Name: sex differences in mouse hippocampal astrocytes after in-vitro ischemia
Uploaded: 2016-10-25T12:30:00
Description: Watch this Scientific Journal Video about Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia at JoVE.com

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

MERK: Denne studien ble gjennomført i samsvar med anbefaling fra Guide for omsorg og bruk av forsøksdyr i National Institute of Health. Dyret protokollen ble godkjent av University of Wisconsin-Madison, Institutional Animal Care og bruk komité. Primær astrocyte Kultur protokoll som er presentert her er adoptert fra protokollene som presenteres av Zhang Y 19 mfl. Og Cengiz P 20 mfl. Med noen modifikasjoner. 1. hippocampus Dissection og astrocyte Kultur <ol><li> Forber...

<ol>
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For å studere kjønnsforskjeller i egenskaper og funksjon av astrocytter under fysiologiske og patologiske tilstander, utarbeidelse av kjønnede primære astrocytter i cellekultur er et viktig verktøy for å utnytte. I denne studien rapporterer vi en svært effektiv og reproduserbar metode for å kultur et høyanriket homogen befolkning på kjønnede hippocampus astrocytter fra nyfødt (P0-P2) C57Bl / 6 (villtype) eller K19F (GFAP null) museunger in vitro. Etablering av denne metodikken hjelper etterforskerne...

Astrocytter er en av de viktigste nøkkelspillere i sentralnervesystemet (CNS). Økende bevis indikerer at rollene som astrocytter er mer enn å gi nevronale støtte. Faktisk kan rollene til astrocytter under fysiologiske betingelser være meget komplekse, for eksempel styring av migrasjon av utviklings aksoner 1, regulerer CNS blodstrøm 2, å opprettholde pH homeostase av den synaptiske interstitiell væske 3, og som deltar i blod-hjerne-barrieren 4 og synaptisk transmisjon fem. Under patologiske tilstander, astrocytter svare på skade med en prosess som kalles reaktiv astrogliosis hvori morfologien, antall, plassering, topografi (med hensyn til avstand fra fornærmelse) og funksjon av astrocyttene kan endre seg i en heterogen måte 6,7. Astrogliosis sett følgende neonatal hypoksisk iskemisk encefalopati kanskje bidrar til sykelighet og dødelighet av nyfødte <sopp&gt; 8. Nylige kliniske og eksperimentelle studier indikerer at graden av hjerneskade synes å være kjønnsavhengig, og at de mannlige nyfødte er mer utsatt for virkningen av hypoksi / ischemi (HI) -relaterte hjerneskade, noe som resulterer i mer alvorlige nevrologiske utfall sammenlignet kvinner med sammenlignbare hjerneskader 9-11. Selv om lokalisering av skaden er avhengig av fosterets alder og varigheten og alvorlighetsgraden av fornærmelse, er hippocampus en av de mest vanlig bevirkes områder i CNS etter betegnelsen neonatal HI, og økt hippocampus astrogliosis er blitt bekreftet ved oppregulering av den Glial Fibrillary Surt Protein (GFAP) 3 d etter neonatal HI 7,10,12,13. Kjønnsforskjeller i astrocyte funksjon ble vist i både nyfødte og voksne gnagere etter cerebral iskemi 14,15. I tillegg ble hann astrocytic mottakelighet for in-vitro-ischemi vist ved øket cell død sammenlignet med kvinnelige kortikale astrocytter i kultur 16. Kjønnsforskjeller start in-utero og fortsetter til døden 17. I løpet av det siste tiåret, har betydningen av å inkludere kjønnene i eksperimentelle forhold i cellekultur og i-vivo studier vært vektlegging av Institute of Medicine og NIH til å søke grunnleggende kunnskap i kjønnsforskjeller sett i fysiologiske og patologiske tilstander 17,18 . Utvikling av pålitelige metoder for å isolere og opprettholde bestandene av sexed hippocampus astrocytter er viktig å forstå den cellulære grunnlag av kjønnsforskjeller i de patologiske konsekvenser av neonatal HI. Denne studien er designet for å gi teknikker for å forberede beriket kjønnsspesifikke hippocampus astrocyttkulturer fra nyfødte mus for å vurdere rollene til GFAP-immunoreaktive astrocytter følgende Oxygen / glukose deprivasjon (OGD) og Reoksygeneringen (Reox), indusereHI i cellekultur miljø. Denne teknikken kan brukes til å teste en hvilken som helst hypotese med hensyn til hippocampus-astrocytter i neonatale hanner og hunner i henhold til normoksisk og iskemiske tilstander.

