Name: assessment of vascular regeneration in the cns using the mouse retina
Uploaded: 2014-06-23T18:45:00
Description: Watch this Scientific Journal Video about Assessment of Vascular Regeneration in the CNS Using the Mouse Retina at JoVE.com

PS besidder en Canada Research Chair i Retinal Cellebiologi og Alcon Research Institute New Investigator Award. Dette arbejde blev støttet af tilskud fra den canadiske Institutes of Health Research (221.478), den canadiske diabetesforening (OG-3-11-3329-PS), naturvidenskab og teknik Forskningsråd Canada (418.637) og instituttet Fighting Blindness Canada. Der blev også leveret af Reseau de Recherche en Santé de la Vision du Québec.

Etik erklæring: Alle dyreforsøg klæber retningslinjer dyrepleje oprettet af Foreningen for Forskning i Vision og Oftalmologi (ARVO) Redegørelse for anvendelsen af dyr i Ophthalmic og Vision Research og Canadian Råd Animal Care. 1.. Oxygen retinopati (OIR) <ol><li> Optag fødselsdato museunger som P0. </li><li> Registrere alle dyrs vægt ved indrejsen i O 2 for at sikre en passende vægt rækkevidde. Bemærk: C57BL / 6 mus ved P17, bør kropsvægten ligge me...

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Hvad er den mest effektive måde at stimulere væksten af ​​nye sunde skibe i nervøs iskæmisk væv? Er det terapeutisk gyldigt at blande sig og accelerere naturligt forekommende vaskulær genvækst? I neuro-iskæmiske patologier såsom iskæmiske retinopatier eller slagtilfælde, er vaskulær degeneration forbundet med nedsat neuronal funktion 35-38. Derfor at modvirke tidlig skade, genindføre regional mikro-cirkulation i den umiddelbare / tidlig segment af sygdommen kan vise sig gavnligt. I en okulær...

