stroke.">Pathophysiology, treatment, and animal and cellular models of human ischemic stroke.</a> Molecular Neurodegeneration. 6 (1), 11 (2011).</li><li>Berezowski, V., Fukuda, A. M., Cecchelli, R., Badaut, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+and+astrocytes:+a+concerto+en+duo+in+ischemic+pathophysiology.">Endothelial cells and astrocytes: a concerto en duo in ischemic pathophysiology.</a> International Journal of Cell Biology. 2012, 176287 (2012).</li><li>Roque, C., Baltazar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Astrocytes+on+the+Injury+Induced+by+In+vitro+Ischemia.">Impact of Astrocytes on the Injury Induced by In vitro Ischemia.</a> Cellular and Molecular Neurobiology. 37 (8), 1521-1528 (2017).</li><li>Ransom, B. R., Ransom, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes:+multitalented+stars+of+the+central+nervous+system.">Astrocytes: multitalented stars of the central nervous system.</a> Methods in Molecular Biology. 814, 3-7 (2012).</li><li>Halassa, M. M., Fellin, T., Haydon, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tripartite+synapse:+roles+for+gliotransmission+in+health+and+disease.">The tripartite synapse: roles for gliotransmission in health and disease.</a> Trends in Molecular Medicine. 13 (2), 54-63 (2007).</li><li>Forder, J. P., Tymianski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postsynaptic+mechanisms+of+excitotoxicity:+Involvement+of+postsynaptic+density+proteins,+radicals,+and+oxidant+molecules.">Postsynaptic mechanisms of excitotoxicity: Involvement of postsynaptic density proteins, radicals, and oxidant molecules.</a> Neuroscience. 158 (1), 293-300 (2009).</li><li>Basic Kes, V., Simundic, A. M., Nikolac, N., Topic, E., Demarin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pro-inflammatory+and+anti-inflammatory+cytokines+in+acute+ischemic+stroke+and+their+relation+to+early+neurological+deficit+and+stroke+outcome.">Pro-inflammatory and anti-inflammatory cytokines in acute ischemic stroke and their relation to early neurological deficit and stroke outcome.</a> Clinical Biochemistry. 41 (16-17), 1330-1334 (2008).</li><li>Gursoy-Ozdemir, Y., Can, A., Dalkara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reperfusion-induced+oxidative/nitrative+injury+to+neurovascular+unit+after+focal+cerebral+ischemia.">Reperfusion-induced oxidative/nitrative injury to neurovascular unit after focal cerebral ischemia.</a> Stroke. 35 (6), 1449-1453 (2004).</li><li>Cekanaviciute, E., 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=Astrocytic+transforming+growth+factor-beta+signaling+reduces+subacute+neuroinflammation+after+stroke+in+mice.">Astrocytic transforming growth factor-beta signaling reduces subacute neuroinflammation after stroke in mice.</a> Glia. 62 (8), 1227-1240 (2014).</li><li>Huang, L., 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=Glial+scar+formation+occurs+in+the+human+brain+after+ischemic+stroke.">Glial scar formation occurs in the human brain after ischemic stroke.</a> International Journal of Medical Sciences. 11 (4), 344-348 (2014).</li><li>Rodriguez-Grande, B., 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=The+acute-phase+protein+PTX3+is+an+essential+mediator+of+glial+scar+formation+and+resolution+of+brain+edema+after+ischemic+injury.">The acute-phase protein PTX3 is an essential mediator of glial scar formation and resolution of brain edema after ischemic injury.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 34 (3), 480-488 (2014).