Name: minimally-invasive technique for injection into rat optic nerve
Uploaded: 2015-05-19T14:00:00.000+00:00
Duration: 7 min 34 s
Description: Watch this Scientific Journal Video about Minimally-invasive Technique for Injection into Rat Optic Nerve at JoVE.com

This study was supported by NeuralStem, Inc., and Johns Hopkins Project RESTORE.

NOTA: Todos os procedimentos com animais foram aprovados pelo Comitê Animal Care e Use o Johns Hopkins. Aparelhos de anestesia exigir uma inspecção anual e calibração, se necessário. 1. Anestesia e Posicionamento <ol><li> Anestesia. <ol><li> Execute todos os procedimentos cirúrgicos sob anestesia com isofluorano 2-3%. Confirme nível adequado de anestesia por pitada dedo do pé e taxa de respiração. Verifique se o rato não se mexe em resposta a uma pitada dedo do pé. NOTA: Um vacilo indica anestesia que é tarde demais e pode exigir anestesia mais longos antes de iniciar ou uma concentração de isofluorano superior. A taxa de respiração inferior a 1 respiração cada 2 s é muito lento o que indica anestesia é demasiado elevada e pode exigir um aligeiramento da concentração de isofluorano. </li><li> Prenda o animal como necessário para evitar o movimento da cabeça durante o procedimento. Coloque uma gota de lidocaína no olho cirúrgico. Use lágrimas artificiais a cada 10 minutos para evitar a secura sob anestesia. Daruma injecção de buprenorfina a 0,01 mg / kg SC pré-operatório e em seguida cada 6-8 horas, conforme necessário. </li></ol></li><li> Posicionamento. <ol><li> Coloque ratos em um quadro estereotáxico e manter aquecido com uma almofada de aquecimento. Molhar a pele do couro cabeludo com álcool tendo o cuidado de evitar a exposição sobre os olhos. Use ferramentas estéreis e técnica estéril para minimizar o risco de infecções pós-operatórias. </li></ol></li></ol> 2. Controle dos olhos <ol><li> Coloque um fio de sutura 4-0 na conjuntiva lateral e amarrá-lo com sutura suficiente para permitir tração suave. </li></ol> 3. Dissecção <ol><li> Dissecção inicial. <ol><li> Adicione uma incisão ~ 1 polegada na pele que recobre o cume orbital usando um bisturi tamanho 10, como mostrado na Figura 1. Retirar a pele e fáscia subjacente e cuidadosamente dissecar longe do painel frontal. Para evitar sangramento excessivo no cirúrgico, evitar o corte vasos sanguíneos enquanto dissecando a fáscia. Use estrategicamente placed pontas de algodão para proporcionar hemostase. </li></ol></li><li> Dissecção mais profundo. NOTA: Com tração suave na conjuntiva puxando o olho para baixo e para fora do soquete, o músculo orbital superior entrará em vista. A fim de expor o nervo óptico, este deve ser cortado do músculo e gordura retro-orbital removido. A gordura pode ser descartado e não deve ser substituído após a injecção. A partir deste ponto, a fáscia nervo óptico deve ser visível como um feixe do nervo óptico em si, juntamente com os vasos sanguíneos envolvidos na dura-máter (Figura 1). <ol><li> Fazer uma pequena incisão na dura usando o bisturi ou com uma agulha de calibre 31 chanfrada para fazer a perfuração menos traumática. </li></ol></li></ol> 4. Pipeta Injector <ol><li> Retire uma micropipeta de vidro para um diâmetro de 50-100 um. Para garantir a estabilidade da montagem, a micropipeta em um micromanipulador e se ligar a uma seringa de Hamilton ligada a uma bomba de infusão. </li><li> Puxe as contas (ou células-tronco)reconstituída num volume de 0,5-1,0 mL na micropipeta retrógrada juntamente com o volume de 0,5 ul de uma solução de metil azul antes e depois. Observação: Uma taxa de infusão definida para 0,5-2 mL por min impede trauma para o nervo óptico. </li></ol> 5. Injeção <ol><li> Diminuir a ponta da micropipeta para o nervo óptico logo acima da dura-máter entalhado. NOTA: Uma pequena, mas rápido movimento da ponta de vidro nos resultados do nervo óptico em menos danos. À medida que a bomba de infusão inicia, o corante azul de metilo deve destacar a área do nervo óptico injectado. O corante deve permanecer localizada dentro do nervo óptico sem vazando no espaço subaracnóide. </li><li> Seguir a segunda banda de azul de metileno para determinar quando a injecção de células estaminais é completa e ligar a bomba de infusão fora como a segunda dose de azul de metileno é injectado. Manter a micropipeta ainda dentro do nervo óptico durante 2 min para cada volume de 1 mL injectada no para prevenir altaejeção de pressão após a retirada da micropipeta. NOTA: Uma abordagem alternativa é conectar a ponta da pipeta a uma seringa cheia de ar que pode ser manipulado manualmente. De qualquer maneira, evitar força excessiva para minimizar o dano para os nervos ópticos. Recomendamos injetando não mais de 2 l de volume, em cada nervo óptico. </li></ol> 6. Acompanhamento <ol><li> Immediate. <ol><li> Quando a injecção estiver concluída, remova a micropipeta, juntamente com todas as pontas de algodão usados ​​para fornecer hemostasia. Suturar a pele com seda 3-0 e remova o fio de sutura conjuntival. </li><li> Mantenha ratos quente sobre uma almofada de aquecimento até que eles emergem da anestesia. Não deixe um animal sem supervisão até que tenha recuperado a consciência suficiente para manter decúbito esternal. Não devolva um animal que tenha sido submetido a cirurgia para a companhia de outros animais até que esteja totalmente recuperado. </li><li> Se o animal está exibindo sinais de dor excessiva, incluindo letargia, ataxia, ou trabalharam breathing, administrar analgesia adequada com buprenorfina intramuscular (0,05 mg / kg) até três vezes ao dia. </li></ol></li><li> Longo prazo. <ol><li> Para a próxima 24-48 h, observe o rato por complicações da cirurgia, incluindo inchaço ou secreção da ferida ou outros sinais de dor, como vocalização, aparência curvada, não-aliciamento, ou não comer. Considere consulta com um veterinário ou a eutanásia ético, conforme necessário. </li></ol></li></ol>