Gennem CNS udvikling, nerver, immunceller og blodkar etablere bemærkelsesværdigt koblede netværk for at sikre tilstrækkelig vævsperfusion og tillade overførsel af sensorisk information 1-5. Fordelingen af vaskulære systemer resulterer i utilstrækkelig iltningen af vævet og kompromitteret metabolisk forsyning og er i stigende grad anerkendt som en vigtig bidragyder til patogenesen af neurodegenerative sygdomme 6. Vaskulær frafald og forværringen af neurovaskulær enhed i hjernen, for eksempel, er forbundet med vaskulær demens, vaskulære læsioner i den hvide substans i hjernen 7 og Alzheimers sygdom med stenose af arterioler og små fartøjer 8. Desuden er nedsat vaskulær barriere funktion menes at bidrage multipel sklerose 9 og amyotrofisk lateral sklerose 10. Direkte relevans for den retinale model beskrevet i denne protokol, blændendesygdomme, såsom diabetisk retinopati 11 og præmatur retinopati 12, er 13, kendetegnet af en fase med tidlig vaskulær degeneration. Den efterfølgende iskæmisk stress på neurovascular nethinden udløser en anden fase af overdreven og patologisk neovaskularisering som sandsynligvis stammer som en kompenserende reaktion genetablere ilt og energiforsyning 14-16. En attraktiv strategi for at overvinde den iskæmiske stress, der er centralt for sygdomsprogression er at genoprette funktionelle vaskulære netværk specifikt i iskæmiske områder af neuro-nethinden (figur 2 og 3). Fremprovokere en kontrolleret angiogene respons kan komme på tværs som kontra-intuitivt for en tilstand, hvor anti-angiogenese behandlinger såsom anti-VEGFs betragtes som tilpassede behandlinger. Alligevel er bevis for gyldigheden af ​​denne tilgang montering. For eksempel, øge &quot;fysiologisk-lignende&quot; vaskulær genvækst i ischemic retinopatier er elegant demonstreret gennem indførelse af endotel præcursorceller 17, hæmning af Müller celle-udtrykte VEGF-induceret nedregulering af andre angiogene faktorer 18, injektion af myeloide stamceller 19, hæmning af NADPH oxidase induceret apoptose 20, stigende kosten ω-3 polyumættede fedtsyrer syre indtag 21 behandling med et carboxyterminalt fragment af tryptophan tRNA-syntetase 22 og direkte indgivelse af VEGF eller FGF-2 til beskyttelse af gliaceller 23. Desuden har vi påvist, at modulerende klassiske neuronale vejledning tidskoder såsom Semaphorins eller Netrins i iskæmiske retinopatier accelererer vaskulær regenerering af sunde fartøjer i nethinden og dermed reducerer patologisk angiogenese 24, 25. Af direkte klinisk relevans, flere af de førnævnte dyreforsøg bevis for, at fremme vaskulær regeneration i den tidlige fase af iskæmisk retinopati kan reducere synstruende præ-retinal neovaskularisering 19, 23, 24, 26, sandsynligvis gennem reduktion af iskæmisk byrde. Udarbejdelse af terapeutiske strategier, der stimulerer regenerering af funktionelle fartøjer fortsat en stor udfordring for vaskulære biologer. Her beskriver vi et eksperimentelt system, der beskæftiger musemodel af ilt-induceret retinopati (OIR) at undersøge strategier til at modulere vaskulære genvækst i nethinden. Udviklet af Smith et al. I 1994 27, denne model fungerer som en proxy for menneskerettigheder proliferative retinopati og består af udsætte P7 museunger til 75% O 2, indtil P12 og derefter re-introducere hvalpene til omgivende rum O 2-spænding (Figur 1). Dette paradigme løst efterligner et scenarie, hvor en tidlig spædbarn er ventileretmed O 2. Eksponeringen af museunger til hyperoxi provokerer degeneration af retina kapillærer og microvasculature, og giver en reproducerbar område af vaso-udslettelse (VO), typisk vurderes ved udrejse fra O 2 på P12, selvom maksimal VO område nås ved 48 timer (P9) efter eksponering til O 2 28. Hos mus, de avaskulære VO zoner spontant regenerere i løbet af ugen efter re-introduktion til rumluft og til sidst VO zoner er fuldstændig re-vaskulariseret (Figur 2). Reintroduktion til rumluft af mus udsat for OIR provokerer også præ-retinal neovaskularisering (NV) (maksimal ved P17), der typisk vurderes at bestemme effektiviteten af ​​anti-angiogene behandling paradigmer. I sin reneste form, det OIR model giver en meget reproducerbar og kvantificerbar redskab til at vurdere ilt-induceret vaskulær degeneration og bestemme omfanget af destruktive præ-retinal neovaskularisering 29-31. <p class = &quot;jove_content&quot;&gt; Forskellige eksplorative behandling paradigmer, der modulerer CNS vaskulær regenerering kan undersøges ved hjælp af OIR model, herunder brug af farmakologiske forbindelser, genterapi, gendeletion og meget mere. Tilbøjelighed en given tilgang til at påvirke vaskulær genvækst vurderes trinvis i vinduet mellem P12 (maksimal VO efter exit fra hyperoxi) og P17 (maksimal NV). Evaluering af behandlingsresultatet på patologisk NV kan hurtigt og nemt bestemmes parallelt og er blevet grundigt beskrevet af Stahl og kolleger 30, 31. Her giver vi en enkel trin-for-trin procedure at undersøge graduering af fysiologiske revaskularisering inden den neurale nethinde ved farmakologiske forbindelser, potentielle lægemidler, virale vektorer eller studere indflydelsen af ​​kandidatgener i transgene eller knockout-mus.

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			<td height="21" style="height:21px;width:639px;">C57Bl/6 mice ((Other strains may be used; angiogenic response varies from one strain to the other)</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:639px;">CD1 nursing mothers</td>
			<td style="width:181px;">Vendor of choice</td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Operating Scissors straight</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">14192</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Dissecting Scissors straight</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">14393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Vannas Eye Scissors</td>
			<td style="width:181px;">Harvard Apparatus</td>
			<td style="width:175px;">72-8483</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Iris Forceps, curved, serrated</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">15915</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Brushes 362R size 0</td>
			<td style="width:181px;">Dynasty</td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Dumont Forceps #3; straight</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">500338</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Surgical Blade, size 10</td>
			<td style="width:181px;">Bard-Parker</td>
			<td style="width:175px;">371110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Rhodamine Griffonia (Bandeiraea) Simplicifolia Lectin I</td>
			<td style="width:181px;">Vector Laboratories, Inc</td>
			<td style="width:175px;">RL-1102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Microscope slides</td>
			<td style="width:181px;">VWR</td>
			<td style="width:175px;">16004-368</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Fluoromount G</td>
			<td style="width:181px;">Electron Microscopy Sciences</td>
			<td style="width:175px;">17984-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Zeiss Axio Observer Z1 Inverted Phase and Fluorescence Microscope</td>
			<td style="width:181px;">Zeiss</td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="42" rowspan="2" style="height:42px;width:639px;">Leica MZ9.5 Stereomicroscope</td>
			<td rowspan="2" style="width:181px;">Leica</td>
			<td rowspan="2" style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Fluorescein isothicyanate-dextran, 70000</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:175px;">46945</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of vascular regeneration in the cns using the mouse retina