</li><li>Cheng, X., 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=Dynamic+Alterations+of+Brain+Injury,+Functional+Recovery,+and+Metabolites+Profile+after+Cerebral+Ischemia/Reperfusion+in+Rats+Contributes+to+Potential+Biomarkers.">Dynamic Alterations of Brain Injury, Functional Recovery, and Metabolites Profile after Cerebral Ischemia/Reperfusion in Rats Contributes to Potential Biomarkers.</a> Journal of Molecular Neuroscience. 70 (5), 667-676 (2020).</li><li>Wang, X., 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=Dual+role+of+intrauterine+immune+challenge+on+neonatal+and+adult+brain+vulnerability+to+hypoxia-ischemia.">Dual role of intrauterine immune challenge on neonatal and adult brain vulnerability to hypoxia-ischemia.</a> Journal of Neuropathology &amp; Experimental Neurology. 66 (6), 552-561 (2007).</li><li>Panahpour, H., Farhoudi, M., Omidi, Y., Mahmoudi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+In+vivo+Assessment+of+Blood-Brain+Barrier+Disruption+in+a+Rat+Model+of+Ischemic+Stroke.">An In vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke.</a> Journal of Visualized Experiments. (133), e57156 (2018).</li><li>Laake, J. H., Haug, F. M., Wieloch, T., Ottersen, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+in+vitro+model+of+ischemia+based+on+hippocampal+slice+cultures+and+propidium+iodide+fluorescence.">A simple in vitro model of ischemia based on hippocampal slice cultures and propidium iodide fluorescence.</a> Brain Research Protocols. 4 (2), 173-184 (1999).</li><li>Cimarosti, H., Kantamneni, S., Henley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischaemia+differentially+regulates+GABA(B)+receptor+subunits+in+organotypic+hippocampal+slice+cultures.">Ischaemia differentially regulates GABA(B) receptor subunits in organotypic hippocampal slice cultures.</a> Neuropharmacology. 56 (8), 1088-1096 (2009).</li><li>Cho, S., 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=Spatiotemporal+evidence+of+apoptosis-mediated+ischemic+injury+in+organotypic+hippocampal+slice+cultures.">Spatiotemporal evidence of apoptosis-mediated ischemic injury in organotypic hippocampal slice cultures.</a> Neurochemistry International. 45 (1), 117-127 (2004).</li><li>Dong, W. Q., Schurr, A., Reid, K. H., Shields, C. B., West, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rat+Hippocampal+Slice+Preparation+as+an+Invitro+Model+of+Ischemia.">The Rat Hippocampal Slice Preparation as an Invitro Model of Ischemia.</a> Stroke. 19 (4), 498-502 (1988).</li><li>Tamura, R., 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=Neuroprotective+effects+of+adenosine+deaminase+in+the+striatum.">Neuroprotective effects of adenosine deaminase in the striatum.</a> Journal of Cerebral Blood Flow and Metabolism. 36 (4), 709-720 (2016).</li><li>Dennis, S. H., 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=Oxygen/Glucose+Deprivation+Induces+a+Reduction+in+Synaptic+AMPA+Receptors+on+Hippocampal+CA3+Neurons+Mediated+by+mGluR1+and+Adenosine+A(3)+Receptors.">Oxygen/Glucose Deprivation Induces a Reduction in Synaptic AMPA Receptors on Hippocampal CA3 Neurons Mediated by mGluR1 and Adenosine A(3) Receptors.</a> Journal of Neuroscience. 31 (33), 11941-11952 (2011).</li><li>Bar El, Y., Kanner, S., Barzilai, A., Hanein, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+changes+in+neuron-astrocyte+networks+in+culture+under+the+effect+of+norepinephrine.">Activity changes in neuron-astrocyte networks in culture under the effect of norepinephrine.</a> PLoS One. 13 (10), (2018).</li><li>Kuszczyk, M. A., 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=Blocking+the+Interaction+between+Apolipoprotein+E+and+A+beta+Reduces+Intraneuronal+Accumulation+of+A+beta+and+Inhibits+Synaptic+Degeneration.">Blocking the Interaction between Apolipoprotein E and A beta Reduces Intraneuronal Accumulation of A beta and Inhibits Synaptic Degeneration.