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	<li>Dahlmann-Noor, A., Vijay, S., Jayaram, H., Limb, A., Khaw, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+approaches+and+future+prospects+for+stem+cell+rescue+and+regeneration+of+the+retina+and+optic+nerve.">Current approaches and future prospects for stem cell rescue and regeneration of the retina and optic nerve.</a> Canadian journal of ophthalmology Journal canadien d'ophtalmologie. 45 (4), 333-341 (2010).</li><li>Quigley, H. A., Iglesia, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+to+replace+the+optic+nerve.">Stem cells to replace the optic nerve.</a> Eye. 18 (11), 1085-1088 (2004).</li><li>Ghaffarieh, A., Levin, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optic+nerve+disease+and+axon+pathophysiology.">Optic nerve disease and axon pathophysiology.</a> International review of neurobiology. 105, 1-17 (2012).</li><li>Fukui, Y., Hayasaka, S., Bedi, K. S., Ozaki, H. S., Takeuchi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+study+of+the+development+of+the+optic+nerve+in+rats+reared+in+the+dark+during+early+postnatal+life.">Quantitative study of the development of the optic nerve in rats reared in the dark during early postnatal life.</a> Journal of anatomy. 174, 37-47 (1991).</li><li>Celestre, P. C., 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=Minimally+invasive+approaches+to+the+cervical+spine.">Minimally invasive approaches to the cervical spine.</a> The Orthopedic clinics of North America. 43 (1), 137-147 (2012).</li><li>Hallas, B. H., Wells, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Technique+for+Multiple+Injections+into+the+Mammalian+Optic+Nerve.">A Novel Technique for Multiple Injections into the Mammalian Optic Nerve.</a> Kopf Carrier. 54, (2001).</li><li>Harvey, A. R., Hellstrom, M., Rodger, J. <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+and+transplantation+in+the+retinofugal+pathway.">Gene therapy and transplantation in the retinofugal pathway.</a> Progress in brain research. 175, 151-161 (2009).</li><li>Adachi-Usami, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optic+neuritis--from+diagnosis+to+optic+nerve+transplantation.">Optic neuritis--from diagnosis to optic nerve transplantation.</a> Nippon Ganka Gakkai zasshi. 104 (12), 841-857 (2000).</li><li>Slater, B. J., Vilson, F. L., Guo, Y., Weinreich, D., Hwang, S., Bernstein, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optic+nerve+inflammation+and+demyelination+in+a+rodent+model+of+nonarteritic+anterior+ischemic+optic+neuropathy.">Optic nerve inflammation and demyelination in a rodent model of nonarteritic anterior ischemic optic neuropathy.</a> Investigative ophthalmology &amp; visual science. 54 (13), 7952-7961 (2013).</li><li>Zarbin, M. A., Arlow, T., Ritch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+nanomedicine+for+vision+restoration.">Regenerative nanomedicine for vision restoration.</a> Mayo Clinic proceedings. 88 (12), 1480-1490 (2013).</li></ol>

