Die Autoren möchten Paola López Pieraldi für die technische Unterstützung zu danken. A.H.M. ist dankbar für die Zuschüsse 8G12MD007600 und U54-NS083924, die diese Publikation unterstützt. Wir danken NIH-NIMHD-G12-MD007583-Stipendium für die Anlage-Unterstützung. D.E.R.A. ist dankbar für die Gemeinschaft von NIHNIGMS-R25GM110513 zur Verfügung gestellt. Wir sind dankbar für die Nutzung des gemeinsamen Instrumentierung und Gewährung der Beihilfe von Dr. Priscila Sanabria für den Einsatz der optischen Bildgebung Anlage des Programms RCMI durch G12MD007583. Darüber hinaus wollen wir danke Jose Padilla für seine herausragende Rolle in Filmen und schneiden das visuelle Protokoll.

<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. 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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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Dieses Protokoll beschreibt die Isolation der Astrozyten aus Ratte Cortex. Bei dieser Methode ist es entscheidend, Kontamination mit anderen zellulären Arten wie Mikroglia, Oligodendrozyten und Fibroblasten zu verringern. Um die Anzahl der Mikroglia zu reduzieren, können mehrere Schritte unternommen werden: Ändern der Medien, Orbital, rütteln und chemischen Behandlungen. Wenn Kultur Reinheit durch Immunfluoreszenz mittels selektiver zellulären Marker oder für die prominentesten Zelle Verunreinigungen bestätigt ist...

