Este estudo foi apoiado pelo Seoul National University Research Grant (800-20140542), o Programa Pioneer Pesquisa da NRF / MEST (2012-0009544) ea Análise de Tecnologia Programa de Inovação Bio-Signal da NRF / MEST (2009-0090895), ea concessão da NRF / MEST (2015M3A9E6028949).

Todos os experimentos em animais foram realizados de acordo com a Associação de Pesquisa em Visão e Oftalmologia Declaração para o uso de animais em Oftalmologia e Vision Research, e as diretrizes e regulamentos estabelecidos pela Universidade Animal Care Institucional e Comitê de Uso Seoul Nacional e Nacional de Seul Hospital Universitário Comitê de Biossegurança. 1. Preparação Kit injeção e Vetores Virais <ol><li> Prepare a seringa microlitro equipado com uma agulha romba 33 G esterilizados usando gás de óxido de etileno. Dilui-se a vectores virais em PBS durante a titulação adequada no tubo de micro (ou seja, 1 x 10 6 TU / mL). Lave a seringa várias vezes com vectores virais para remover qualquer espaço morto na seringa. </li></ol> 2. Injecção sub-retiniana de vetores virais <ol><li> Anestesiar os ratos adultos (ou seja, 6-8 semanas de idade) com uma injecção intraperitoneal de uma mistura de tiletamine zolazepam e (1: 1, 2,25 mg / kg de peso corporal) e cloridrato de xilazina (0,7 mg / kg de peso corporal) ou um regime de anestesia adequada alternativa. </li><li> Dilatar pupilas com um colírio de fenilefrina e tropicamida 0,5% a 0,5%. </li><li> Preparar a seringa microlitro por carregamento com 1,5-2 ul de vectores virais. </li><li> Abra a pálpebra do olho e se projetam para expor o equador para injecção conveniente e focar sob o microscópio cirúrgico. Manter a posição dos olhos salientes até terminar a injecção, ou de deslocamento da agulha pode ocorrer durante a injecção. Para segurar o globo ocular firme, coloque os dedos fora da borda orbital. </li><li> Aplicar uma gota de solução viscoelástica oftálmica para a superfície da córnea. </li><li> Colocar uma pequena tampa deslizante rodada no topo da córnea para visualizar a retina. </li><li> Perfurar um pequeno buraco no ligeira posterior ao limbo usando um estéril 30 G 1/2 agulha para a nova injecção sub-retiniana. Faça o buraco inferior for o olho direito, e superior para o olho esquerdo para a conveniência. NOTA: Se o buraco é feito no temporais ou nasal, é difícil de ser coberta pela pálpebra após a injeção. Punção inicial deve ser feita ligeiramente posterior ao limbo para evitar os vasos do limbo, que correm ao longo do limbo e podem ser facilmente reconhecidas. Tenha cuidado para evitar bater a lente com agulha ao fazer a punção inicial. Não insira todo o bisel da agulha para evitar a perfuração da lente. </li><li> Inserir a agulha romba 33 g de microlitro seringa através do orifício pré-furado e aproximar-se a agulha no espaço sub-retiniano até o ponto em que é sentida a resistência moderada. NOTA: Para a injeção sub-retiniana, o melhor ângulo de abordagem da agulha é de cerca de 45 graus contra plano da íris, ea agulha sem corte deve ser empurrado posteriormente em direção à área peripapilar. Quadrado pontilhado indicou o trajeto da agulha sugerido através da cavidade vítrea para o injectio subretinaln na Figura 1. NOTA: Não haverá resistência sentiu quando perfuração (ou passagem) as camadas da retina como eles são muito moles. Assim, a primeira sensação de resistência leve indica que a agulha já tiver tocado na camada de EPR e a inserção da agulha deve ser interrompido. Tenha cuidado para não penetrar o tecido escleral com pressão