Gli autori vogliono ringraziare Paola López Pieraldi per l'assistenza tecnica. A.H.M è grato per le borse di studio 8G12MD007600 e U54-NS083924 supportato questa pubblicazione. Ringraziamo NIH-NIMHD-G12-MD007583 borsa di studio per il supporto della struttura. D.E.R.A. è grata per la borsa di studio fornito da NIHNIGMS-R25GM110513. Siamo grati per l'uso della zona di strumentazione comune e concedere l'aiuto del dottor Priscila Sanabria per l'uso della struttura Imaging ottico del programma RCMI da G12MD007583. Inoltre, vogliamo ringraziare Jose Padilla per il suo ruolo eccezionale in riprese e montaggio il protocollo visual.

<ol>
	<li>Goldstein, L. B., Bertels, C., Davis, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interrater+reliability+of+the+NIH+stroke+scale.">Interrater reliability of the NIH stroke scale.</a> Arch Neurol. 46 (6), 660-662 (1989).</li><li>Hinkle, J. L., Guanci, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+ischemic+stroke+review.">Acute ischemic stroke review.</a> J Neurosci Nurs. 39 (5), 285-293 (2007).</li><li>Kassner, A., Merali, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Blood-Brain+Barrier+Disruption+in+Stroke.">Assessment of Blood-Brain Barrier Disruption in Stroke.</a> Stroke. 46 (11), 3310-3315 (2015).</li><li>Moskowitz, M. A., Lo, E. H., Iadecola, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+science+of+stroke:+mechanisms+in+search+of+treatments.">The science of stroke: mechanisms in search of treatments.</a> Neuron. 67 (2), 181-198 (2010).</li><li>Ben Haim, L., Carrillo-de Sauvage, M. A., Ceyzeriat, K., Escartin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elusive+roles+for+reactive+astrocytes+in+neurodegenerative+diseases.">Elusive roles for reactive astrocytes in neurodegenerative diseases.</a> Front Cell Neurosci. 9, 278 (2015).</li><li>Broderick, J., 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=Guidelines+for+the+Management+of+Spontaneous+Intracerebral+Hemorrhage+in+Adults+2007+Update:+A+Guideline+From+the+American+Heart+Association/American+Stroke+Association+Stroke+Council,+High+Blood+Pressure+Research+Council,+and+the+Quality+of+Care+and+Outcomes+in+Research+Interdisciplinary+Working+Group:+The+American+Academy+of+Neurology+affirms+the+value+of+this+guideline+as+an+educational+tool+for+neurologists.">Guidelines for the Management of Spontaneous Intracerebral Hemorrhage in Adults 2007 Update: A Guideline From the American Heart Association/American Stroke Association Stroke Council, High Blood Pressure Research Council, and the Quality of Care and Outcomes in Research Interdisciplinary Working Group: The American Academy of Neurology affirms the value of this guideline as an educational tool for neurologists.</a> Stroke. 38 (6), 2001-2023 (2007).</li><li>Wang, 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=Oxygen-glucose+deprivation+induced+glial+scar-like+change+in+astrocytes.">Oxygen-glucose deprivation induced glial scar-like change in astrocytes.</a> PLoS One. 7 (5), e37574 (2012).</li><li>Sofroniew, M. 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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=HB-EGF+affects+astrocyte+morphology,+proliferation,+differentiation,+and+the+expression+of+intermediate+filament+proteins.">HB-EGF affects astrocyte morphology, proliferation, differentiation, and the expression of intermediate filament proteins.</a> J Neurochem. 128 (6), 878-889 (2014).</li><li>Robinson, C., Apgar, C., Shapiro, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+Hypertrophy+Contributes+to+Aberrant+Neurogenesis+after+Traumatic+Brain+Injury.">Astrocyte Hypertrophy Contributes to Aberrant Neurogenesis after Traumatic Brain Injury.</a> Neural Plast. , 1347987 (2016).</li><li>Brekke, E., Berger, H. R., Wideroe, M., Sonnewald, U., Morken, T. 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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. 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Questo protocollo descrive l'isolamento degli astrociti da cortecce di ratto. In questo metodo, è fondamentale per ridurre la contaminazione con altri tipi cellulari come microglia, oligodendrociti e fibroblasti. Per ridurre il numero di microglia, si possono adottare diverse misure: cambiando la media, agitazione orbitale e trattamenti chimici. Una volta che la purezza di cultura è confermata dall'immunofluorescenza usando gli indicatori cellulari selettivi o per i contaminanti più prominenti di cella, gli esperiment...

