Diese Studie wurde von der Seoul National University Research Grant (800-20140542) unterstützt, der Pioneer Research Program der NRF / MEST (2012-0009544) und die Bio-Signalanalyse-Technologie-Innovations-Programm der NRF / MEST (2009-0090895), und die Gewährung von NRF / MEST (2015M3A9E6028949).

Alle Tierversuche wurden in Übereinstimmung mit der Association for Research in Vision and Ophthalmology Erklärung für die Nutzung von Tieren in Ophthalmic und Vision Research durchgeführt und die Richtlinien und Vorschriften von der Seoul National University Institutional Animal Care und Verwenden Committee und Seoul National dargelegt Universitätsklinikum Biosafety Committee. 1. Vorbereiten Injection Kit und Virale Vektoren <ol><li> Bereiten Sie die Mikroliter-Spritze mit einer 33 G stumpfen Nadel sterilisiert mit Ethylenoxid-Gas ausgestattet. Verdünnt den viralen Vektoren in PBS zur adäquaten Titer im Mikroröhre (dh 1 × 10 6 TU / ul). Spülen Sie die Spritze mehrmals mit viralen Vektoren, jede toten Raum in der Spritze zu entfernen. </li></ol> 2. Subretinale Injektion von viralen Vektoren <ol><li> Betäuben den erwachsenen Mäusen (dh, 6-8 Wochen alt) mit einer intraperitonealen Injektion der Mischung aus tiletamine und Zolazepam (1: 1, 2,25 mg / kg Körpergewicht) und Xylazin-Hydrochlorid (0,7 mg / kg Körpergewicht) oder eine alternative geeignete Anästhesie Regimes. </li><li> Erweitern sich die Schüler mit einem Auge Tropfen Phenylephrin 0,5% und 0,5% Tropicamid. </li><li> Bereiten Sie den Mikroliterspritze durch das Laden mit 1,5 bis 2 &amp; mgr; l von viralen Vektoren. </li><li> Öffnen Sie das Augenlid und stehen den Blick auf den Äquator zum bequemen Injektion freizulegen und sich auf unter dem Operationsmikroskop. Aufrechterhaltung der vorstehende Augenposition bis Beendigung der Injektion oder Verschiebung der Nadel während der Injektion ein. Um den Augapfel fest zu halten, legen Sie die Finger außerhalb des Augenhöhlenrand. </li><li> Einen Tropfen Augen viskoelastische Lösung, um die Hornhautoberfläche. </li><li> Setzen Sie einen kleinen runden Deckglas auf der Oberseite der Hornhaut, um die Netzhaut zu visualisieren. </li><li> Stechen ein kleines Loch an leichten hinter dem Limbus mit einer sterilen Nadel 30 G 1/2 für die weitere subretinale Injektion. Machen Sie das Loch unterlegen for das rechte Auge und überlegen für das linke Auge für die Bequemlichkeit. HINWEIS: Wenn das Loch an zeitliche oder Nasen gemacht, ist es schwer, durch das Augenlid nach der Injektion bedeckt sein. Punktion sollte etwas hinter dem Limbus, die Limbusgefäßen, die entlang des Limbus ausgeführt und kann leicht erkannt werden, zu vermeiden. Seien Sie vorsichtig, schlagen Sie das Objektiv mit Nadel und gleichzeitig die Punktion zu vermeiden. Nicht die gesamte Kegel der Nadel legen nicht auf Linsenpunktion zu vermeiden. </li><li> Legen Sie die 33 G stumpfen Nadel der Mikroliterspritze durch die Pre-Stichloch und nähern sich die Nadel in den subretinalen Raum bis zu dem Punkt, wenn mild Widerstand zu spüren ist. HINWEIS: Bei den subretinalen Injektion, der beste Ansatz Winkel der Nadel ist etwa 45 Grad gegen Iris Ebene und die stumpfe Nadel sollte nach hinten in Richtung peripapillären Bereich geschoben werden. Gepunktete Quadrat angezeigt die vorgeschlagene Nadel Weg über den Glaskörperraum für den subretinalen injection in 1. HINWEIS: Es wird kein Widerstand fühlte, als Piercing (oder Weitergabe) die Netzhautschichten, wie sie sind sehr weich. Somit wird das erste Gefühl einer leichten Widerstand zeigt an, dass die Nadel die RPE-Schicht bereits berührt wurde und die Einführung der Nadel gestoppt werden sollte. Achten Sie darauf, Skleragewebe mit Überdruck durchdringen, weil