Schlaganfall ist definiert als "eine akute neurologische Dysfunktion vaskulären Ursprungs mit plötzlichen oder schnelle Entwicklung der Symptome und Zeichen, entsprechend der Beteiligung der Schwerpunkte im Gehirn"1,2. Es gibt zwei Arten von Schlaganfällen: ischämische und hämorrhagische. Wenn vaskuläre Dysfunktion verursacht wird, entweder durch ein Aneurysma oder eine arteriovenöse Fehlfunktion, begleitet durch eine Schwächung mit hinteren Ruptur einer Arterie ist dies hämorrhagischer Schlaganfall3 bezeichnet, die in den meisten Fällen zum Tod führt. Wenn ein Thrombus oder eine Lungenembolie Blutfluss behindert, verursacht eine vorübergehende Entziehung von Sauerstoff und Glukose zu einer Region des Gehirns, ischämischen Schlaganfall4heißt es. Bei Nichtbeachtung nähren Zellen um das betroffene Gebiet oder ischämischen Kern führt zu eine homöostatische und metabolische Ungleichgewicht, energetische Dysfunktion, neuronalen Tod und Entzündung5, die eine lebenslange Behinderung für Patienten6auslösen können.
Ischämischen Schlaganfall ist eine multifaktorielle Verletzungen, an denen verschiedene Arten von Zellen, die reagieren und üben ihre Wirkungen zu unterschiedlichen Zeitpunkten. Viele Interaktionen erstellen ein schwierigen Umfelds um das Verhalten einzelner Zellen untersuchen. Also, wie wir den Beitrag von einem bestimmten Zelltyp unter einem komplexen Umfeld zu studieren? Eine anerkannter in-vitro- Modell der Ischämie besteht aus auszusetzen Zellen Sauerstoff und Glukose Deprivation (OGD), für einen bestimmten Zeitraum, gefolgt von der Wiederherstellung der Zellen zu einer inklusieve Umgebung. Dieses System simuliert einen ischämischen Schlaganfall gefolgt von Blut Reperfusion. Bei dieser Methode sind Zellen oder Geweben eine Glukose-freie Medien in einem Umfeld von Sauerstoff, mit einer spezialisierten hypoxischen Kammer gesäubert ausgesetzt. Die OGD Inkubationszeit variiert von wenigen Minuten bis zu 24 Stunden, abhängig von der Hypothese, die getestet werden will. Studien haben gezeigt, dass je nach den Zeiten der OGD und inklusieve Umwelt, bestimmten Phänotypen eines Schlaganfalls (d.h.akute oder subchronische) erreicht werden. Primäre isolierte Astrozyten, OGD mit hinteren Restaurierung inklusieve Bedingungen ausgesetzt ist ein gut untersuchten zellulären Modell, Schlaganfall in-vitro-7zu imitieren. OGD ist möglich, die unabhängige molekularen Mechanismen der isolierten Zellen unter einem Schlaganfall-ähnliche Umgebung zu offenbaren.
Mit zunehmender unser Wissen der Astrozyten Biologie deutlich geworden, dass sie entscheidend für die Aufrechterhaltung der Synapsen und Aufrechterhaltung neuronaler Plastizität, Reparatur und Entwicklung8sind. Unter normalen Bedingungen Astrozyten freizugeben und reagieren auf Zytokine, Chemokine, Wachstumsfaktoren und Gliotransmitters, metabolic-Balance und Homöostase innerhalb von Synapsen5,9zu halten. In akuten Neuroinflammation, wie ischämischen Schlaganfall können diese Zellen reaktive werden, zeigen eine langfristige Überexpression des Glial Fibrillary sauren Protein (GFAP) und zeigen Hypertrophie in ihrer Morphologie5,10, 11 , 12. wie der ischämischen Infarkt entwickelt, die Homöostase von Astrozyten bereitgestellt wird betroffen, bei normalen Glutamat-Aufnahme, Energiestoffwechsel, Austausch von aktiven Molekülen und antioxidative Aktivität13.
Reaktivierte Astrozyten vermehren um das Infarkt-Gewebe während Leukozyten in Richtung der lesioned Bereich14migrieren. Astrocytic Verbreitung kann mit Hilfe von Markern wie wuchernden Cell nuclear Antigen (PCNA), Ki67 und Bromodeoxyuridine (BrdU)15gemessen werden. Diese proliferative Reaktion wird in einer zeitabhängigen Weise erzeugt und es hilft bilden die glial Narbe, ein Array von irreversibel reaktive Astrocyten entlang das Parenchym der beschädigten Seite nach einer Verletzung-9. Eine der ersten Funktionen dieser Narbe ist die Immunzelle Extravasation aus diesem Bereich zu begrenzen. Studien haben jedoch gezeigt, dass die Narbe ein physisches Hindernis für Axone wird zu erweitern, wie sie release-Moleküle axonale Wachstum hemmen, und erstellen Sie eine physische Barriere, die verhindert, dass Axone erweitern um den verletzten Bereich16. Dennoch gibt es wissenschaftliche Beweise dafür, dass nach einer Rückenmarksverletzung ganz glialen Narbenbildung zu vermeiden die Regeneration der Axone17beeinträchtigen kann. So, der Kontext, in der die spezifischen astrocytic Reaktion gemessen wird, muss auf dem Rahmen der Verletzung studierte berücksichtigt werden.
Die vorgestellte Methode kann angewendet werden, um die individuelle Funktion der Astrozyten nach Glukose Sauerstoffmangel zu untersuchen und es kann geändert werden, je nach Fragestellung, die der Prüfer beantworten will. Z. B. neben der morphologischen Veränderungen und die Marker OGD zeitversetzt zum Ausdruck gebracht, die Überstände von Astrozyten OGD ausgesetzt können analysiert werden, weiter um lösliche Faktoren von diesen Zellen freigegeben wird, oder als konditionierten Medium zur Beurteilung zu identifizieren sein Wirkung in anderen Gehirnzellen. Dieser Ansatz ermöglicht Untersuchungen zur Reaktivität von Astrozyten, die die Aufklärung der Faktoren führen könnte, die Regeln und modulieren ihre Antwort in einem Szenario mit ischämischen Schlaganfall.

