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 los animales procedimientos fueron aprobados por el Comité de Cuidado de Animales y el empleo de Johns Hopkins. Máquinas de anestesia requieren inspección anual y calibración según sea necesario. 1. Anestesia y Posicionamiento <ol><li> Anestesia. <ol><li> Realizar todos los procedimientos quirúrgicos con anestesia con isofluorano 2-3%. Confirmar nivel adecuado de anestesia por pizca dedo del pie y ritmo respiratorio. Compruebe que la rata no se inmuta en respuesta a una pizca dedo del pie. NOTA: Un estremecimiento indica la anestesia que es demasiado tarde y puede requerir anestesia más largos antes de comenzar o una concentración isofluorano superior. Una tasa de respiración menos de 1 respiración cada 2 seg es demasiado lento que indica la anestesia es demasiado alta y puede requerir aligeramiento de la concentración de isofluorano. </li><li> Asegure el animal si es necesario para evitar el movimiento de la cabeza durante el procedimiento. Coloque una gota de lidocaína en el ojo quirúrgico. Use lágrimas artificiales cada 10 minutos para evitar la sequedad mientras que bajo anestesia. Daruna inyección de buprenorfina de 0,01 mg / kg SC pre-operativamente y luego cada 6-8 horas según sea necesario. </li></ol></li><li> Posicionamiento. <ol><li> Coloque las ratas en un marco estereotáxico y mantenga caliente con una almohadilla térmica. Humedezca la piel del cuero cabelludo con el cuidado de alcohol para evitar la exposición en los ojos. Utilice herramientas estériles y una técnica estéril para minimizar el riesgo de infecciones postoperatorias. </li></ol></li></ol> 2. Control de los ojos <ol><li> Coloque una sutura 4-0 en la conjuntiva lateral y lo atan con suficiente sutura para permitir una tracción suave. </li></ol> 3. Disección <ol><li> Disección inicial. <ol><li> Hacer una incisión ~ 1 pulgada en la piel que cubre la cresta orbital utilizando un tamaño de 10 bisturí como se muestra en la Figura 1. Retraer la piel y la fascia subyacente y cuidadosamente diseccionar la fascia. Para prevenir el sangrado excesivo en el quirúrgico, evitar cortar los vasos sanguíneos, mientras que la disección de la fascia. Utilizar estratégicamente plconsejos de Aced algodón para proporcionar hemostasia. </li></ol></li><li> Disección más profundo. NOTA: Con tracción suave en la conjuntiva del ojo tirando hacia abajo y fuera de la cuenca, el músculo orbital superior vendrá a la vista. Con el fin de exponer el nervio óptico, este músculo se debe cortar la grasa y retro-orbital retira. La grasa puede ser desechado y no debe ser sustituido después de la inyección. Desde este punto, la fascia nervio óptico debe ser visible como un haz del nervio óptico en sí, junto con los vasos sanguíneos envueltos en la duramadre (Figura 1). <ol><li> Hacer una pequeña incisión en la duramadre utilizando el bisturí o una aguja biselada de calibre 31 para hacer la perforación menos traumática. </li></ol></li></ol> 4. Pipetear Inyector <ol><li> Tire de una micropipeta de vidrio de un diámetro de 50-100 micras. Para proporcionar estabilidad, montar la micropipeta en un micromanipulador y adjuntar a una jeringa Hamilton conectado a una bomba de infusión. </li><li> Tire hacia arriba de los granos (o células madre)reconstituida en un volumen de 0,5-1,0 l en la micropipeta retrógrado junto con 0,5 l de volumen de solución de azul de metilo antes y después. NOTA: Una tasa de infusión de 0,5 a 2 l por minuto impide traumatismo en el nervio óptico. </li></ol> 5. Inyección <ol><li> Baje la punta de la micropipeta en el nervio óptico justo encima de la duramadre mellado. NOTA: Una pequeña, pero a paso ligero movimiento de la punta de vidrio en los resultados del nervio óptico en el menor daño. Cuando comienza la bomba de infusión, el colorante azul de metilo debe resaltar el área del nervio óptico inyectado. El tinte debe permanecer localizada dentro del nervio óptico sin fugas en el espacio subaracnoideo. </li><li> Sigue la segunda banda de azul de metilo para determinar cuando la inyección de células madre es completa y girar la bomba de infusión fuera como se inyecta la segunda dosis de azul de metilo. Mantenga la micropipeta todavía dentro del nervio óptico durante 2 min para cada volumen 1 l se inyecta en alta para evitareyección de presión tras la retirada de la micropipeta. NOTA: Un enfoque alternativo es para conectar la punta de la pipeta a una jeringa llena de aire que puede ser manipulado manualmente. De cualquier manera, evitar una fuerza excesiva para minimizar el daño a los nervios ópticos. Se recomienda la inyección de no más de 2 l de volumen en cada nervio óptico. </li></ol> 6. Seguimiento <ol><li> Inmediato. <ol><li> Cuando la inyección, retire la micropipeta junto con las puntas de algodón utilizados para proporcionar la hemostasia. Suturar la piel con seda 3-0 y saque la sutura conjuntival. </li><li> Mantenga ratas caliente en una almohadilla eléctrica hasta que emergen de la anestesia. No deje a un animal sin vigilancia hasta que se haya recuperado el conocimiento suficiente para mantener decúbito esternal. No devuelva un animal que ha sido sometido a cirugía para la compañía de otros animales hasta que se recupere totalmente. </li><li> Si el animal está mostrando signos de dolor, incluyendo excesiva letargo, ataxia, o respirando trabajadog, administrar analgesia adecuada con buprenorfina intramuscular (0,05 mg / kg) hasta tres veces diarias. </li></ol></li><li> A largo plazo. <ol><li> Para el próximo 24 a 48 horas, se observe la rata de complicaciones de la cirugía, incluyendo inflamación o secreción de la herida u otros signos de dolor como la vocalización, la apariencia encorvada, sin preparación, o no comer. Considere la posibilidad de consultar con un veterinario o la eutanasia ética como sea necesario. </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.