The authors have no meaningful disclosures to report.

Direct injection into the optic nerve of stem cells or other products intended to facilitate regeneration provides a convenient model compared to other means of injections into the CNS. This technique takes less time, requires less total anesthesia, avoids drilling or removing skull or bone tissue, reduces complications rates and allows for more rapid recovery following surgery. 
The most critical steps in this protocol include: 1. Adequate hemostasis in the surgical field to allow clear visualization of the optic nerve bundle, 2. Precise measurement of the pipette tip to allow easy insertion into the optic nerve with minimal trauma, 3. Tight control of eye using conjunctival suture which allows optimal exposure of the optic nerve. 
An alternative lateral approach to the optic nerve has been described previously which also allows injection directly into the optic nerve without dissection into the skull bone.6 In our experience, the lateral view is more limited to the very proximal optic nerve just behind the retina. In our superior approach, a larger portion of the optic nerve is visible allowing injection into both the proximal and mid-optic nerves. 
The major limitation of optic nerve injections is based on the rat&#8217;s limited use of vision. Rats can function well with their other senses of touch, hearing and smell and do not necessarily depend on vision as much as humans. Therefore, behavioral studies of rat vision have been limited to instinctual optokinetic reflexes and do not reliably correlate with an intact optic nerve. 
However, pathological assessments of axonal count and integrity, and myelination can provide useful information about the benefit of regenerative therapies. Furthermore, the pathological changes in the optic nerve are likely to represent changes in other parts of the CNS such as the brain and spinal cord that are more difficult to access. 
The most common current and future application of this procedure is for placement of regenerative stem cells into damage optic nerves. Various disorders of the optic nerve including trauma,7 inflammatory,8 and vascular9 etiologies can potentially benefit from regenerative strategies. Localized injections of small molecules and nanoparticles10 into the optic nerve are also being evaluated for regenerative potential.

O nervo óptico oferece uma localização ideal para o sistema nervoso central (SNC) de pesquisa regenerativa incluindo condições oftalmológicas como a neurite óptica, glaucoma e trauma. As injecções de uma variedade de células estaminais quer demonstraram eficácia ou mostraram-se promissores na substituição de mielina perdido, aumentando a contagem axonal e / ou prevenção de doenças degenerativas. 1,2 O nervo óptico humano contém aproximadamente 1,2 milhões de axónios que viajam paralelas da retina para o quiasma com um diâmetro de cerca de 3,0-3,5 mm. 3 Para modelar doenças humanas em laboratório, o rato tem sido utilizado frequentemente. O nervo óptico de rato adulto contém cerca de 100.000 axónios dentro de um diâmetro de aproximadamente 0,5 mm. 4 Uma das principais limitações na pesquisa no SNC regenerativo é desossada acesso directo. As complicações e riscos cirúrgicos para o animal são maiores quando o crânio ou vértebras são removidos. Similar aos benefícios deabordagens minimamente invasivas da coluna vertebral, 5 injeções diretas do nervo óptico sem abrir o crânio de oferta reduzida complicações e uma recuperação mais rápida. Esta técnica tem sido utilizada em estudos anteriores. 6 Neste manuscrito e vídeo acompanhante, que demonstram um procedimento minimamente invasivo para injectar células estaminais no nervo óptico de rato.

