Bu çalışma Seul Ulusal Üniversitesi Araştırma Grant (800-20140542) tarafından desteklenen, NTG / EBTB Pioneer Araştırma Programı (2012-0009544) ve NRF / EBTB Bio-Sinyal Analizi Teknoloji İnovasyon Programı (2009-0090895), ve NRF / EBTB Hibe (2015M3A9E6028949).

Hayvanlar üzerinde deneyler tüm Oftalmik ve Vizyon Araştırma Hayvanların Kullanım Vizyon ve Oftalmoloji Tablosunda Araştırma Derneği göre yapılan ve kurallar ve yönetmelikler Seul Ulusal Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Komitesi ve Seul Ulusal öngördüğü edildi Üniversite Hastanesi Biyogüvenlik Komitesi. 1. Enjeksiyon Kit ve Viral Vektörler hazırlanması <ol><li> Etilen oksit gazı ile sterilize edilmiş bir 33 G, kör bir iğne ile donatılmış mikrolitre şırınga hazırlayın. Mikro tüp (yani, 1 x 10 6 TU / ml) &#39;de yeterli bir titre PBS içinde viral vektörleri ile seyreltilir. Şırınga herhangi ölü boşluğu kaldırmak için enjektörü viral vektörler ile birkaç kez yıkayın. </li></ol> Viral vektörler 2. Subretinal Enjeksiyon <ol><li> Tiletami karışımının intraperitoneal enjeksiyonu ile - (8 haftalık, yani, 6), yetişkin fareler anesteziKD ve zolazepam (1: 1, 2.25 mg / kg vücut ağırlığı) ve ksilazin hidroklorür (0.7 mg / kg vücut ağırlığı) ya da alternatif olarak uygun anestezi rejimi. </li><li> Fenilefrin% 0.5 göz damlası ile öğrenciler dilate ve% 0.5 tropikamid. </li><li> Viral vektörlerin 2 ul - 1.5 ile yüklenerek mikrolitre şırınga hazırlayın. </li><li> Göz kapağı açın ve uygun enjeksiyon için ekvator ortaya çıkarmak ve işletim mikroskop altında odaklanmak göz çıkıntı. Enjeksiyon sırasında oluşabilecek iğne enjeksiyonu veya deplasman bitirme kadar çıkıntı göz pozisyonunu koruyun. Sıkıca göz küresi tutun, yörünge jant dışında parmaklarını yerleştirin. </li><li> Kornea yüzeyine oftalmik viskoelastik bir çözelti damla uygulanır. </li><li> Retina görselleştirmek için kornea üstünde küçük bir yuvarlak kapak slayt yerleştirin. </li><li> Daha fazla subretinal enjeksiyon için steril 30 G 1/2 iğne kullanılarak limbusa hafif posterior küçük bir delik delmek. Fo delik aşağı olunr sağ göz ve rahatlık için sol göz için üstün. NOT: delik zamansal ya da nazal yapılır ise, enjeksiyondan sonra göz kapağında kapsamına zor. İlk ponksiyon hafifçe yapılan limbusun boyunca çalışacak ve kolayca tanınabilir limbal damarları önlemek için limbusa posteriorunda edilmelidir. İlk ponksiyon yapılırken iğne ile lensi çarpmamak için dikkatli olun. Lens ponksiyon önlemek için iğnenin tüm konik takmayın. </li><li> Önceden delinmiş delikten mikrolitre şırınga 33 G künt iğne yerleştirin ve hafif direnç hissedilir zaman noktaya kadar subretinal boşluğa iğne yaklaşım. NOT: subretinal enjeksiyon için, iğnenin en iyi yaklaşım açısı iris düzleminin karşı yaklaşık 45 derece, ve künt bir iğne peripapiller alana doğru posterior itti olmalıdır. Noktalı kare subretinal injectio için vitreus boşluğuna genelinde önerilen iğne yolu belirtilenŞekil 1 &#39;de: n. NOT: Onlar çok yumuşak olduğu gibi delici (veya geçen) retina katmanları zaman direnç hissettim olacak. Böylece, hafif direnç ilk duygu iğne zaten RPE dokundu ve iğnenin takılması durdurulması gerektiğini belirtir. İğne potansiyel subretinal alana konulmalıdır çünkü aşırı basınç skleral