El nervio óptico es un lugar ideal para el sistema nervioso central (SNC) la investigación regenerativa incluyendo condiciones oftalmológicas tales como neuritis óptica, glaucoma y el trauma. Las inyecciones de una variedad de células madre han demostrado la eficacia o mostrado promesa en la sustitución de la mielina perdida, aumentar el conteo de axonal y / o la prevención de enfermedades degenerativas. 1,2 El nervio óptico humano contiene aproximadamente 1,2 millones de axones paralelas que viajan desde la retina hasta el quiasma con un diámetro de aproximadamente 3,0-3,5 mm. 3 Para modelar enfermedades humanas en el laboratorio, la rata se ha usado con frecuencia. El nervio óptico de la rata adulta contiene aproximadamente 100.000 axones dentro de un diámetro de aproximadamente 0,5 mm. 4 Una de las principales limitaciones en el SNC investigación regenerativa es el acceso sin hueso directa. Las complicaciones y riesgos quirúrgicos para el animal son más altas cuando se quitan el cráneo o vértebras. Similar a los beneficios deabordajes mínimamente invasivos en la columna vertebral, 5 inyecciones nervio óptico directos sin necesidad de abrir las oferta reducida complicaciones cráneo y una recuperación más rápida. Esta técnica se ha utilizado en estudios previos. 6 En este manuscrito y vídeo que acompaña, se demuestra un procedimiento mínimamente invasivo para inyectar células madre en el nervio óptico de la rata.

<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.

A la conclusión del experimento, las ratas se sacrificaron y se perfundieron con 4% de paraformaldehído. Los nervios ópticos fueron cuidadosamente disecados y montados para seccionamiento criostato. La figura 2 muestra un ejemplo de un nervio óptico de rata entero a baja potencia en el que se inyectó colorante azul de Evans con el fin de visualizar el sitio. La flecha identifica la ubicación precisa de la inyección. Esta disección se realiza dentro de unos pocos minutos de la inyección, como se indica por la difusión restringida del colorante por el nervio. En otras inyecciones, hemos observado una lenta difusión de tinte hacia el quiasma óptico en el transcurso de varias horas. <img alt="Figura 1" src="/files/ftp_upload/52249/52249fig1highres.jpg" /> Figura 1:. Campo de vista quirúrgico La rata se coloca para acceder con el ojo izquierdo en esta figura. Se hace una incisión por encima de la cresta orbital y la fasciael tejido se diseca hacia abajo detrás del ojo. Una sutura conjuntival permite que el operador aplica una suave tracción que tira del nervio óptico intracraneal a la vista sin necesidad de abrir el cráneo. <img alt="Figura 2" src="/files/ftp_upload/52249/52249fig2highres.jpg" /> Figura 2:. Disección bruto de nervio óptico inyectado Utilizando colorante azul de Evans, el sitio de inyección en el nervio óptico puede ser groseramente visualizó bajo un microscopio de disección. En esta imagen, el nervio óptico se cortó en el sitio de inyección para mostrar el colorante incrustado dentro del tejido del nervio 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 mínimamente invasiva para la inyección en nervio óptico de la rata

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 vistas.  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. 

Video: 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.

Los métodos para la manipulación experimental después de la transección del nervio óptico en el SNC de los 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 ...

Investigación

Neurociencias

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.

Inyección intravítrea para el establecimiento de modelos Enfermedades Oculares

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

Biología

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.

Rata Modelo de neuropatía óptica isquémica posterior inducida por Fotoquímicamente

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.

Un método alternativo de inyección y validado para acceder al espacio subretiniano<i&gt; vía</i&gt; Un enfoque posterior transescleral

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.

El modelo de roedor de la anterior no arterítica neuropatía óptica isquémica (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.

Tomografía de coherencia óptica ultraalta resolución ratón para facilitar la inyección Intraocular en investigación de retina la terapia génica

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 inyección directa del nervio vago para ratas

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.

Abordaje transconjuntival para inyección en el nervio óptico de la rata

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 optimizada de inyección intracameral en ratas para reducir el daño tisular

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 destacado: Un nuevo protocolo para inyecciones intracamerales para mejorar la precisión en la oftalmología 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 mínimamente invasiva para la inyección en nervio óptico de la rata

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