die Nadel in das Potenzial subretinalen Raum platziert werden. Wenn die Nadel in die Lederhautgewebe durch übermäßige Macht, tritt sie in den äußeren Augen orbitalen Bereiche ohne Widerstand. (Dieser Schritt ist kritisch) </li></ol><img alt="Abbildung 1" src="/files/ftp_upload/53030/53030fig1.jpg" /> Abbildung 1. Schematische Darstellung der Subretinale Injection. H &amp; E gefärbten Querschnitt der Maus Auge Darstellung der Strukturen mit einer Nadel Weg für den subretinalen Injektion markiert (eine gepunktete Quadrat). Vergrößerung: 40x, Maßstabsbalken:<html> 500 &amp; mgr; m. <a href="https://www.jove.com/files/ftp_upload/53030/53030fig1large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <ol start="9"><li> Injizieren Sie den viralen Vektoren (dh 1 x 10 6 TU / ul) vorsichtig in den subretinalen Raum ohne Zittern um unerwünschte Gewebeschäden zu vermeiden und vorsichtig ziehen Sie die Nadel. Halten Sie den Augapfel fest während der Injektion, wie in 2.4 beschrieben. </li><li> Beachten Sie die Bildung von subretinalen Bläschen nach Injektion unter Operationsmikroskop, um sicherzustellen, gibt es keine Netzhautblutungen. </li></ol><img alt="Figur 2" src="/files/ftp_upload/53030/53030fig2.jpg" /> Abbildung 2. Subretinale Belb Formation ohne Netzhautblutungen. Mikroskopische Ansicht des subretinalen belb nach dem subretinalen Injektion unter Operationsmikroskop zeigt erfolgreiche bleb Bildung ohne Netzhautblutungen.m / files / ftp_upload / 53030 / 53030fig2large.jpg &quot;target =&quot; _ blank &quot;&gt; Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen. <ol start="11"><li> Schließen Sie vorsichtig das Augenlid auf die Injektionsstelle für selbstdicht decken. Bringen Sie die Maus mit nach Hause Käfig und am Leben zu erhalten, bis die Auswertung. Hinweis: Nach dem die Forscher erhalten den Verfahren verwendet werden, können sie die wiederholbare Ergebnisse mit Schnellverfahren durch Überspringen Sie diese Schritte erhalten (2,5, 6 und 10) </li></ol> 3. Bewertung der Wirksamkeit von Gentransfer in Retinapigmentepithel <ol start="1"><li> Opfern die Mäuse in der CO 2 Kammer. Entkernen die Augen mit einer Schere. </li><li> Fixieren die Augen in der 4% Paraformaldehyd-Lösung bei 4 ° C für mindestens 1 Stunde bis einige Tage. </li><li> Besorgen Sie sich die Hornhaut mit feinen Pinzetten und Punktion der Hornhaut mit Mikro-Schere. Entfernen Sie die Hornhaut nach scissoring entlang des Limbus. Nehmen Sie den Objektiv mit einer Pinzette. </li><li> Schneiden Sie die Ziliarkörper mit micro-Schere ermöglicht Netzhautablösung. Ziehen Sie die gesamte Netzhaut von der RPE / Aderhaut / Lederhaut-Komplex. Schneiden Sie den RPE / Aderhaut / Lederhaut-Komplex radial von der Peripherie zum Zentrum in der Nähe des Sehnervs mehrmals, um 4 bis 8 Blätter für flache Montage machen. </li><li> Klappen Sie den RPE / Aderhaut / Lederhaut-Komplex zu RPE Seite nach unten zu machen, und schneiden Sie die verbleibenden Gewebe an der skleralen Seite einschließlich der Muskeln, Bindehaut und Sehnerv. Dieser Schritt ist wichtig, um eine flache RPE / Aderhaut / Lederhaut-Komplex zu machen, weil der verbleibende Gewebe macht die komplexe uneben. </li><li> Inkubieren Sie die Anlage mit primären und sekundären Antikörpers für Ihre spezielle Forschungszwecke (optional, siehe die Referenzen für detaillierte Methoden). Flach montieren RPE / Aderhaut / Lederhaut-Komplexe auf dem Objektträger und absorbieren die PBS mit einem saugfähigen Schwamm. </li><li> In 30 ul Montagelösung und legen Sie das Deckglas. Beachten Sie die Probe für die Wirksamkeit von Vektor-Lieferung unter Fluoreszenzmikroskop. Bewahren Sie die Proben inder Kühlschrank zur weiteren Beobachtung. </li></ol>