excessiva porque a agulha deve ser colocada no potencial espaço sub-retiniano. Se a agulha perfura o tecido escleral por excesso de poder, ele entra nos espaços orbitais extra-oculares sem resistência. (Esta etapa é crítica) </li></ol><img alt="Figura 1" src="/files/ftp_upload/53030/53030fig1.jpg" /> Figura 1. Diagrama esquemático da injeção sub-retiniana. H &amp; E corte manchado do olho do rato que descreve as estruturas com um trajeto da agulha para a injeção sub-retiniana marcados (um quadrado pontilhada). Ampliação: 40X, Barra de escala:<html> 500 mm. <a href="https://www.jove.com/files/ftp_upload/53030/53030fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <ol start="9"><li> Injectar os vectores virais (por exemplo, 1 x 10 6 TU / ul) suavemente para o espaço sub-retiniano sem tremor para evitar danos nos tecidos não desejados e retirar a agulha suavemente. Segurar firmemente o globo ocular durante a injecção, tal como descrito em 2.4. </li><li> Observar a formação de bolha subretinal após a injecção sob microscópio cirúrgico para se certificar de que não há sangramento na retina. </li></ol><img alt="Figura 2" src="/files/ftp_upload/53030/53030fig2.jpg" /> Figura 2. Formação Subretinal Belb sem retina Hemorragia. Vista microscópica de belb subretinal após a injeção sub-retiniana sob o microscópio de operação mostra a formação de bolha bem sucedido sem hemorragia retiniana.m / files / ftp_upload / 53030 / &quot;target =&quot; _ blank 53030fig2large.jpg &quot;&gt; Clique aqui para ver uma versão maior desta figura. <ol start="11"><li> Com cuidado, feche a pálpebra para cobrir o local da injecção de auto-selagem. Retornar os ratos para gaiola e manter viva até a avaliação. NOTA: Depois que os pesquisadores se acostumar com os procedimentos, eles podem obter os resultados repetíveis com procedimentos rápidos saltando etapas (2,5, 6 e 10) </li></ol> 3. Avaliação da Eficácia da Entrega Gene em Retinal Pigment Epithelium <ol start="1"><li> Sacrifício dos ratinhos na câmara de CO 2. Enuclear os olhos com uma tesoura. </li><li> Fixar os olhos na solução de paraformaldeído a 4% a 4 ° C durante um mínimo de 1 hora a alguns dias. </li><li> Agarre a córnea com uma pinça fina e perfurar a córnea com micro-tesoura. Remover a córnea após tesoura ao longo do limbo. Retire a lente com uma pinça. </li><li> Apare o corpo ciliar com mICRO-tesoura, permitindo o descolamento de retina. Puxe cuidadosamente toda a retina do / coróide / complexo esclera RPE. Cortar o RPE / coróide / complexo escleral radialmente a partir da periferia para o centro perto do nervo óptico várias vezes para fazer 4-8 folhas para montagem plana. </li><li> Virar o RPE / coróide / complexo escleral para fazer lado RPE para baixo, e aparar todos os tecidos restantes na parte lateral escleral incluindo músculos, conjuntiva e nervo óptico. Este passo é importante fazer uma RPE / coróide / complexo escleral plana porque qualquer tecido remanescente torna o complexo desigual. </li><li> Incubar o complexo com o anticorpo primário e secundário para seus fins específicos de investigação (opcional, consulte as referências para os métodos detalhados). Plano de montagem do EPR / coróide / complexos esclerais sobre a lâmina de vidro e absorver o PBS com uma esponja absorvente. </li><li> Adicionar 30 ml de solução de montagem e coloque a cobertura deslizante. Observar a amostra para a eficácia da entrega do vetor sob o microscópio fluoresceína. Mantenha as amostras emo refrigerador para posterior observação. </li></ol>