doku nüfuz için dikkatli olun. İğne aşırı güç tarafından skleral doku deler, bu direnç olmadan göz dışı yörünge alanlarda girer. (Bu adım önemlidir) </li></ol><img alt="Figür 1" src="/files/ftp_upload/53030/53030fig1.jpg" /> Subretinal Enjeksiyon Şekil 1. Şematik. H &amp; E işaretli subretinal enjeksiyon için bir iğne yolunun olan yapıları resmeden fare göz lekeli kesiti (noktalı kare). Büyütme: 40X, Ölçek çubuğu:<html> 500 mikron. <a href="https://www.jove.com/files/ftp_upload/53030/53030fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <ol start="9"><li> İstenmeyen doku hasarını önlemek ve yavaşça iğneyi çekilme titreme olmadan subretinal boşluğa yavaşça viral vektörler (örneğin, 1 x 10 6 TU / ul) enjekte edilir. 2.4&#39;te tarif edildiği gibi enjeksiyon sırasında sıkıca gözün tutun. </li><li> Hiçbir retina kanaması olduğundan emin olmak için, işletim mikroskop altında enjekte edildikten sonra subretinal bleb oluşumunu gözlemleyin. </li></ol><img alt="Şekil 2," src="/files/ftp_upload/53030/53030fig2.jpg" /> Şekil 2. Retina Kanama olmadan Subretinal BELB Formasyonu. Işletim mikroskop altında subretinal enjeksiyondan sonra subretinal BELB mikroskopik görünümü retinal kanama olmadan başarılı bleb oluşumu göstermektedir.m / files / ftp_upload / 53030 / 53030fig2large.jpg &quot;target =&quot; _ blank &quot;&gt; bu rakamın daha büyük bir versiyonunu görmek için lütfen buraya tıklayınız. <ol start="11"><li> Yavaşça kendini sızdırmazlık için enjeksiyon yerinde kapsayacak şekilde göz kapaklarını kapatın. Ev kafesine fareler dönün ve değerlendirme kadar canlı tutmak. NOT: Araştırmacılar prosedürlere alışması sonra, aşağıdaki adımları atlayarak hızlı prosedürlere tekrarlanabilir sonuçlar elde (2.5, 6, ve 10) olabilir </li></ol> Retina Pigment Epitel Gen Teslimat Etkinliğinin Değerlendirilmesi 3. <ol start="1"><li> CO2 odası içinde fareler kurban. Makas kullanılarak gözleri enükleasyon. </li><li> Birkaç gün için en az 1 saat boyunca 4 ° C &#39;de% 4 paraformaldehit solüsyonunda gözleri düzeltildi. </li><li> Ince forseps ile kornea tut ve mikro-makas ile kornea delinme. Limbusun boyunca makaslama sonra kornea çıkarın. Forseps ile lensi çıkarın. </li><li> M siliyer cisim Trimretina dekolmanı izin ICRO-makas. Yavaşça RPE / koroid / sklera karmaşık bütün retinanın çekin. Düz montaj için 4 ila 8 yaprakları yapmak için optik sinir birkaç kez yakın merkeze çevresinden radyal RPE / koroid / sklera kompleksi kesin. </li><li> Aşağı RPE tarafı yapmak ve kas, konjonktiva ve optik sinir dahil skleral tarafında kalan tüm dokuları kesmek için RPE / koroid / sklera kompleksi çevirin. Kalan doku, karmaşık düzensiz hale getirir, çünkü bu adım, düz RPE / koroid / sklera kompleksi yapmak önemlidir. </li><li> (Ayrıntılı yöntemleri için referanslara bakın, isteğe bağlı) özel araştırma amaçlı birincil ve ikincil antikor ile kompleks inkübe edin. Cam slayt RPE / koroid / sklera kompleksleri Düz montaj ve emici bir sünger ile PBS emer. </li><li> Montaj çözümü 30 ul ekleyin ve kapağını kaydırarak yerleştirin. Floresein mikroskop altında vektör teslim etkinliği için örnek gözlemleyin. Örnekleri tutunDaha fazla gözlem için buzdolabı. </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.

