Cette étude a été soutenue par le Grant Université nationale de Séoul de recherche (800-20140542), le Programme de Pioneer Research de la NRF / MEST (2012-0009544), et le Programme Bio-Signal Analysis Technology Innovation de la NRF / MEST (2009-0090895), et la subvention de la NRF / MEST (2015M3A9E6028949).

Toutes les expériences sur les animaux ont été effectuées en conformité avec l&#39;Association pour la recherche en vision et ophtalmologie Déclaration pour l&#39;utilisation d&#39;animaux dans ophtalmique et Vision Research, et les directives et règlements énoncés par le Comité nationale de Séoul soin et l&#39;utilisation des animaux et institutionnelle Université nationale de Séoul Comité de biosécurité de l&#39;hôpital universitaire. 1. Préparation Kit d&#39;injection et vecteurs viraux <ol><li> Préparer la seringue microlitre équipé d&#39;une aiguille émoussée 33 G stérilisée en utilisant du gaz d&#39;oxyde d&#39;éthylène. Diluer les vecteurs viraux dans PBS pour le titre adéquat dans le micro tube (soit 1 x 10 6 TU / pi). Rincer la seringue à plusieurs reprises avec des vecteurs viraux pour éliminer tout espace vide dans la seringue. </li></ol> 2. Injection sous-rétinienne de vecteurs viraux <ol><li> Anesthésier les souris adultes (par exemple, 6 - 8 semaines) par une injection intraperitoneale du mélange de tiletamiet ne zolazepam (1: 1, 2,25 mg / kg de poids corporel) et de chlorhydrate de xylazine (0,7 mg / kg de poids corporel) ou un régime d&#39;anesthésie adapté alternative. </li><li> Dilater les élèves avec un collyre de phényléphrine 0,5% et tropicamide 0,5%. </li><li> Préparer la seringue microlitre en chargeant avec 1,5 à 2 pi de vecteurs viraux. </li><li> Ouvrir la paupière et l&#39;oeil en saillie pour exposer l&#39;équateur pour injection pratique et se concentrer sur la sous microscope opératoire. Maintenir la position de l&#39;oeil en saillie jusqu&#39;à terminer l&#39;injection, ou le déplacement de l&#39;aiguille peut se produire lors de l&#39;injection. Pour tenir le globe oculaire fermement, placer les doigts à l&#39;extérieur du rebord orbitaire. </li><li> Appliquer une goutte de solution viscoélastique ophtalmique à la surface de la cornée. </li><li> Placer une petite lamelle ronde sur le haut de la cornée afin de visualiser la rétine. </li><li> Percer un petit trou à peu en arrière du limbe en utilisant une aiguille stérile 30 G 1/2 pour la poursuite de l&#39;injection sous-rétinien. Faire le trou inférieur for l&#39;œil droit, et supérieur pour l&#39;oeil gauche pour la commodité. NOTE: Si le trou est fait au temporelle ou nasale, il est difficile d&#39;être couvert par la paupière après l&#39;injection. Ponction initiale devrait être rendue légèrement derrière le limbe pour éviter les vaisseaux limbiques qui courent le long du limbe et peut être facilement reconnus. Soyez prudent pour éviter de heurter la lentille avec l&#39;aiguille tout en rendant la ponction initiale. Ne pas insérer l&#39;ensemble du biseau de l&#39;aiguille pour éviter lentille crevaison. </li><li> Placez l&#39;aiguille émoussée 33 G de microlitre seringue à travers le trou pré-percé et l&#39;approche de l&#39;aiguille dans l&#39;espace sous-rétinien jusqu&#39;au moment où une légère résistance se fait sentir. NOTE: Pour l&#39;injection sous-rétinien, le meilleur angle d&#39;approche de l&#39;aiguille est d&#39;environ 45 degrés contre iris avion, et l&#39;aiguille émoussée doit être poussé en arrière vers la zone péripapillaire. Carré en pointillés indique la voie de l&#39;aiguille suggéré dans la cavité vitréenne pour la sous-rétinien injection sur la figure 1. NOTE: Il n&#39;y aura pas de résistance ressentie lorsque perçage (ou passage) les couches de la rétine car ils sont très doux. Ainsi, le premier sentiment de légère résistance indique que l&#39;aiguille a déjà touché la couche RPE et l&#39;insertion de l&#39;aiguille doit être arrêté. Attention à ne pas pénétrer dans le tissu scléral avec une pression excessive parce que l&#39;aiguille doit être