<ol>
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(79), (2013).</li><li>Wert, K. J., Skeie, J. M., Davis, R. J., Tsang, S. H., Mahajan, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subretinal+injection+of+gene+therapy+vectors+and+stem+cells+in+the+perinatal+mouse+eye.">Subretinal injection of gene therapy vectors and stem cells in the perinatal mouse eye.</a> J Vis Exp. (69), (2012).</li><li>Eberle, D., Santos-Ferreira, T., Grahl, S., Ader, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subretinal+transplantation+of+MACS+purified+photoreceptor+precursor+cells+into+the+adult+mouse+retina.">Subretinal transplantation of MACS purified photoreceptor precursor cells into the adult mouse retina.</a> J Vis Exp. (84), e50932 (2014).</li><li>Sahel, J. A., Roska, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+for+blindness.">Gene therapy for blindness.</a> Annu Rev Neurosci. 36, 467-488 (2013).</li><li>Allocca, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+adeno-associated+virus+serotypes+efficiently+transduce+murine+photoreceptors.">Novel adeno-associated virus serotypes efficiently transduce murine photoreceptors.</a> J Virol. 81 (20), 11372-11380 (2007).</li><li>Takahashi, K., 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=Sustained+transduction+of+ocular+cells+with+a+bovine+immunodeficiency+viral+vector.">Sustained transduction of ocular cells with a bovine immunodeficiency viral vector.</a> Hum Gene Ther. 13 (11), 1305-1316 (2002).</li><li>Rolling, F., 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=Gene+therapeutic+prospects+in+early+onset+of+severe+retinal+dystrophy:+restoration+of+vision+in+RPE65+Briard+dogs+using+an+AAV+serotype+4+vector+that+specifically+targets+the+retinal+pigmented+epithelium.">Gene therapeutic prospects in early onset of severe retinal dystrophy: restoration of vision in RPE65 Briard dogs using an AAV serotype 4 vector that specifically targets the retinal pigmented epithelium.</a> Bull Mem Acad R Med Belg. 161 (10-12), 497-508 (2006).</li><li>Le Meur, G., 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=Restoration+of+vision+in+RPE65-deficient+Briard+dogs+using+an+AAV+serotype+4+vector+that+specifically+targets+the+retinal+pigmented+epithelium.">Restoration of vision in RPE65-deficient Briard dogs using an AAV serotype 4 vector that specifically targets the retinal pigmented epithelium.</a> Gene Ther. 14 (4), 292-303 (2007).</li><li>Alexander, J. J., Hauswirth, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+viral+vectors+and+the+retina.">Adeno-associated viral vectors and the retina.</a> Adv Exp Med Biol. 613, 121-128 (2008).</li><li>Puche, N., 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=Genetic+and+environmental+factors+associated+with+reticular+pseudodrusen+in+age-related+macular+degeneration.">Genetic and environmental factors associated with reticular pseudodrusen in age-related macular degeneration.</a> Retina. 33 (5), 998-1004 (2013).</li><li>Campochiaro, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+for+neovascular+age-related+macular+degeneration.">Gene transfer for neovascular age-related macular degeneration.</a> Hum Gene Ther. 22 (5), 523-529 (2011).</li><li>Park, S. W., 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=Intracellular+amyloid+beta+alters+the+tight+junction+of+retinal+pigment+epithelium+in+5XFAD+mice.">Intracellular amyloid beta alters the tight junction of retinal pigment epithelium in 5XFAD mice.</a> Neurobiol Aging. 35 (9), 2013-2020 (2014).</li><li>Shin, 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=Intracellular+cleavage+of+amyloid+beta+by+a+viral+protease+NIa+prevents+amyloid+beta-mediated+cytotoxicity.">Intracellular cleavage of amyloid beta by a viral protease NIa prevents amyloid beta-mediated cytotoxicity.</a> PLoS One. 9 (6), e98650 (2014).</li></ol>

The authors have nothing to disclose.