</a> American Journal of Pathology. 182 (5), 1750-1768 (2013).</li><li>Skaper, S. D., Facci, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+Nervous+System+Neuron-Glia+co-Culture+Models+and+Application+to+Neuroprotective+Agents.">Central Nervous System Neuron-Glia co-Culture Models and Application to Neuroprotective Agents.</a> Methods in Molecular Biology. 1727, 63-80 (2018).</li><li>Fang, A., 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=Effects+of+astrocyte+on+neuronal+outgrowth+in+a+layered+3D+structure.">Effects of astrocyte on neuronal outgrowth in a layered 3D structure.</a> BioMedical Engineering OnLine. 18 (1), 74 (2019).</li><li>Fernando, G., Yamila, R., Cesar, G. J., Ramon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+Effects+of+neuroEPO+Using+an+In+vitro+Model+of+Stroke.">Neuroprotective Effects of neuroEPO Using an In vitro Model of Stroke.</a> Behavioral Sciences. 8 (2), (2018).</li><li>Zhao, L. R., Willing, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+endogenous+capacity+to+repair+a+stroke-damaged+brain:+An+evolving+field+for+stroke+research.">Enhancing endogenous capacity to repair a stroke-damaged brain: An evolving field for stroke research.</a> Progress in Neurobiology. 163, 5-26 (2018).</li><li>Crandall, J. E., Jacobson, M., Kosik, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogenesis+of+microtubule-associated+protein+2+(MAP2)+in+embryonic+mouse+cortex.">Ontogenesis of microtubule-associated protein 2 (MAP2) in embryonic mouse cortex.</a> Brain Research. 393 (1), 127-133 (1986).</li><li>Chamak, B., Fellous, A., Glowinski, J., Prochiantz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAP2+expression+and+neuritic+outgrowth+and+branching+are+coregulated+through+region-specific+neuro-astroglial+interactions.">MAP2 expression and neuritic outgrowth and branching are coregulated through region-specific neuro-astroglial interactions.</a> Journal of Neuroscience. 7 (10), 3163-3170 (1987).</li><li>Almeida, A., Medina, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+for+the+isolation+of+metabolically+active+mitochondria+from+rat+neurons+and+astrocytes+in+primary+culture.">A rapid method for the isolation of metabolically active mitochondria from rat neurons and astrocytes in primary culture.</a> Brain Research Protocols. 2 (3), 209-214 (1998).</li><li>Bessa, A., 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=GPER:+A+new+tool+to+protect+dopaminergic+neurons.">GPER: A new tool to protect dopaminergic neurons.</a> Biochim Biophys Acta. 1852 (10), 2035-2041 (2015).</li><li>De Simone, U., Caloni, F., Gribaldo, L., Coccini, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Co-culture+Model+of+Neurons+and+Astrocytes+to+Test+Acute+Cytotoxicity+of+Neurotoxic+Compounds.">Human Co-culture Model of Neurons and Astrocytes to Test Acute Cytotoxicity of Neurotoxic Compounds.</a> International Journal of Toxicology. 36 (6), 463-477 (2017).</li><li>Yang, L., Shah, K. K., Abbruscato, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+model+of+ischemic+stroke.">An in vitro model of ischemic stroke.</a> Methods in Molecular Biology. 814, 451-466 (2012).</li><li>Holloway, P. M., Gavins, F. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+Ischemic+Stroke+In+Vitro:+Status+Quo+and+Future+Perspectives.">Modeling Ischemic Stroke In Vitro: Status Quo and Future Perspectives.</a> Stroke. 47 (2), 561-569 (2016).</li><li>Rossi, D. J., Brady, J. D., Mohr, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+metabolism+and+signaling+during+brain+ischemia.">Astrocyte metabolism and signaling during brain ischemia.</a> Nature Neuroscience. 10 (11), 1377-1386 (2007).</li></ol>