<table><tbody><tr><td>Lewis rat</td><td>Charles River</td><td>4</td><td>Any rat strain will work.</td></tr><tr><td>Anesthesia machine</td><td>Surgivet</td><td>CDS9000</td><td>CDS 9000 Small Animal Anesthesia Machine - Pole Mount</td></tr><tr><td>Infusion pump</td><td>Stoelting</td><td>53129</td><td></td></tr><tr><td>Dissection microscope</td><td>National Optical</td><td>409-411-1105</td><td></td></tr><tr><td>Fiber-optic light source</td><td>Fisher Scientific</td><td>12-562-21</td><td></td></tr><tr><td>Dissection and Stereotaxic Instrument</td><td>Stoelting</td><td>51400</td><td></td></tr><tr><td>Pipette Puller</td><td>Kopf</td><td>750</td><td></td></tr><tr><td>Pipettes</td><td>World Precision Instruments</td><td>18150-6</td><td></td></tr><tr><td>Disposable scalpel blades</td><td>Harvard Apparatus</td><td>810-15-021</td><td></td></tr><tr><td>Iridectomy scissors</td><td>Electron Microscopy Sciences</td><td>Uniband LA-4XF</td><td></td></tr></tbody></table>

minimally-invasive technique for injection into rat optic nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.

Na conclusão da experiência, os ratos foram sacrificados e perfundidos com paraformaldeído a 4%. Os nervos ópticos foram cuidadosamente dissecados e montados para seccionamento criostático. A Figura 2 mostra um exemplo de um nervo óptico de rato inteiro com baixa potência, em que o corante azul de Evans foi injectada a fim de visualizar o local. A seta identifica a localização precisa da injecção. Esta dissecção foi realizada dentro de alguns minutos da injecção, tal como indicado pela difusão restrita do corante para o nervo. Em outras injeções, temos observado uma lenta difusão de corante para o quiasma ao longo de várias horas. <img alt="Figura 1" src="/files/ftp_upload/52249/52249fig1highres.jpg" /> Figura 1:. Campo cirúrgico de vista O rato é posicionado com a acessar o olho esquerdo nesta figura. Uma incisão é feita acima do cume orbital ea fascialtecido é dissecado para baixo por trás do olho. Uma sutura conjuntival permite que o operador aplica tracção suave que puxa o nervo óptico sem intracraniana em vista a abertura do crânio. <img alt="Figura 2" src="/files/ftp_upload/52249/52249fig2highres.jpg" /> Figura 2:. Dissecção macroscópica do nervo óptico injectado Utilizando corante azul de Evans, o local da injecção no nervo óptico pode ser grosseiramente visualizados sob um microscópio de dissecção. Nesta imagem, o nervo óptico foi cortado no local da injecção para mostrar o corante incorporado no interior do tecido do nervo óptico.

Watch this Scientific Journal Video about Minimally-invasive Technique for Injection into Rat Optic Nerve at JoVE.com

Minimally-invasive Technique for Injection into Rat Optic Nerve

Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid.

Técnica minimamente invasiva para a injeção em Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central ...

minimally-invasive-technique-for-injection-into-rat-optic-nerve

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

9.5K visualizações.  Johns Hopkins University. Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid. 

Vídeo: Minimally-invasive Technique for Injection into Rat Optic Nerve

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

Métodos para manipulações experimentais após a transecção do nervo óptico no sistema nervoso central de mamíferos