Ictus è definito come "un'acuta disfunzione neurologica di origine vascolare con sviluppo rapido o improvviso dei sintomi e dei segni, corrispondenti alla partecipazione delle zone focali del cervello"1,2. Ci sono due tipi di ictus: ischemico ed emorragico. Quando la disfunzione vascolare è causata da un aneurisma o un malfunzionamento arterovenoso, accompagnato da indebolimento con posteriore rottura di un'arteria, questo è definito ictus emorragico3 che, nella maggior parte dei casi conduce alla morte. Quando un trombo o un embolo ostacola il flusso di sangue, causando una temporanea privazione di ossigeno e glucosio per una regione del cervello, si chiama colpo ischemico4. Inadempimento di nutrire le cellule intorno alla zona interessata o core ischemico conduce ad un squilibrio omeostatico e metabolico, disfunzione energetica, morte neuronale e l'infiammazione5, che può indurre una disabilità permanente per pazienti6.
L'ictus ischemico è una ferita multifattoriale che coinvolge diversi tipi di cellule che reagiscono ed esercitano i loro effetti in diversi momenti. Molte interazioni creano un ambiente difficile per studiare il comportamento delle singole celle. Così, come noi a studiare il contributo di un tipo specifico delle cellule in un ambiente così complesso? Un modello accettato in vitro di ischemia consiste di esponendo le cellule a deprivazione di ossigeno e glucosio (OGD), per un certo periodo, seguito dal restauro delle cellule ad un ambiente normoxic. Questo sistema simula un colpo ischemico seguito da riperfusione di sangue. In questo metodo, le cellule o i tessuti sono esposti a una media glucosio-liberi in un ambiente che ha eliminato l'inceppo di ossigeno, utilizzando una camera ipossica specializzata. Il tempo di incubazione di OGD può variare da pochi minuti fino a 24 h, a seconda l'ipotesi che vuole essere testato. Studi hanno dimostrato che in base ai tempi di OGD e normoxic ambiente, fenotipi specifici del colpo (cioè, acuta o subcronica) può essere realizzato. Astrociti isolati, esposti a OGD con restauro posteriore a condizioni di normossia, è un modello di cellulare ben studiato per simulare la corsa in vitro7. Utilizzando OGD è possibile svelare i meccanismi molecolari indipendenti delle cellule isolate in un ambiente di colpo-come.
Come aumenta la nostra conoscenza della biologia di astrociti, è diventato evidente che sono cruciali per mantenere la sinapsi ed il sostenimento di riparazione, di sviluppo e plasticità neurale8. In condizioni normali, gli astrociti rilasciare e rispondere alle citochine, chemochine, fattori di crescita e gliotrasmettitori, mantenendo l'equilibrio metabolico e l'omeostasi nelle sinapsi5,9. In neuroinflammation acuta, come l'ictus ischemico, queste cellule possono diventano reattive, mostrare una lungo termine sovraespressione della proteina acida fibrillare gliale (GFAP) e mostrano ipertrofia nella loro morfologia5,10, 11 , 12. come si sviluppa l'infarto ischemico, l'omeostasi fornito da astrociti diventa affetto, per quanto riguarda l'assorbimento normale del glutammato, metabolismo energetico, scambio di molecole attive e antiossidante attività13.
Gli astrociti riattivati proliferano intorno al tessuto di infarto mentre leucociti migrano verso la zona leso14. Proliferazione astrocytic può essere misurata usando gli indicatori come antigene nucleare delle cellule di proliferazione (PCNA), Ki67 e bromodeossiuridina (BrdU)15. Questa risposta proliferativa viene generata in un modo dipendente dal tempo e aiuta a formare la cicatrice gliale, matrice di astrociti reattivi irreversibilmente lungo il parenchima del sito danneggiato dopo un infortunio9. Una delle funzioni iniziali di questa cicatrice è di limitare lo stravaso delle cellule immuni da questa zona. Tuttavia, gli studi hanno mostrato che la cicatrice diventa un impedimento fisico per gli assoni estendere, come essi rilasciare molecole inibendo la crescita assonale e creare una barriera fisica che impedisce che gli assoni che si estende intorno alla zona danneggiata16. Tuttavia, ci è prove scientifiche che dimostrano che dopo una lesione del midollo spinale, completamente prevenire la formazione di cicatrice gliale può alterare la rigenerazione di assoni17. Così, il contesto in cui viene misurata la risposta astrocytic specifica, deve essere considerato al momento il quadro della ferita ha studiato.
La metodologia presentata può essere applicata per studiare la funzione individualizzata dei astrocytes dopo privazione di ossigeno del glucosio e può essere modificato a seconda delle domande che lo sperimentatore vuole rispondere. Ad esempio, oltre il cambiamento morfologico e gli indicatori espressi in tempi diversi di OGD, i surnatanti dagli astrociti esposti a OGD possono essere analizzati ulteriormente per identificare fattori solubili rilasciati da queste cellule, o usato come un media condizionati per valutare la effetto in altre cellule del cervello. Questo approccio permette studi sulla reattività di astrociti che potrebbe portare alla delucidazione dei fattori che regolano e modulano la loro risposta in uno scenario di ictus ischemico.