The authors declare that they have no conflict of interests.

The method here described consists of the astrocyte and neuron isolation from rat embryonic cortical tissue, allowing the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures. It was adapted from a previous study of our group5, where the cortical neuron&#8211;glia and neuron-enriched embryonic cultures isolation were described and the two cultures characterized. Using these cultures, Roque et al. found that astrocytes play a key role in responding to an ischemic damage a...

According to data from the World Health Organization, about 5.5 million people die every year from ischemic stroke1. This condition is characterized by the interruption of blood flow to a certain brain region, resulting in a reversible or irreversible loss in the supply of oxygen and nutrients to the tissue, which alters tissue function and leads to mitochondrial dysfunction, calcium dysregulation, glutamate excitotoxicity, inflammation and cell loss2,3.
Apart from vascular cells, neuronal and glial cells are involved in the pathophysiology of the ischemic stroke4. In particular, astrocytes are essential to the maintenance of neurons and recently were shown to play a critical role in the response to the ischemic lesion5. This type of glial cell performs functions of structural support, defence against oxidative stress, synthesis of neurotransmitters, stabilization of cell-cell communication, among others6. Along with neurons, astrocytes play a direct role in synaptic transmission, regulating the release of molecules such as adenosine triphosphate, gamma-aminobutyric acid and glutamate7. Part of the injury induced by ischemia is caused by the excessive release of glutamate and its accumulation at the synaptic cleft, leading to the overactivation of N-methyl-D-aspartate receptors, activating downstream signalling cascades, ultimately resulting in excitotoxicity8. Since astrocytes are able to remove glutamate from the synaptic cleft and convert it into glutamine, they are crucial in defending against excitotoxicity, thereby exerting a neuroprotective effect on ischemia. These cells also play a role in ischemia-induced neuroinflammation. After the ischemic insult, activated astrocytes undergo morphologic changes (hypertrophy), proliferate, and show an increase in glial fibrillary acidic protein (GFAP) expression. They can become reactive (astrogliosis), releasing pro-inflammatory cytokines such as tumour necrosis factor-&#945;, interleukin-1&#945; and interleukin-1&#946;, and producing free radicals, including nitric oxide and superoxide, which in turn can induce neuronal death9,10. In contrast, reactive astrocytes may also play a neuroprotective effect, since they release anti-inflammatory cytokines, such as transforming growth factor-&#946;, that is upregulated after stroke11. Moreover, they can generate a glial scar, which can limit tissue regeneration by inhibiting axonal sprouting; however, this glial scar can isolate the injury site from viable tissue, thus preventing a cascading wave of uncontrolled tissue damage12,13.
Thus, it is imperative to establish models that allow studying neuron-glia interactions under an ischemic injury in order to find therapeutic strategies that limit or reverse the effects of ischemic injury. Compared to other models used to study ischemic injury, namely in vivo models14,15,16, organotypic cultures17,18,19 and acute brain slices20,21,22, primary cell cultures are less complex, which makes possible the study of individual contributions of each cell type in the pathophysiology of ischemic stroke and how each cell type responds to possible therapeutic targets. Typically, in order to study the interactions between neuron-enriched cultures and astrocyte-enriched cultures, neurons and glial cells of postnatal origin are used23,24, or postnatal glial cells and embryonic neurons25,26. Herein is proposed a simple approach to establish neuron- or astrocyte-enriched cultures and neuron-glia cultures from the same tissue. These primary cells are obtained from rat embryonic cortex, a region frequently affected by stroke27,28. Moreover, the dissociation of the tissue is performed only by a mechanical procedure. Therefore, this protocol allows isolating cells in the same stage of development, in a fast and inexpensive way, and with high performance and reproducibility.
The crosstalk between astrocytes and neurons can be explored using a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes seeded in multiwell plates. Small paraffin spheres can be used to ensure the separation of the two cell cultures. This approach allows independent manipulation of each cell type before they are brought into contact. For example, it is possible to silence a specific gene in astrocytes and see how it can influence the neuronal vulnerability or protection against ischemic-induced damage. An established method to induce ischemic-like conditions in vitro is oxygen and glucose deprivation (OGD)3, which consists in replacing the regular atmosphere (95% air and 5% CO2) by an anoxic atmosphere (95% N2 and 5% CO2), associated with the omission of glucose.
The method described is suitable for studying the interactions between neurons and astrocytes in the context of ischemic stroke, in a simple, fast, reproducible and inexpensive way.