In diesem Video-Artikel beschrieben wir die Limbus-Ansatz subretinalen Injektionstechnik im Detail mit den repräsentativen Ergebnissen der RPE / Aderhaut / Lederhaut Flachmontage. Dies ist eine einfache und bequeme Technik zum subretinalen Injektion von viralen Vektoren in RPE. Direkte Visualisierung bleb Bildung während der Injektion ist ein wichtiger Schritt für die pünktliche Lieferung für die Anfänger. Es gibt einige subretinalen Injektionstechniken in Blatt eingeführt of Visualized Experiments 8-10.</su...

In der Augenheilkunde hat die Gentherapie als Behandlungsmethode in monogenen geerbt Retinopathien entstanden. Es vererbt Retinopathien mit Genen im retinalen Pigmentepithel (RPE), einschließlich Leber angeborene amurosis 1,2, Retinitis pigmentosa 3 und choroideremia 4 verbunden. Das Forschungsfeld der Gentherapie erweitert in beiden präklinischen Studien und klinischen Studien unter Verwendung von viralen Vektoren, wie Adeno-assoziierte Virus (AAV), Lentiviren (LV) und Adenovirus (Ad) 5. Verschiedene virale Vektoren haben unterschiedliche Tropismus in der Netzhaut. Für eine sichere und wirksame Gentherapie, virale Vektoren sollten sorgfältig entsprechend Zielzellen und Zielgene ausgewählt werden. Die Route der Genübertragung ist auch wichtig für eine effektive Genübertragung an Zielzellen, so sollte es sorgfältig und ausgewählt werden. Die beiden am häufigsten verwendeten Methoden zur intraokularen Abgabe von viralen Vektoren sind SUBRETinal Einspritzung und intravitreale Injektion 6. Letztere, intravitreale Injektion, wurde weit verbreitet für die Arzneimittelabgabe verwendet werden, um CNV in feuchten altersbedingten Makuladegeneration (AMD) und Makulaödem bei diabetischer Retinopathie 7 behandeln. Intravitreale Route bietet Exposition viraler Vektoren zu glasigen und inneren Retina, sondern die Diffusion der Vektoren äußeren Netzhaut begrenzt ist. Auf der anderen Seite stellt die subretinale Route direkte Lieferung von viralen Vektoren zum Potential Raum zwischen Retina und RPE, Induzieren einer lokalisierten Bläschen. Daher wird subretinalen Injektion momentan als ein effizienter Weg für die gezielte Photorezeptorzellen und RPE. In Bezug auf die chirurgischen Ansatz, Pars plana als einen sicheren Bereich für die intravitreale Injektion gewählt, um Netzhautschäden in menschlichen Patienten zu vermeiden. Durch einfaches Ändern dieses Konzept für Mäuse, wir könnten virale Vektoren subretinal oder intravireally via Limbus-Ansatz zu injizieren. In diesem VideoArtikel zeigen wir eine einfache und bequeme Methode der subretinalen Injektion von viralen Vektoren in Mäuse RPE. Nach Einzelpunktion bei posterior zu Limbus mit einer 30 G 1/2 Nadel wird ein 33 G stumpfen Nadel ausgestattet Mikroliterspritze in den subretinalen Raum über den Limbus Einstichstelle eingeführt. Die viralen Vektoren 1,5-2 ul Volumen auf das Potential Raum zwischen Retina und RPE Induzieren subretinalen Bläschen injiziert. Dieser Vorgang kann unter direkter Sicht mit Operationsmikroskop durchgeführt werden. Wiederholte Praxis werden reproduzierbare Ergebnisse auch ohne direkte Visualisierung der Bläschenbildung zu gewährleisten. Dies soll dazu dienen, um eine genaue und zeitsparende Experimente zur subretinalen Gentransfer in Mäusen RPE durchzuführen.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>TWEEZERS DUMONT #5 11cm DUMOSTAR 0.1 x 0.06 mm TIPS</td>
			<td>WPI</td>
			<td>500233</td>
			<td></td>
		</tr>
		<tr>
			<td>VANNAS Scissors S/S, 105mm</td>
			<td>WPI</td>
			<td>555583S</td>
			<td></td>
		</tr>
		<tr>
			<td>33G Blunt needle</td>
			<td>WPI</td>
			<td>NF33BL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoFil Syringe, 10 microliter&#160;</td>
			<td>WPI</td>
			<td>NANOFIL</td>
			<td></td>
		</tr>
		<tr>
			<td>RPE-KIT</td>
			<td>WPI</td>
			<td>RPE-KIT</td>
			<td>For easy one hand injection</td>
		</tr>
		<tr>
			<td>30Gx1/2 (0.3mmx 13mm) BD PrecisionGlideTM Needle</td>
			<td>BD</td>
			<td>305107</td>
			<td>Initial puncture for subretinal injection</td>
		</tr>
		<tr>
			<td>Microscope Cover Glasses (No. 1 3 mm diameter)</td>
			<td>Warner Instruments</td>
			<td>64-0720&#160; (CS-3R)</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica operating microscope</td>
			<td>Leica</td>
			<td>LM M80</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoresecein microscope</td>
			<td>Nikon</td>
			<td>Eclipse 80i</td>
			<td></td>
		</tr>
		<tr>
			<td>Lentivirus</td>
			<td>Thermo scientific</td>
			<td>TMO.LV-Ctr</td>
			<td>Used to dilute vectors</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010-015</td>
			<td>Used to dilute vectors</td>
		</tr>
		<tr>
			<td>Troperin (Phenylephrin 0.5%-Tropicamide 0.5%)</td>
			<td>Hanmi</td>
			<td></td>
			<td>For dilation</td>
		</tr>
		<tr>
			<td>Proparacaine Hydrochloride Ophthalmic Solution USP, 0.5% (Sterile)</td>
			<td>Bausch&#38;Lomb</td>
			<td></td>
			<td>For topical anesthesia</td>
		</tr>
		<tr>
			<td>Healon GV OVD</td>
			<td>Abbott Medical Optics Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zoletil 50 (tiletamine hypochloride and zolazepam hypochloride)</td>
			<td>Virbac</td>
			<td></td>
			<td>For general anesthesia</td>
		</tr>
		<tr>
			<td>Rompun&#174; injection (Xylazine HCl)</td>
			<td>Bayer</td>
			<td></td>
			<td>For general anesthesia</td>
		</tr>
	</tbody>
</table>