<ol>
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The authors have nothing to disclose.

Neste artigo de vídeo, descrevemos o limbo abordagem técnica de injeção sub-retiniana em detalhe com resultados representativos de RPE / coróide / escleral flat-mount. Esta é uma técnica fácil e conveniente para a injecção sub-retiniana de vectores virais em EPR. A visualização direta da formação de bolha durante a injeção é um passo importante para a entrega correta para os novatos. Existem algumas técnicas de injeção subretinal introduzidas no Journal of Experiments Visualized 8-10. Espa...

Em oftalmologia, terapia genética surgiu como a modalidade de tratamento em retinopatias hereditárias monogênicas. Há retinopatias herdados associados com genes no epitélio pigmentar da retina (RPE), incluindo Leber amurosis congênita 1,2, retinite pigmentosa 3, 4 e choroideremia. O campo da terapia genética de pesquisa está em expansão em ambos os estudos pré-clínicos e clínicos que utilizam vectores virais tais como vírus adeno -associated (AAV), lentivírus (LV) e adenovírus (Ad) 5. Diferentes vectores virais possuem tropismo diferente na retina. Para uma terapia génica eficaz e segura, vectores virais devem ser cuidadosamente seleccionados de acordo com as células alvo e genes alvo. A via de entrega de genes, também é importante para a entrega de genes a células alvo eficaz, assim, deve ser cuidadosamente escolhida bem. Os dois métodos mais comuns para entrega intra-ocular de vectores virais são subretinal injeção e injeção intravítrea 6. Este último, injecção intravítrea, tem sido amplamente utilizada para a entrega de drogas para tratar a neovascularização coroidal em molhado degeneração macular relacionada com a idade (AMD) e o edema macular em retinopatia diabética 7. Via intravítrea proporciona uma exposição de vectores virais para a retina interna e vítreo, mas a difusão dos vectores a retina externa é limitada. Por outro lado, a via sub-retiniano fornece a entrega directa de vectores virais para o espaço potencial entre retina e EPR, induzindo uma vesícula localizada. Portanto, a injeção sub-retiniana é actualmente considerado como uma rota mais eficiente para o direcionamento de células fotorreceptoras e RPE. Em termos de abordagem cirúrgica, pars plana é escolhida como uma área segura para injeção intravítrea para evitar danos na retina em pacientes humanos. Por simplesmente modificar essa abordagem para ratos, poderíamos injetar vetores virais subretinally ou intravireally via abordagem de limbo. Neste vídeoartigo, demonstramos um método fácil e conveniente de injecção sub-retiniana de vectores virais em ratinhos EPR. Após a punção único no limbo posterior para com uma agulha 30 G 1/2, 33 g de uma agulha romba microlitro equipado seringa é inserida no espaço sub-retiniano através do local de punção limbo. Os vectores virais de 1,5-2 ul de volume são injectados para o espaço potencial entre retina e EPR induzir bolhas subretinal. Este procedimento pode ser realizado sob visualização directa utilizando microscópio cirúrgico. A prática repetida irá garantir resultados replicáveis ​​mesmo sem a visualização direta da formação de bolha. Isso vai ajudar os pesquisadores a realizar experimentos precisos e que economizam tempo para entrega gene em camundongos subretinal 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.

Para avaliar a eficácia da injecção sub-retinal na transdução de genes virais por este protocolo, foi utilizada vectores LV disponíveis comercialmente com o promotor de CMV expressando GFP e RFP para o indicador. Os olhos foram enucleados após o período de tempo adequado de acordo com a finalidade da investigação. Para os resultados representativos, os olhos foram enucleados 10 semanas e 20 semanas após a injeção sub-retiniana. Após a remoção completa da retina utilizando o método descrito acima, o plan...

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.

Limbo Abordagem-Subretinal injeção de vetores virais para terapia genética em ratos 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 ...

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

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Neuroscience

20.9K visualizações.  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.

Vídeo: 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.

Modelos experimentais para estudo da fisiologia epitélio pigmentar da retina e Fisiopatologia

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

Neurociência

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.

Injeção intracraniana da adeno-associado vetores virais

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.

Dissecação de um olho do rato para um monte inteiro do epitélio pigmentar da retina

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.

Injeção estereotáxica de um vetor viral para manipulação genética condicional no Cabo de Rato Spinal

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 Viral intracerebroventricular do Neonatal do rato do cérebro para a transdução neuronal persistente e generalizada

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.

Um protocolo para a terapia de décadas transcraniana em ratos

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 reprogramação direta de células gliais residentes em interneurônios por injeção intracerebral de vetores virais

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.

Cultivo de longo prazo, sem soro de explantes de retina de rato organotipo com epitélio pigmento de retina intacto

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.

Injeção de Constructo Viral (AAV) no Colículo Superior de Camundongos para Imagem com Cálcio

Entrega de punção lombar de AAV9 em ratos juvenis para terapia gênica do 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.

Autor em destaque: Avaliando a eficácia da terapia gênica intratecal em ratos juvenis

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.

Entrega de vetor intratecal em ratos juvenis por injeção de cisterna lombar

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 ...