<table>
	<tbody>
		<tr>
			<td>24 -well culture plates</td>
			<td>Thermo Fischer Scientific</td>
			<td>142475</td>
			<td></td>
		</tr>
		<tr>
			<td>95% N2/5% CO2 gas cylinder</td>
			<td>ArL&#237;quido</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse conjugated to Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A11001</td>
			<td>1/1000 dilution; incubation period - 1 h at room temperature</td>
		</tr>
		<tr>
			<td>Anti-rabbit conjugated to Alexa Fluor 546</td>
			<td>Invitrogen</td>
			<td>A11010</td>
			<td>1/1000 dilution; incubation period - 1 h at room temperature</td>
		</tr>
		<tr>
			<td>B27 supplement (50x)</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Dako Fluorescence Mounting Medium</td>
			<td>Dako</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose anhydrous</td>
			<td>Fisher Scientific</td>
			<td>G/0450/60</td>
			<td>3.4 g/L</td>
		</tr>
		<tr>
			<td>Epifluorescence microscope</td>
			<td>Zeiss</td>
			<td>AxioObserver Z1x</td>
			<td>63x objective</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Biochrom</td>
			<td>S0615</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Sigma-Aldrich</td>
			<td>G1272</td>
			<td>120 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>G8415</td>
			<td>25&#181;M</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G3126</td>
			<td>0.5 mM</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H1399</td>
			<td>2 &#181;M; incubation period - 10 min at room temperature</td>
		</tr>
		<tr>
			<td>Hypoxia incubation chamber</td>
			<td>Stemcell Technologies</td>
			<td>27310</td>
			<td>Chamber used for OGD induction</td>
		</tr>
		<tr>
			<td>Insulin from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>I5500</td>
			<td>5 mg/L</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Sigma-Aldrich</td>
			<td>K-002</td>
			<td>87.5 mg/Kg</td>
		</tr>
		<tr>
			<td>Minimum Essential Medium Eagle medium</td>
			<td>Sigma-Aldrich</td>
			<td>M0268</td>
			<td>warm up to 37 &#176;C before use</td>
		</tr>
		<tr>
			<td>Mouse Anti-MAP2</td>
			<td>Santa Cruz Biotechnology</td>
			<td>Sc-74421</td>
			<td>1/500 dilution; incubation period overnight at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td>warm up to 37 &#176;C before use</td>
		</tr>
		<tr>
			<td>Paraffin pastilles for histology</td>
			<td>Sigma-Aldrich</td>
			<td>1.07164</td>
			<td>Solidification point 56-58&#176;C</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma -Aldrich</td>
			<td>P6148</td>
			<td>4% in PBS</td>
		</tr>
		<tr>
			<td>Penicilin/Streptomycin</td>
			<td>Biochrom</td>
			<td>A 2213</td>
			<td>penicillin (12U/mL) /streptomycin (12&#181;g/mL)</td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P1024</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Anti-GFAP</td>
			<td>DAKO</td>
			<td>Z0334</td>
			<td>1/2000 dilution; incubation period overnight at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Sodium hydrogen carbonate</td>
			<td>Fisher Scientific</td>
			<td>S/4240/60</td>
			<td>2.2g/L</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Sigma-Aldrich</td>
			<td>X1126</td>
			<td>12 mg/Kg</td>
		</tr>
	</tbody>
</table>

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.

To characterize the cultures, immunocytochemistry to assess the number of cells that expressed GFAP or MAP2, widely used markers of astrocytes and neurons (Figure 2), was performed in each type of cortical culture. This analysis revealed that astrocyte-enriched cultures presented 97% of the cells expressing GFAP (Figure 2A). Regarding the neuron-enriched culture 78% of the cells expressed MAP2, 4% of the cells expressed GFAP, and 18% of the cells were both GFAP ...

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

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 ...