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

Neurociência

Intravitreous Injection for Establishing Ocular Diseases Model

Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as direct application of local treatment. This video introduces how to use simple and inexpensive tools to finish the intravitreous injection procedure. Use of a 1 ml syringe, instead of a hemilton syringe, is used. Practical tips for how to make appropriate injection needles using glass pipettes with perfect tips, and how to easily connect the syringe needle with the glass pipette tightly together, are given.
To conduct a good intravitreous injection, there are three aspects to be observed: 1) injection site should not disrupt retina structure; 2) bleeding should be avoided to reduce the risk of infection; 3) lens should be untouched to avoid traumatic cataract. In brief, the most important point is to reduce the interruption of normal ocular structure. To avoid interruption of retina, the superior nasal region of rat eye was chosen. Also, the puncture point of the needle was at the par planar, which was about 1.5 mm from the limbal region of the rat eye. A small amount of vitreous is gently pushed out through the puncture hole to reduce the intraocular pressure before injection. With the 45&deg; injection angle, it is less likely to cause traumatic cataract in the rat eye, thus avoiding related complications and influence from lenticular factors. In this operation, there was no cutting of the conjunctiva and ocular muscle, no bleeding. With quick and minor injury, a successful intravitreous injection can be done in minutes. 
The injection set outlined in this particular protocol is specific for intravitreous injection. However, the methods and materials presented here can also be used for other injection procedures in drug delivery to the brain, spinal cord or other organs in small mammals.

Intravitreous injection is a widely used technique in visual sciences research for ocular diseases or as direct application of local treatment. This video demonstrated a protocol for intravitreous injection using a 1ml syringe with glass pipette. Useful tips about avoiding massive bleeding and lens damage are given.

Injeção intravítrea para o Estabelecimento de Modelo de Doenças Ocular

Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as ...

Biologia

Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history have&#160;remained poorly understood. Recently, we developed a reliable, reproducible animal model of PION and tested the treatment effect of some neurotrophic factors in this model1. The purpose of this video is to demonstrate our photochemically induced model of posterior ischemic optic neuropathy, and to evaluate its effects with retrograde labeling of retinal ganglion cells. Following surgical exposure of the posterior optic nerve, a photosensitizing dye, erythrosin B, is intravenously injected and a laser beam is focused onto the optic nerve surface. Photochemical interaction of erythrosin B and the laser during irradiation damages the vascular endothelium, prompting microvascular occlusion mediated by platelet thrombosis and edematous compression. The resulting ischemic injury yields a gradual but pronounced retinal ganglion cell dieback, owing to a loss of axonal input - a remote, injury-induced and clinically relevant outcome. Thus, this model provides a novel platform to study the pathophysiologic course of PION,&#160;and can be further optimized for testing therapeutic approaches for optic neuropathies as well as other CNS ischemic diseases.

The goal of this protocol is to photochemically induce ischemic injury to the posterior optic nerve in rat. This model is critical to studies of the pathophysiology of posterior ischemic optic neuropathy, and therapeutic approaches for this and other optic neuropathies, as well as of other CNS ischemic diseases.

Modelo de Rato Induzida fotoquimicamente posterior neuropatia óptica isquémica

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history ...

Medicina

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to the interphotoreceptor/subretinal compartment that has direct exposure to photoreceptors and the retinal pigment epithelium (RPE). Subretinal injections of plasminogen as well as recent preclinical and clinical trials have demonstrated safety and/or efficacy of delivering viral vectors and stem cells to individuals with advanced retinal disease. Mouse models of retinal disease, particularly hereditary retinal dystrophies, are essential for testing these therapies. The most common injection procedure in rodents is to use small transcorneal or transcleral incisions with an anterior approach to the retina. With this approach, the injection needle penetrates the neurosensory retina disrupting the underlying RPE and on insertion can easily nick the lens, causing lens opacification and impairment of noninvasive imaging. Accessing the subretinal space via a transcleral, posterior approach avoids these problems: the needle crosses the sclera approximately 0.5 mm from the optic nerve, without retinal penetration and avoids disrupting the vitreous. Collateral damage is limited to that associated with the focal sclerotomy and the effects of a transient, serous retinal detachment. The simplicity of the method minimizes ocular injury, ensures rapid retinal reattachment and recovery, and has a low failure rate. The minimal damage to the retina and RPE allows for clear assessment of the efficacy and direct effects of the therapeutic agents themselves. This manuscript describes a novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, stem cells or induced pluripotent stem (iPS) cells to the subretinal space in mice with high efficacy, minimal damage, and fast recovery.