placé dans l&#39;espace sous-rétinien potentiel. Si l&#39;aiguille perce le tissu scléral par la puissance excessive, il entre dans les espaces extra-orbitales sans résistance. (Cette étape est essentielle) </li></ol><img alt="Figure 1" src="/files/ftp_upload/53030/53030fig1.jpg" /> Figure 1. Schéma de l&#39;injection sous-rétinienne. H &amp; E de section teinté de l&#39;œil de la souris représentant les structures avec une voie d&#39;aiguilles pour l&#39;injection sous-rétinienne marquée (un carré pointillé). Grossissement: 40X, Barre d&#39;échelle:<html> 500 um. <a href="https://www.jove.com/files/ftp_upload/53030/53030fig1large.jpg" target="_blank">S&#39;il vous plaît, cliquez ici pour voir une version plus grande de cette figure.</a> <ol start="9"><li> Injecter les vecteurs viraux (par exemple, 1 x 10 6 TU / pi) doucement dans l&#39;espace sous-rétinien, sans tremblements d&#39;éviter des dommages aux tissus non désirées et retirer délicatement l&#39;aiguille. Tenez fermement le globe oculaire pendant l&#39;injection comme décrit dans 2.4. </li><li> Observer la formation de bulle sous-rétinien après l&#39;injection sous microscope opératoire pour vous assurer qu&#39;il n&#39;y a pas de saignement de la rétine. </li></ol><img alt="Figure 2" src="/files/ftp_upload/53030/53030fig2.jpg" /> Figure 2. Formation sous-rétinienne Belb sans hémorragie rétinienne. Vue microscopique de belb sous-rétinien après l&#39;injection sous-rétinien sous microscope opératoire montre succès la formation de bulle sans hémorragie rétinienne.m / files / ftp_upload / 53030 / 53030fig2large.jpg &quot;target =&quot; _ blank &quot;&gt; S&#39;il vous plaît cliquez ici pour voir une version plus grande de cette figure. <ol start="11"><li> Fermez doucement la paupière pour recouvrir le site d&#39;injection pour l&#39;auto-étanchéité. Retourner les souris à la maison cage et garder en vie jusqu&#39;à ce que l&#39;évaluation. NOTE: Après les chercheurs obtiennent utilisés pour les procédures, ils peuvent obtenir des résultats reproductibles avec des procédures rapides en sautant les étapes (2,5, 6 et 10) </li></ol> 3. Evaluation de l&#39;efficacité de Gene Livraison en épithélium pigmentaire rétinien <ol start="1"><li> Sacrifiez la souris dans la chambre de CO 2. Énucléer les yeux avec des ciseaux. </li><li> Fixer les yeux dans la solution de paraformaldehyde à 4% à 4 ° C pendant au moins une heure à quelques jours. </li><li> Prenez la cornée avec des pinces fines et à la perforation de la cornée avec des micro-ciseaux. Retirer la cornée après ciseaux le long du limbe. Retirez la lentille avec une pince. </li><li> Coupez le corps ciliaire avec mICRO-ciseaux permettant décollement de la rétine. Tirez doucement sur l&#39;ensemble de la rétine / choroïde / sclérotique complexe de l&#39;EPR. Couper le complexe RPE / choroïde / scléral radialement de la périphérie vers le centre, près du nerf optique à plusieurs reprises de faire 4 à 8 feuilles pour montage à plat. </li><li> Retournez le RPE / choroïde / complexe scléral pour faire face RPE vers le bas, et couper tous les tissus restants sur le côté scléral y compris les muscles, de la conjonctive et du nerf optique. Cette étape est importante pour faire un / choroïde / scléral complexe de l&#39;EPR à plat parce que tout tissu restant inégale rend le complexe. </li><li> Incuber le complexe avec l&#39;anticorps primaire et secondaire pour vos fins de recherche spécifiques (en option, voir les références pour les méthodes détaillées). Flat-monter les RPE / choroïde / sclérotique complexes sur la lame de verre et d&#39;absorber l&#39;PBS avec une éponge absorbant. </li><li> Ajouter 30 pi de solution de montage et placer la lame de couverture. Observer l&#39;échantillon pour l&#39;efficacité de la prestation de vecteur au microscope fluorescéine. Gardez les échantillons dansle réfrigérateur pour observation. </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. 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The authors have nothing to disclose.