Bu video makalede, biz RPE / koroid / sklera düz montaj temsilcisi sonuçları ile detaylı limbal-yaklaşım subretinal enjeksiyon tekniği nitelendirdi. Bu RPE viral vektörlerin retina altı enjeksiyon için kolay ve uygun bir tekniktir. Enjeksiyon sırasında kabarcık oluşumu doğrudan görselleştirme yeni başlayanlar için doğru bir teslimat için önemli bir adımdır. Visualized Experiments 8-10 Journal tanıtılan bazı subretinal enjeksiyon teknikleri vardır. Subretinal alan retina ve RPE aras...

Oftalmolojide, gen terapisi monogenik miras retinopatilerin tedavi yöntemi olarak ortaya çıkmıştır. Leber konjenital amurosis 1,2, retinitis pigmentosa 3 ve Koroideremi 4 olmak üzere retina pigment epiteli (RPE) genlerin ilişkili kalıtsal retinopatiler vardır. gen tedavisi araştırma alanı preklinik çalışmalarda ve adeno virüsü enzimi yüksek yoğunluklu lipoproteinlerde (AAV), lentivirüs (LV) ve adenovirüs (Ad) gibi viral vektörler kullanılarak klinik çalışmalarda hem de genişliyor 5. Farklı viral vektörler retina farklı tropizm ihtiva. Güvenli ve etkili bir gen terapisi için viral vektörler, dikkatli bir şekilde hedef hücreler ve hedef genlerin uygun olarak seçilmelidir. etkili bir gen verici hedef hücrelere gen teslim yolu bu nedenle, dikkatli bir şekilde da seçilmelidir, da önemlidir. viral vektörlerin göz içi teslimat için en yaygın iki yöntemler subret edilirinal enjeksiyon ve intravitreal enjeksiyon 6. İkincisi, intravitreal enjeksiyon, yaygın diyabetik retinopati 7 ıslak yaşa bağlı makula dejenerasyonu (YBMD) ve maküla ödemi koroid neovaskülarizasyon tedavisi için ilaç verilmesi için kullanılır olmuştur. İntravitreal rota vitreus ve iç retina viral vektörlerin pozlama sağlar, ancak dış retinaya vektörlerin difüzyon sınırlıdır. Öte yandan, alt retinal yol lokalize bir kabarcık indükleyici, retina ve RPE arasındaki potansiyel boşluğa viral vektörlerin doğrudan dağıtım sağlar. Bu nedenle, alt retinal enjeksiyon anda fotoreseptör hücreleri ve RPE hedefleme için daha verimli bir yol olarak kabul edilir. Cerrahi yaklaşım açısından, plana insan hastalarda retina hasarı önlemek için intravitreal enjeksiyon için güvenli bir alan olarak seçilmiştir pars. Sadece farelere bu yaklaşımı değiştirerek, biz limbal yaklaşımla subretinally veya intravireally viral vektörlerin enjekte edebilir. Bu videodamakale, biz fareler RPE viral vektörlerin subretinal enjeksiyonu kolay ve uygun bir yöntem ortaya koymaktadır. 30 G 1/2 iğne ile limbusa posterior tekli delme sonra, 33 G iğne künt donanımlı mikrolitre şırınga limbal ponksiyon sitesi üzerinden subretinal boşluğa yerleştirilir. 1.5 viral vektörler - 2 ul hacminde, retina ve RPE subretinal kabarcıkları indükleyici arasındaki potansiyel boşluğa enjekte edilir. Bu prosedür, cerrahi mikroskop kullanılarak doğrudan görüş altında gerçekleştirilebilir. Tekrarlanan uygulama bile kabarcık oluşumu doğrudan görselleştirme olmadan tekrarlanabilir sonuçları garanti edecektir. Bu fareler RPE subretinal gen aktarımı için doğru ve zaman kazandıran deneyler gerçekleştirmek için araştırmacıların yardımcı olacaktır.

<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>
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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.

Bu protokol ile viral gen transdüksiyon üzerinde subretinal enjeksiyonunun etkinliğini değerlendirmek için, göstergesi GFP ve RFP hem ifade CMV promotör ile piyasada mevcut LV vektörleri kullanılır. Gözler araştırma amacına göre uygun süre sonra iki parçaya ayrıldı. Temsilcisi sonuçları için gözler 10 hafta ve subretinal enjeksiyondan sonra 20 hafta parçaya ayrıldı. Yukarıda tarif edilen yöntem kullanılarak, retina tamamen uzaklaştırıldıktan sonra, RPE / koroid / sklera kompleksinin düz...

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.

Fareler Retina Pigment Epitel Gen Tedavisi için Viral Vektörler Limbal Yaklaşım-Subretinal Enjeksiyon

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 Görüntüleme.  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.

Video: 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.

Retina Pigment Epitel Fizyoloji ve Patofizyoloji Çalışma Deneysel Modeller

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

Nörobilim

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.

Adeno-ilişkili Viral Vektörler İntrakranial Enjeksiyon

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.

Retina pigment epiteli Tüm Mount Fare Göz Diseksiyon

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.

Fare Omurilik Koşullu Gen Manipülasyon için bir Viral Vector Stereotaksik Enjeksiyon

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.

Kalıcı ve yaygın Nöronal İletim için Yenidoğan Fare Beyin intraserooroventriküler Viral Enjeksiyon

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.

Farelerde Transkraniyal Photobiomodulation tedavisi için bir iletişim kuralı

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 doğrudan reprogramming Resident glial hücreler Interneurons viral vektörler ıntracerebral enjeksiyon ile

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.

Sağlam Retina Pigment Epitel ile Organotipik Fare Retina Eksplantlarının Uzun Süreli, Serumsuz Ekimi

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 ...

CNS Gen Tedavisi için Juvenil Sıçanlarda AAV9'un Lomber Delinme Verilmesi

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.

Yazar Spotlight: Juvenil Sıçanlarda İntratekal Gen Terapisi Etkinliğinin Değerlendirilmesi

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.

Juvenil Sıçanlarda Lomber Sarnıç Enjeksiyonu ile İntratekal Vektör Verilmesi

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 ...