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

sex differences in mouse hippocampal astrocytes after in-vitro ischemia

Astrogliosis følgende hypoksi / iskemi (HI) -relaterte hjerneskade spiller en rolle i økt sykelighet og dødelighet hos nyfødte. Nyere kliniske studier indikerer at graden av hjerneskade synes å være sex avhengig, og at de mannlige nyfødte er mer utsatt for virkningene av HI-relaterte hjerneskade, noe som resulterer i mer alvorlige nevrologiske utfall sammenlignet med kvinner med tilsvarhjerneskader. Utviklingen av pålitelige metoder for å isolere og opprettholde høyanriket populasjoner av sexed hippocampus astrocytter er viktig å forstå den cellulære grunnlag av kjønnsforskjeller i de patologiske konsekvenser av neonatal HI. I denne studien, beskriver vi en fremgangsmåte for å lage kjønnsspesifikke hippocampus astrocyttkulturer som er utsatt for en modell for in-vitro-ischemi, oksygen-glukosemangel, etterfulgt av reoksygenering. Etterfølgende reaktiv astrogliosis ble undersøkt av immunostaining for Glial Fibrillary Surt Protein (GFAP) og S100B. Denne metoden gir et nyttig verktøy for å studere rollen til hann- og hunn hippocampus astrocytter følgende neonatal HI, hver for seg.

Forstå roller kjønnede astrocyttkulturer fungerer under fysiologiske eller patofysiologiske tilstander har blitt umåtelig belyst ved å dyrke disse cellene under in vitro betingelser. Det viktige aspektet med å utføre seksuelle dyrkning, er å bestemme kjønn av musen pup før bruk. Vi har fastslått kjønn på mus genetisk ved PCR og ved visuell vurdering (figur 2) 16. Metodikken for kjønnsbestemmelse ved hjelp av PCR ble vedtatt med endringer ...

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

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrocytter er en av de viktigste nøkkelspillere i sentralnervesystemet (CNS). Her rapporterer vi en praktisk metode for sexed hippocampus astrocyte kultur protokoll for å studere mekanismene bak astrocyte funksjon i mannlige og kvinnelige nyfødte valper etter in vitro iskemi.

Kjønnsforskjeller i Muse Hippokampale Astrocytter etter<em&gt; In-Vitro</em&gt; iskemi

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

Research

JoVE Journal

Neuroscience

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Dual Elektrofysiologiske Opptak av Synaptically-fremkalt Astroglial og Neuronal Responses i akutt hippocampus Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Isolation and Culture of Mouse Cortical Astrocytes

Isolasjon og kultur av mus Kortikale Astrocytes

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

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Modellering astrocytom Patogenese<em&gt; In Vitro</em&gt; Og<em&gt; In Vivo</em&gt; Bruke kortikal astrocytter eller nevrale stamceller fra Betinget, Genetisk konstruert Mus

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

Immunohistokjemisk Visualisering av hippocampus Neuron aktivitet Etter Spatial læring i en musemodell for nevrologiske lidelser

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we ...

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

Protokoll for Tredimensjonal Confocal Morfometrisk Analyse av Astrocytter

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

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

Tuning i Hippocampal Theta Band<em&gt; I Vitro</em&gt;: Metoder for opptak fra isolert gnagere Septohippocampal Circuit

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent ...

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes

In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markører for å individualisere kortikale mus astrocytter

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

Neuronal Differensiering fra mus embryonale stamceller In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Horizontal Hippocampal Slices of the Mouse Brain

Horisontale Hippocampal skiver av musen hjernen

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...