Dans cet article, vidéo, nous avons décrit la technique d&#39;injection sous-rétinien limbique-approche en détail avec des résultats représentatifs de RPE / choroïde / scléral plate-mount. Ceci est une technique facile et pratique pour l&#39;injection sous-rétinien de vecteurs viraux en RPE. La visualisation directe de la formation de bulle pendant l&#39;injection est une étape importante pour la livraison précis pour les débutants. Il existe des techniques d&#39;injection sous-rétiniens introduites dans le...

En ophtalmologie, la thérapie génique a émergé comme la modalité de traitement dans les rétinopathies héréditaires monogéniques. Il ya rétinopathies héréditaires associés à des gènes de l&#39;épithélium pigmentaire rétinien (EPR) dont Leber amurosis congénitale 1,2, la rétinite pigmentaire 3 et 4 choroideremia. Le domaine de la thérapie génique de la recherche est en pleine expansion dans les deux études précliniques et des essais cliniques utilisant des vecteurs viraux tels que le virus adéno -associated (AAV), lentivirus (LV) et l&#39;adénovirus (Ad) 5. Différents vecteurs viraux ont un tropisme différent dans la rétine. Pour une thérapie génique efficace et sans danger, les vecteurs viraux doivent être soigneusement sélectionnées en fonction des cellules cibles et des gènes cibles. La voie d&#39;administration de gènes est également important pour la délivrance de gènes efficace à des cellules cibles, par conséquent, il doit être choisi avec soin aussi bien. Les deux méthodes les plus courantes pour la livraison intra-oculaire de vecteurs viraux sont subretInal injection et injection intravitréenne 6. Ce dernier, l&#39;injection intravitréenne, a été largement utilisé pour l&#39;administration de médicaments pour traiter la néovascularisation choroïdienne dans humide de la dégénérescence maculaire liée à l&#39;âge (DMLA) et de l&#39;œdème maculaire dans la rétinopathie diabétique 7. Voie intravitréenne offre une exposition à des vecteurs viraux vitré et la rétine interne, mais la diffusion des vecteurs de rétine externe est limitée. D&#39;autre part, la voie sous-rétinien fournit plus directement de vecteurs viraux à l&#39;espace potentiel entre la rétine et RPE, induisant une bulle localisée. Par conséquent, l&#39;injection sous-rétinien est actuellement considéré comme une voie plus efficace pour cibler des cellules photoréceptrices et RPE. En termes d&#39;approche chirurgicale, pars plana est choisie comme une zone de sécurité pour injection intravitréenne pour éviter d&#39;endommager la rétine chez des patients humains. En modifiant simplement cette approche à des souris, nous pourrions injecter vecteurs viraux sous-rétinienne ou intravireally via approche limbique. Dans cette vidéoarticle, nous démontrons une méthode facile et pratique de l&#39;injection sous-rétinien de vecteurs viraux dans des souris RPE. Après la ponction unique au postérieure à limbe avec une aiguille 30 G 1/2, un 33 G aiguille émoussée microlitre équipée seringue est insérée dans l&#39;espace sous-rétinien via le site de ponction limbique. Les vecteurs viraux de 1,5 à 2 volume ul sont injectés à l&#39;espace potentiel entre la rétine et RPE induire bulles sous-rétiniens. Cette procédure peut être réalisée sous visualisation directe utilisant microscope chirurgical. Pratique répétée garantira des résultats reproductibles, même sans la visualisation directe de la formation de vésicules. Cela aidera les chercheurs à réaliser des expériences précises et gagner du temps pour la livraison de gènes sous-rétinien chez la souris RPE.

<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.

Pour évaluer l&#39;efficacité de l&#39;injection sous-rétinienne sur la transduction du gène viral par ce protocole, nous avons utilisé des vecteurs disponibles dans le commerce BT avec le promoteur du CMV exprimant à la fois la GFP et DP pour l&#39;indicateur. Les yeux ont été énucléés, après la période de temps appropriée selon le but de la recherche. Pour les résultats représentatifs, les yeux ont été énucléés 10 semaines et 20 semaines après l&#39;injection sous-rétinien. Après l&#39;élimina...

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.