<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

Ischämischen Schlaganfall ist eine komplexe-Hirn-Trauma durch einen Thrombus oder Lungenembolie behindern Blutfluss in Teilen des Gehirns verursacht. Dies führt zur Entziehung von Sauerstoff und Glukose, die Energie Versagen und neuronalen Tod verursacht. Nach eine Beleidigung des ischämischen Schlaganfalls Astrozyten werden reaktive und vermehren sich um die Verletzung Aufstellungsort, wie es sich entwickelt. In diesem Szenario ist es schwierig, den spezifischen Beitrag der Astrozyten, die Region des Gehirns ausgesetzt Ischämie zu studieren. Dieser Artikel stellt daher eine Methode zur primären Astrozyten Reaktivität und Verbreitung unter einem in Vitro Modell einer Ischämie-ähnlichen Umgebung, genannt Glukose Sauerstoffmangel (OGD) zu studieren. Astrozyten wurden isoliert von 1-4 Tage-alten Neugeborenen Ratten und die Anzahl der unspezifischen astrocytic Zellen wurde anhand Astrozyten selektive Marker Glial Fibrillary sauren Protein (GFAP) und nukleare Färbung. Der Zeitraum, in dem Astrozyten OGD Zustand ausgesetzt sind, sowie der Sauerstoffgehalt anpassbar, denen sie ausgesetzt sind. Diese Flexibilität ermöglicht es Wissenschaftlern, die Dauer der ischämischen-ähnlichen Zustand in verschiedene Gruppen von Zellen in Vitrozu charakterisieren. Dieser Artikel beschreibt die Zeitrahmen von OGD, die Astrozyten Reaktivität, hypertrophe Morphologie und Verbreitung gemessen durch Immunfluoreszenz mit wuchernden Cell Nuclear Antigen (PCNA) induzieren. Neben der Verbreitung Astrozyten Energie- und oxidativen Stress zu unterziehen, und reagieren auf OGD durch Loslassen lösliche Faktoren in der Zelle-Medium. Dieses Medium kann gesammelt und verwendet, um die Auswirkungen von Molekülen veröffentlicht von Astrozyten im neuronalen Primärkulturen ohne Zell-Zell-Interaktion zu analysieren. Zusammenfassend lässt sich sagen diese Primärzelle Kulturmodells effizient einsetzbar zu verstehen, die Rolle der isolierten Astrozyten auf Verletzungen.

Eines der Hauptanliegen der astrocytic Primärkultur ist das Vorhandensein von anderen Zellen wie Neuronen, Oligodendrozyten, Fibroblasten und Mikroglia. In Abbildung 1isolierte Zellen von Ratte Cortex hatten Medien Änderungen alle 3 Tage und waren entweder unbehandelt oder behandelt mit hinzugefügt LME für 1 h 24 h später, Zellen wurden Immunostained für GFAP und counterstained mit DAPI. Unbehandelte Zellen zeigte einen Durchschnitt von 39 % nicht-GFAP ...

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

Ischämischen Schlaganfall ist schwer zu lernen ist ein komplexer Vorgang, in dem der spezifische Beitrag der Astrozyten, die betroffene Hirnregion Glukose Sauerstoffmangel (OGD) ausgesetzt. Dieser Artikel stellt eine Methode zur isolierte Astrozyten zu erhalten und ihre Reaktivität und Verbreitung unter OGD Bedingungen zu studieren.

Überwachung der Astrozyten Reaktivität und Proliferation in Vitro unter ischämischen-ähnlichen Bedingungen

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

Research

JoVE Journal

Neuroscience

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

9.8K Aufrufe.  Universidad Central del Caribe. Ischämischen Schlaganfall ist schwer zu lernen ist ein komplexer Vorgang, in dem der spezifische Beitrag der Astrozyten, die betroffene Hirnregion Glukose Sauerstoffmangel (OGD) ausgesetzt. Dieser Artikel stellt eine Methode zur isolierte Astrozyten zu erhalten und ihre Reaktivität und Verbreitung unter OGD Bedingungen zu studieren.

Serie: 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.

Eine Neuronale und Astrocyte Co-Kultur-Assay für High Content Analyse der Neurotoxizität

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

Forschung

Biologie

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.

Isolation und Kultur von Maus Kortikale Astrozyten

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

Neurowissenschaften

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.

Dreidimensionale Gewebezüchtungen ausgerichtet Astrozyten Netzwerke Nervensystem Regeneration zu rekapitulieren Entwicklungsmechanismen

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

Biotechnik

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.

Protokoll zur dreidimensionalen konfokalen morphometrische Analyse der Astrozyten

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.

Geschlechtsunterschiede in Mäuse Hippocampus Astrozyten nach<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.

In VivoKultivierung-wie Murine Astrozyten über das schnelle, einfache und kostengünstige AWESAM-Protokoll

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- Assays zur Beurteilung der Blut-Hirn-Schranke Mesh-artigen Gefäßbildung und -störung

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

Medizin

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.

Ein Zellkulturmodell zum Studium der Rolle von Neuron-Glia-Interaktionen in Ischämie

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.

Primärkulturen von Rattenastrozyten und Mikroglia und ihre Verwendung bei der Untersuchung der amyotrophen Lateralsklerose

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.

Isolierung und direkte neuronale Reprogrammierung von Maus-Astrozyten

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