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

Um método de injeção Alternativa e validado para acessar o espaço sub-retiniano<i&gt; via</i&gt; Uma abordagem transcleral Posterior

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy (rNAION)

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their lifetime. NAION results in optic nerve edema, selective loss of the retinal ganglion cell neurons (RGCs) and atrophy of the optic nerve. A rodent model of NAION that expresses most NAION features and sequelae has been developed, which is applicable to both rats and mice. This model utilizes a focal laser application of 532 nm wavelength to illuminate a photoactive dye, Rose Bengal (RB), to cause capillary damage and leakage at the targeted anterior optic nerve (the laminar region). After rNAION induction, there is an early optic nerve ischemia, optic nerve edema, and intraneural inflammation, followed by selective RGC and axonal loss. Since the optic nerve is a CNS white matter tract, the rNAION model is applicable to mechanistic studies of selective white matter ischemia, as well as neuroprotective analyses and short and long-term mechanisms of glial and neuronal response to ischemia.

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy rNAION

The following report describes how to replicate the rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION), using the appropriate dye, contact lens and laser parameters. We also reveal the appropriate steps for evaluating the rNAION lesion in vivo.

O modelo de roedor de nonarterítica óptica isquêmica anterior Neuropatia (rNAION)

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

Tomografia de coerência óptica Mouse ultra alta resolução para auxiliar a injeção intra-ocular em pesquisa de terapia de genes da retina

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

Sheath-Preserving Optic Nerve Transection in Rats to Assess Axon Regeneration and Interventions Targeting the Retinal Ganglion Cell Axon

Retinal ganglion cell (RGC) axons converge at the optic nerve head to convey visual information from the retina to the brain. Pathologies such as glaucoma, trauma, and ischemic optic neuropathies injure RGC axons, disrupt transmission of visual stimuli, and cause vision loss. Animal models simulating RGC axon injury include optic nerve crush and transection paradigms. Each of these models has inherent advantages and disadvantages. An optic nerve crush is generally less severe than a transection and can be used to assay axon regeneration across the lesion site. However, differences in crush force and duration can affect tissue responses, resulting in variable reproducibility and lesion completeness. With optic nerve transection, there is a severe and reproducible injury that completely lesions all axons. However, transecting the optic nerve dramatically alters the blood brain barrier by violating the optic nerve sheath, exposing the optic nerve to the peripheral environment. Moreover, regeneration beyond a transection site cannot be assessed without reapposing the cut nerve ends. Furthermore, distinct degenerative changes and cellular pathways are activated by either a crush or transection injury.
The method described here incorporates the advantages of both optic nerve crush and transection models while mitigating the disadvantages. Hydrostatic pressure delivered into the optic nerve by microinjection completely transects the optic nerve while maintaining the integrity of the optic nerve sheath. The transected optic nerve ends are reapposed to allow for axon regeneration assays. A potential limitation of this method is the inability to visualize the complete transection, a potential source of variability. However, visual confirmation that the visible portion of the optic nerve has been transected is indicative of a complete optic nerve transection with 90-95% success. This method could be applied to assess axon regeneration promoting strategies in a transection model or investigate interventions that target the axonal compartments.

This protocol describes an optic nerve transection that preserves the optic nerve sheath in rats. Hydrostatic pressure from microinjections into the optic nerve generate a complete transection, allowing for sutureless reapposition of the transected optic nerve ends and direct targeting of the axonal compartment in a transection model.

Retinal ganglion cell (RGC) axons converge at the optic nerve head to convey visual information from the retina to the brain. Pathologies such as ...

Direct Vagus Nerve Injection Protocol For Rats

There is a relative abundance of strategies and methodologies to facilitate drug delivery to the central nervous system. However, drug delivery directly to the peripheral nervous system is less common, with fewer detailed methods publications available to aid researchers. Here, we describe a direct nerve injection method for peripheral nervous system drug delivery, using the vagus nerve as a model nerve. This method can be used in the treatment of autonomic nervous system disorders through targeting of the left vagus nerve, although this general injection method can be extrapolated to injection of other nerves with minor modification. This method explains all critical steps involved in the procedure involving microsurgery in anesthetized adult rats under a dissecting microscope. The use of a tracking dye is described to facilitate the monitoring of injection fidelity in real time. Illustrations of successful and failed injections are provided. If carried out properly, direct vagus nerve injections can be conducted in a safe manner that is well-tolerated by the rat without post-delivery complications. For example, once the surgeons were trained in this method, six out of six rats were successfully injected without any complications. This method of direct nerve injection for preclinical rat studies is capable of delivering agents (including but not limited to gene therapy) to peripheral nerves.