Limbal injection Approche-sous-rétinienne de vecteurs viraux pour la thérapie génique chez la souris épithélium pigmentaire rétinien

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

20.9K vues.  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.

Vidéo: 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.

Modèles expérimentaux pour l&#39;étude de la physiologie épithélium pigmentaire rétinien et physiopathologie

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

Recherche

Neurosciences

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.

Injection intracrânienne des vecteurs adéno-associés virale

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.

Dissection d&#39;un oeil de la souris pour un mont entiers de l&#39;épithélium pigmentaire rétinien

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

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.

Injection stéréotaxique d&#39;un vecteur viral pour la manipulation génétique conditionnelle dans la moelle épinière de souris

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.

Injection virale intracérébro du cerveau de souris nouveau-né pour la transduction neuronale persistante et généralisée

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

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.

Un protocole pour la thérapie Photobiomodulation transcrânienne chez la souris

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 Reprogrammation directe des cellules gliales résidentes en interneurones par injection intracérébrale de vecteurs viraux

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

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina&#160;with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2&#8211;9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day.
The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.

The protocol describes organotypic explants of mouse neuroretina, cultivated together with its retinal pigment epithelium (RPE), in R16 defined medium, free of serum and antibiotics. This method is relatively simple to perform, less expensive, and time-consuming when compared to in vivo experiments, and can be adapted to numerous experimental applications.

Culture à long terme et sans sérum d’explants organotypiques de la rétine de souris avec un épithélium pigmentaire rétinien intact

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of ...

Viral Construct (AAV) Injection in Mouse Superior Colliculus for Calcium Imaging

Begin by securing the anesthetized mouse onto a stereotaxic frame with ear bars. Apply ophthalmic ointment on the mouse's eyes to prevent drying, and maintain the body temperature of the mouse at 37 degrees Celsius with a thermostatic heating pad. After shaving the fur and sterilizing the surgical site, inject lidocaine, tilidine, and meloxicam.Once the skin over the SC is removed, clean the skull with a cotton swab. Adjust the stereotaxic frame until the skull surface is flat. Using a drill, create a whole 0.5 millimeter lateral, and 0.42 millimeter anterior to the lambda, until the dura is exposed.Inject an adeno-associated virus, or AAV, expressing GCaMP6m, at depths of one millimeter and 1.6 millimeters from the Lambda using a beveled glass micro-pipette. After the injection, slowly withdraw the injector and proceed with the implantation of the head plate.

Injection de construction virale (AAV) dans un colliculus supérieur de souris pour l’imagerie calcique

Administration par ponction lombaire d’AAV9 chez des rats juvéniles pour la thérapie génique du SNC

Intrathecal Vector Delivery in Juvenile Rats via Lumbar Cistern Injection

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a toxic gene, or provide a gene whose product can modulate the biology of the disease. The delivery method for the therapeutic vector can take many forms, ranging from intravenous infusion for systemic delivery to direct injection into the target tissue. For neurodegenerative disorders, it is often desirable to skew transduction towards the brain and/or spinal cord. The least invasive approach to target the entire central nervous system involves injection into the cerebrospinal fluid (CSF), allowing the therapeutic to reach a large fraction of the central nervous system. The safest approach to deliver a vector into the CSF is the lumbar intrathecal injection, where a needle is introduced into the lumbar cistern of the spinal cord. This technique, also known as a lumbar puncture, has been widely used in neonatal and adult rodents and in large animal models. While the technique is similar across species and developmental stages, subtle differences in size, structure, and elasticity of tissues surrounding the intrathecal space require accommodations in the approach. This article describes a method for performing lumbar puncture in juvenile rats to deliver an adeno-associated serotype 9 vector. Here, 25-35 &#181;L of vector were injected into the lumbar cistern, and a green fluorescent protein (GFP) reporter was used to evaluate the transduction profile resulting from each injection. The benefits and challenges of this approach are discussed.

Pleins feux sur l’auteur : Évaluation de l’efficacité de la thérapie génique intrathécale chez les rats juvéniles

A surgical procedure is described to perform injections into the lumbar cistern of the juvenile rat. This approach has been used for the intrathecal delivery of gene therapy vectors, but it is anticipated that this approach can be used for a variety of therapeutics, including cells and drugs.

Administration intrathécale de vecteurs chez des rats juvéniles par injection dans une citerne lombaire

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a ...