limbal approach-subretinal injection of viral vectors for gene therapy in mice retinal pigment epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Um die Wirksamkeit der subretinalen Injektion auf virale Gen-Transduktion durch dieses Protokoll zu bewerten, haben wir im Handel erhältlich LV Vektoren mit CMV-Promotors exprimieren, sowohl GFP und RFP für den Indikator. Augen wurden nach der jeweiligen Zeitperiode nach dem Forschungszweck ausgeschält. Für die repräsentative Ergebnisse wurden die Augen 10 Wochen und 20 Wochen nach der subretinalen Injektion enukleiert. Nach vollständiger Entfernung der Netzhaut mit dem oben beschriebenen Verfahren wurde die Wohnu...

Watch this Scientific Journal Video about Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium at JoVE.com

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

Limbus-Ansatz-Subretinale Injektion von viralen Vektoren für die Gentherapie bei Mäusen Retinapigmentepithel

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

limbal-approach-subretinal-injection-viral-vectors-for-gene-therapy

Research

JoVE Journal

Neuroscience

21.0K Aufrufe.  Seoul National University College of Medicine. Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

Serie: Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

Experimentelle Modelle für das Studium der retinalen Pigmentepithelzellen Physiologie und Pathophysiologie

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

Forschung

Neurowissenschaften

Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an injecting needle produces during its trajectory is a major concern especially in the mouse brain for not only the brain is small but also sometimes multiple injections are needed.  We show here a method to produce glass capillary needles with a 50-&mu;m lumen which significantly reduces the brain damage and allows a precise targeting into the rodent brain. This method allows a delivery of small volumes (from 20 to 100 nl), reduces bleeding risks, and minimizes passive diffusion of drugs into the brain parenchyma. By using different size of capillary glass tubes, or changing the needle lumen, several types of substances and cells can be injected. Microinjections with a glass capillary tube represent a significant improvement in injection techniques and deep brain targeting with minimal collateral damage in small rodents.