<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

Ictus ischemico è una lesione complessa del cervello causata da un trombo o embolo che ostruisce il flusso di sangue alle parti del cervello. Questo porta alla privazione di ossigeno e glucosio, che causa guasto di energia e morte neuronale. Dopo un insulto di colpo ischemico, astrociti diventano reattivi e proliferano attorno al sito di lesione, come si sviluppa. In questo scenario, è difficile studiare il contributo specifico dei astrocytes per la regione del cervello esposto ad ischemia. Di conseguenza, questo articolo introduce una metodologia per studiare la reattività Astrocita primario e proliferazione sotto un modello in vitro di un ambiente di ischemia-simile, chiamato privazione di ossigeno glucosio (OGD). Gli astrociti sono stati isolati da 1-4 giorno-vecchi ratti neonatali e il numero di cellule astrocytic aspecifica è stata valutata utilizzando Astrocita marcatore selettivo della proteina acida fibrillare gliale (GFAP) e macchiatura nucleare. Il periodo in cui gli astrociti sono sottoposti alla condizione OGD può essere personalizzato, così come la percentuale di ossigeno a che sono esposti. Questa flessibilità permette agli scienziati di caratterizzare la durata della condizione ischemica-come in diversi gruppi di cellule in vitro. Questo articolo discute i tempi di consegna di OGD che inducono la reattività di astrociti, morfologia ipertrofica e proliferazione come misurato dall'immunofluorescenza usando proliferando Cell Nuclear Antigen (PCNA). Oltre alla proliferazione, astrociti subiscono l'energia e lo sforzo ossidativo e rispondono agli OGD attraverso il rilascio di fattori solubili nel mezzo delle cellule. Questo mezzo possa essere raccolte e utilizzato per analizzare gli effetti delle molecole rilasciate dai astrocytes in colture neuronali primarie senza interazione cellula--cellula. In sintesi, questo modello della coltura cellulare primaria in modo efficiente consente di comprendere il ruolo degli astrociti isolati dopo un danno.

Una delle preoccupazioni principali della cultura astrocytic primario è la presenza di altre cellule come i neuroni, oligodendrociti, fibroblasti e microglia. Nella Figura 1, cellule isolate da cortecce di ratto avevano preparato cambia ogni 3 giorni e sono stati o non trattata o trattata con aggiunto LME per 1 h. 24 h più successivamente, le cellule immunostained per GFAP e controcolorati con DAPI. Le cellule non trattate hanno mostrato una media di 39% no...

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

Ictus ischemico è un evento complesso in cui il contributo specifico degli astrociti nella regione colpite del cervello esposto alla privazione di ossigeno glucosio (OGD) è difficile da studiare. Questo articolo introduce una metodologia per ottenere gli astrociti isolati e studiare la loro reattività e la proliferazione in condizioni OGD.

Monitoraggio del Astrocyte reattività e proliferazione in Vitro in condizioni ischemiche-come

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

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

9.8K Views.  Universidad Central del Caribe. Ictus ischemico è un evento complesso in cui il contributo specifico degli astrociti nella regione colpite del cervello esposto alla privazione di ossigeno glucosio (OGD) è difficile da studiare. Questo articolo introduce una metodologia per ottenere gli astrociti isolati e studiare la loro reattività e la proliferazione in condizioni 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.

Un neuronale e astrociti Co-Cultura Assay per l&#39;analisi dei contenuti elevati di neurotossicità

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

Ricerca

Biologia

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.

Isolamento e la coltura di astrociti corticali mouse

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

Neuroscienze

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.

Tessutale tridimensionale allineato Astrocyte reti per ricapitolare i meccanismi dello sviluppo e facilitare la rigenerazione del sistema nervoso

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

Bioingegneria

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.

Protocollo per tridimensionale confocale morfometrica Analisi di astrociti

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.

Le differenze di sesso in ippocampali di topo astrociti dopo<em&gt; In-Vitro</em&gt; 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.

Coltura In Vivo-piace murini astrociti utilizzando il protocollo AWESAM veloce, semplice e poco costoso

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.

In vitro dosaggi per valutare la formazione e la rottura del vaso come la barriera emato-cerebrale

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

Medicina

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.

Un modello di coltura cellulare per studiare il ruolo delle interazioni Neuron-Glia in Ischemia

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.

Colture primarie di astrociti e microglia di ratto e loro utilizzo nello studio della sclerosi laterale amiotrofica

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.

Isolamento e riprogrammazione neuronale diretta degli astrociti di topo

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

Monitoraggio del Astrocyte reattività e proliferazione in Vitro in condizioni ischemiche-come

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