Gnavere nethinden er måske den mest tilgængelige pattedyr system til at undersøge neurovaskulær samspil inden for det centrale nervesystem (CNS). Det er i stigende grad anerkendt, at flere neurodegenerative sygdomme som Alzheimers, multipel sklerose, og amyotrofisk lateral sklerose indeholder elementer vaskulær kompromis. Desuden er de mest fremtrædende årsager til blindhed hos pædiatriske og arbejdsvilkår aldersgrupper (præmatur retinopati og diabetisk retinopati, henholdsvis) er kendetegnet ved vaskulær degeneration og svigt af fysiologisk vaskulær genvækst. Formålet med denne tekniske rapport er at give en detaljeret protokol til at studere CNS vaskulær regenerering i nethinden. Fremgangsmåden kan anvendes til at belyse molekylære mekanismer, der fører til svigt af vaskulær vækst efter iskæmisk skade. Desuden kan udforskes potentielle terapeutiske modaliteter for at fremskynde og genoprette sunde vaskulære plexus. Resultaterne obtained ved hjælp af den beskrevne fremgangsmåde kan give terapeutiske muligheder for iskæmiske retinopatier som den, diabetes eller præmaturitet og muligvis gavne andre vaskulære forstyrrelser i centralnervesystemet.

Den OIR model er almindeligt anvendt til at studere ilt-induceret vaskulær degeneration og iskæmi-induceret patologisk neovaskularisering i nethinden og har været medvirkende i udviklingen af nuværende tidspunkt er ansat anti-angiogenese behandlinger for øjensygdomme 27, 29, 30.. Resultaterne er opnået ved hjælp af denne model kan løseligt ekstrapoleres til iskæmiske retinopatier såsom proliferativ diabetisk retinopati og retinopati hos præmature 30. Her præ...

Watch this Scientific Journal Video about Assessment of Vascular Regeneration in the CNS Using the Mouse Retina at JoVE.com

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

Det gnaver nethinden har længe været anerkendt som en tilgængelig vindue til hjernen. I denne tekniske papir giver vi en protokol, der anvender musemodel af oxygen-induceret retinopati at undersøge de mekanismer, der fører til svigt af vaskulær regenerering inden for det centrale nervesystem efter iskæmisk skade. Det beskrevne system kan også udnyttes til at undersøge strategier til at fremme genvækst af funktionelle blodkar i nethinden og centralnervesystemet.

Vurdering af Vascular Regeneration i CNS Brug af musen Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Research

JoVE Journal

Neuroscience

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

In vivo Electroporation of Developing Mouse Retina

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

Hurtig Mikro-iontophoresis af glutamat og GABA: et nyttigt redskab til Undersøge Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

Hornhindens Micropocket assay: En model af angiogenese i mus Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Imaging Ca<sup&gt; 2+</sup&gt; Dynamics i Cone Lysmodtageren Axon Terminals i Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

Brug af elektroretinogrammet at vurdere Funktion i gnaver Retina og beskyttende virkninger af fjernbetjening Limb iskæmisk prækonditionering

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Mus mikrokirurgi Infusion Teknik for Målrettet Stof Levering i CNS<i&gt; via</i&gt; Den interne Arteria carotis

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Behandlende pattedyr Axon regenerering: In Vivo elektroporation af voksen mus dorsalrods Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Kraften i Interstimulus Interval for vurdering af tidsmæssige forarbejdes i Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

Transpupillær to-foton In vivo Billeddannelse af musens nethinde

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...