In this article, we show a method to make glass capillary needles with a 50-&mu;m lumen. This technique significantly reduces the brain damage, minimizes passive diffusion of drugs and allows a precise targeting into the rodent brain.

Targeting von Deep Brain Strukturen mit Mikroinjektionen für die Lieferung von Drogen, virale Vektoren oder Zelltransplantaten

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an ...

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

Intrakranielle Injektion von Adeno-assoziierte virale Vektoren

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

Präparation einer Maus Eye für einen ganzen Berg von retinalen Pigmentepithel

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Erweiterung der embryonalen und adulten neuralen Stammzellen<em&gt; In Utero</em&gt; Elektroporation oder Viral stereotaktische Injektion

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Stereotaktische Injektion eines viralen Vektors für Conditional Gene Manipulation im Maus-Rückenmark

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

Intracerebroventricular Viral Injektion der Neonatal Maus Gehirn für anhaltenden und verbreiteten Neuronale Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

Entwicklung eines raffinierten Protokolls für Trans-skleralen subretinalen Transplantation von menschlichen retinalen Pigment Epithelzellen in Ratte Augen

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

A Protocol for Transcranial Photobiomodulation Therapy in Mice

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide range of neurological and psychiatric disorders, and memory enhancement in age-related cognitive decline and neurodegenerative diseases. We describe a laboratory protocol for transcranial photobiomodulation therapy (PBMT) in mice. Aged BALB/c mice (18 months old) are treated with a 660 nm laser transcranially, once daily for 2 weeks. Laser transmittance data shows that approximately 1% of the incident red light on the scalp reaches a 1 mm depth from the cortical surface, penetrating the dorsal hippocampus. Treatment outcomes are assessed by two methods: a Barnes maze test, which is a hippocampus-dependent spatial learning and memory task evaluation, and measuring hippocampal ATP levels, which is used as a bioenergetics index. The results from the Barnes task show an enhancement of the spatial memory in laser-treated aged mice when compared with age-matched controls. Biochemical analysis after laser treatment indicates increased hippocampal ATP levels. We postulate that the enhancement of memory performance is potentially due to an improvement in hippocampal energy metabolism induced by the red laser treatment. The observations in mice could be extended to other animal models since this protocol could potentially be adapted to other species frequently used in translational neuroscience, such as rabbit, cat, dog, or monkey. Transcranial photobiomodulation is a safe and cost-effective modality which may be a promising therapeutic approach in age-related cognitive impairment.

Photobiomodulation therapy is an innovative noninvasive modality for the treatment of a wide range of neurological and psychiatric disorders and can also improve healthy brain function. This protocol includes a step-by-step guide to performing brain photobiomodulation in mice by transcranial light delivery, which can be adapted for use in other laboratory rodents.

Ein Protokoll für die transkranielle Photobiomodulation Therapie bei Mäusen

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide ...

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative cell replacement therapies while also creating tools to study cell fate in situ. To date, it has been possible to obtain neurons via in vivo reprogramming, but the precise phenotype of these neurons or how they mature has not been analyzed in detail. In this protocol, we describe a more efficient conversion and cell-specific identification of the in vivo reprogrammed neurons, using an AAV-based viral vector system. We also provide a protocol for functional assessment of the reprogrammed cells&#8217; neuronal maturation. By injecting flip-excision (FLEX) vectors, containing the reprogramming and synapsin-driven reporter genes&#160;to specific cell types in the brain that serve as the target for cell reprogramming. This technique allows for the easy identification of newly reprogrammed neurons. Results show that the obtained reprogrammed neurons functionally mature over time, receive synaptic contacts and show electrophysiological properties of different types of interneurons. Using the transcription factors Ascl1, Lmx1a and Nurr1, the majority of the reprogrammed cells have properties of fast-spiking, parvalbumin-containing interneurons.

This protocol aims at generating directly reprogrammed interneurons in vivo, using an AAV-based viral system in the brain and a FLEX synapsin-driven GFP reporter, which allows for cell identification and further analysis in vivo.

In Vivo Direkte Reprogrammierung von Resident Glial Cells in Interneurons durch Intracerebral Injection of Viral Vectors

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...