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue and results in neuronal loss. Numerous evidences suggest that the interaction between glia and urinal cells exert the pressure effects after an ischemic event. In order to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment.Here we present a simple approach to isolate astrocytes and neurons from rat embryonic cortex, and that by using specific cultural medium allows the establishment of neuron or astrocyte-enriched cultures or neo-glia cultures with high yield and reproducibility. To study the crosstalk between astrocytes and neurons, we propose an approach, based on a co-culture system, in which neurons cultured in cover slips are maintained in contact with a monolayer of astrocytes plated in multi-well plates. The two cultures are maintained a part by small paraffin spheres.This approach allows independent manipulation and the application of specific treatment to each cell type, which represents an advantage in many studies. To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.Start with rat embryonic cortex insulation. The embryos were obtained from a family of rats at 15 days of gestation and are placed in a sterile Falcon tube containing PBS. Still inside the yolk sachet embryos are placed inside a petri dish containing cold PBS.With the help of scissors and tweezers, the yolk sac is broken, and the embryo is removed and placed in another petri dish containing cold PBS over an ice pack. It is necessary to be very careful when breaking the yolk sac not to damage the embryo. Position the embryo under a dissecting microscope, gently fix the embryo using a twizzer.The initial incision should be parallel to the cortex. Be careful not to decapitate the animal. The scalp and are carefully removed not to damage the cortical brain tissue.This next incision separates the cortex. Remove the blood vessels present in the tissue. Finally, using a pipette, transfer the cortical tissue to a Falcon tube with PBS.The single cell suspension is obtained through mechanical digestion of the cortical brain tissue using tips with decreasing diameter. Make sure that the tissue is well homogenized After the digestion, centrifuge the material at 400 Gs for three minutes. Discard the supernatant, and resuspend the sediment in culture medium, previously warmed to 37 degrees.Calculate the total number of cells present in the suspension using a Neubauer chamber and preparate the dilution for the adequate cell density. Finally, seed the cells in the multi-well and incubate at 37 degrees. In order to prepare the material for the co-culture system, heat the paraffin in a heating block until it becomes liquid.Then, with the help of a stereo glass Pasteur pipette prepare small spheres over the cover slips that were previously placed in a multi-well, and coated with Poly-D-Lysine. 24 hours before the two cultures are brought in contact, change the culture medium of neurons and astrocytes to supplement it NBM with or without heat inactivated FBS. When the two cultures are ready to use, transfer the neuron, seated in the cover slips with paraffin spheres to wells containing astrocytes using a tweezers previously immersed in 70%ethanol.After placing both cell types in contact, wait eight to 12 hours, and then start the different stimuli and procedures. The oxygen and glucose deprivation is performed in a culture with seven days of growth. Remove the culture medium, and wash the cells two times with HBSS medium without glucose supplementation.Be sure that all the medium containing glucose is washed. Seal the Hypoxia Chamber and add the gas mix containing 95%nitrogen and 5%carbon dioxide for four minute with a flow of 20 liters for minutes in order to replace the oxygen present inside the chamber. Then, stop the flow and place the Hypoxia Chamber in an incubator at 37 degrees.After the period of oxygen and glucose deprivation, replace the medium, HBSS without glucose supplementation with the appropriate culture medium for the remaining procedures and incubate the cells at 37 degrees. In order to characterize the type of cortical culture, we performed immunohistochemistry in the three types of cortical cultures to assess the number of cells that expressed GFPA or MAP2, which are markers widely used for astrocytic and neuronal cells respectively. These analysis revealed that when this protocol, we were able to obtain a pure enriched culture of astrocytes with about 97%of the cells expressing GFAP.Regarding the neuron-enriched culture, we verified about 78%of the cells expressing MAP2. However we identify about 18%of GFAP and MAP2 negative cells. For the neuron-glia cortical culture, It was observed that about 49%of the cells are MAP2 positive.31%are GFAP positive. And 20%they're not liable for GFAP nor MAP2. Seven days after the cortical culture establishment, the neuron-glia culture and the neuron-enriched culture were subjected to an OGD procedure for 4 and 6 hours.After this procedure, cells were labeled with MAP2 and then the number of MAP2 positive cells were quantified using fluorescence microscopy. In neuron-glia culture, it was observed a decrease of about 30%in the number of the neurons, when the culture was submitted to an OGD period of 4 hours. After an OGD period of 6 hours, the extension of the lesion increases, reaching about 60%of neuronal loss.Regarding the enriched culture of neurons exposed to OGD, it was observed about 41%and 64%decrease in the number of MAP2 cells. Moreover, it was observed that in the neuron-enriched culture, there was a slight increase in the injury extension when compared to the neuron-glia culture also exposed to OGD during 4 hours. In conclusion, here represent an in vitro model to study ischemic stroke established in a simple, fast and expensive and reproducible way.Additionally, the method described also allows to implement neurons and astrocyte-enriched primary cultures, but also a neuron-glia culture. Thus, providing a great in-vitro model for modeling several brain diseases with a higher level of complexity, than immortalized cell lines and pure neuronal or glial cultures.

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Neuroscience

9.1K visualizações.  Universidade da Beira Interior. O AVC isquêmico é uma condição clínica caracterizada pela hipoperfusão do tecido cerebral e resulta em perda neuronal. Numerosas evidências sugerem que a interação entre glia e células urinárias exercem os efeitos de pressão após um evento isquêmico. Para explorar potenciais mecanismos de proteção, é importante desenvolver modelos que permitam estudar interações neurônio-glia em um ambiente isquêmico.Aqui apresentamos uma abordagem simples para isolar astrócitos e...