We present a protocol for direct vagus nerve injection in rats, enabling drug delivery directly into the nerve without post-injection complications. This method applies to preclinical neurological studies involving autonomic nervous system manipulation. It can be used for direct nerve injection for other nerves in rats and other species, with necessary modifications.

Protocolo de injeção direta do nervo vago para ratos

There is a relative abundance of strategies and methodologies to facilitate drug delivery to the central nervous system. However, drug delivery directly ...

Transconjunctival Approach for Injection into the Rat Optic Nerve

The rat serves as an important model for studying disorders of the optic nerve, including heritable, traumatic, neoplastic, and autoimmune conditions. Accessing the rat optic nerve for experimental manipulations and injections is challenging due to the small size of the orbit and the vascularity of the surrounding orbital tissues. Previous techniques have involved a cutaneous incision, which carries a higher risk of infection and requires wound closure. This study aims to describe a unique, less invasive, and potentially more efficient approach to accessing the rat optic nerve. A transconjunctival incision, along with a rat spinal cord hook, is used to isolate the optic nerve for experimental injections. In these experiments, an adenoviral vector is injected into the optic nerve with the goal of using this technique for direct gene therapy delivery in the future. This technique can also be applied by investigators requiring access to the rat optic nerve for other experimental studies, including drug delivery and optic nerve injury research.

This protocol describes a unique technique using a transconjunctival approach to access the rat optic nerve for injections.

Abordagem transconjuntival para injeção no nervo óptico de rato

The rat serves as an important model for studying disorders of the optic nerve, including heritable, traumatic, neoplastic, and autoimmune conditions. ...

<meta charset="utf8"></head><body>Técnica de injeção intracameral de rato otimizada para reduzir o dano tecidual

Intracameral Injection in Rats with Low Risk of Adverse Effects

Intracameral injection is a standard administration routine in ophthalmology. The application of intracameral injection in rodents for research is challenging due to the limiting dimensions and anatomy of the eye, including the small aqueous humor volume, the lens curvature, and lens thickness. Potential damage during intracameral injections introduces adverse effects and experimental variability. This protocol describes a procedure for intracameral injection in rats, allowing precision and reproducibility.
Sprague-Dawley rats were used as experimental models. Since the lens position in rats protrudes into the anterior chamber, injecting from the periphery, as done in humans, is unfavorable. Therefore, an incision is created in the central corneal region using a 31 gauge 0.8 mm stiletto blade to form a self-sealing tunnel into the anterior chamber. An incision at&#160;an angle close to the flat allows to create a long tunnel, which minimizes the loss of aqueous humor and shallowing of the anterior chamber. A 34 gauge nanoneedle is inserted into the tunnel for injection. This enables penetration with minimal friction resistance and avoids touching the lens. Injection of trypan-blue allows&#160;visualization&#160;by slit microscopy the presence of the dye in the anterior chamber and exclude leakage. Bioavailability to the corneal endothelial layer is demonstrated by injection of Hoechst dye, which stained the nuclei of corneal endothelial cells after injection.
In conclusion, this protocol implements a procedure for accurate intracameral injection in rats. This procedure may be used for intracameral delivery of various drugs and compounds in experimental rat models, increasing the efficiency and reproducibility of ophthalmic research.

<meta charset="utf8"></head><body>Autor em destaque: um novo protocolo para injeções intracamerais para aumentar a precisão na oftalmologia de roedores

This protocol describes a technique for intracameral injection in rats using a central corneal incision and a long tunnel into the anterior chamber. This injection method minimizes the risk of inducing inadvertent tissue damage and thereby improves precision and reproducibility.

Técnica minimamente invasiva para a injeção em Rat Optic Nerve

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