Nuestra investigación es apoyada por el Programa de Investigación Intramural del NIH, el Instituto Nacional del Ojo.

Todos los equipos y los reactivos utilizados son estériles. Todos los experimentos con animales fueron aprobados por el Cuidado de Animales y el empleo Comisión (ACUC) en el NEI / NIH (protocolo de estudio en animales NEI-570), y se realizaron de acuerdo a las directrices del NIH y los reglamentos. 1. Etiquetado de referencia retrógrada de las células ganglionares retinianas (CGR) en el Día 1 El propósito de este procedimiento consiste en identificar a las células ganglionares de la retina retrógrada mediante la inyección de un tinte trazador neuronal en el colículo superior de los ratones tres días antes de la lesión del nervio óptico aplastar. El colorante se forma retrógrada tomadas por las células ganglionares de la retina y proporciona un indicador de la CGR de vida (Figura 1). Este enfoque produce el etiquetado reproducible de las CGR viables con poca variación 1-5. <ol><li> Profundamente anestesiar el ratón. Limpiar el cabello en la parte superior de la cabeza. Coloque el ratón en un pequeño aparato estereotáxico. Oinment de protección se aplica tanto a los ojos. La desinfección de la piel de la cabeza tres veces cada uno con un yodóforo y un lavado de alcohol al 70%. </li><li> Hacer una incisión en la piel para exponer el cráneo, y mantenerlo seco y limpio con 3% de peróxido de hidrógeno. </li><li> Identificar y marcar el bregma. Taladrar un agujero por encima del colículo superior a 2,9 mm por detrás del bregma y 0,5 mm lateral a la línea media de cada hemisferio 7. Durante la perforación, aplicar solución salina al sitio donde se perfora el agujero para evitar lesiones de calor secundario. </li><li> El uso de un dispositivo de medición estereotáxica y una jeringa Hamilton, inyectar el trazador de tinta FluoroGold neural (1 l de 3% Fluorocromo en solución salina) muy lentamente en el colículo superior de cada hemisferio, a una profundidad de 1,6 mm de la superficie ósea del cerebro. </li><li> Sutura de la incisión. Una inyección subcutánea de la buprenorfina para la analgesia. </li><li> Mueve el ratón a un lugar caliente y seco y el monitor hasta que es capaz de mantener una postura erguida, y devolverlo a su jaula. Durante los tres primeros días después del procedimiento de etiquetado, analgésicos sistémicos (por ejemplo, buprenorfina) y un ungüento antibiótico tópico se dan dos veces al día y el ratón es monitoreado de cerca. </li></ol> 2. Lesión por aplastamiento del nervio óptico en el día 4 En este procedimiento, se aplicará una lesión por aplastamiento del nervio óptico que causa un daño primario a los axones (Figura 2), lo que conducirá a una muerte gradual de las células ganglionares de la retina. <ol><li> Tres días después de la aplicación de tinte trazador, el ratón está profundamente anestesiados. Utilizando un microscopio de disección, la conjuntiva del ojo se realiza una incisión alrededor de la posición de las cuatro con un par de tijeras de primavera. </li><li> Desviar suavemente los músculos de la órbita y los puso a un lado. Exponer el nervio óptico blanco en su salida del globo ocular. Tenga mucho cuidado de no dañar los vasos sanguíneos. </li><li> Con la ayuda de un par de pinzas de acción de la Cruz-, aplicar una lesión por aplastamiento del nervio óptico a unos 2 mm del globo ocular durante unos 3 segundos. Tenga mucho cuidado de no dañar la arteria oftálmica para causar sangrado. </li><li> Después de la terminación de la lesión por aplastamiento, se sutura la incisión. </li><li> Antes de que el ratón se recupera de la anestesia, la inyección subcutánea de la buprenorfina para la analgesia. </li><li> Mueve el ratón a un lugar caliente y seco y el monitor, hasta que es capaz de mantener una postura erguida y devolverlo a su jaula. Durante los primeros tres días después del procedimiento de lesión por aplastamiento, analgésicos sistémicos (por ejemplo, buprenorfina) y un ungüento antibiótico tópico se dan dos veces al día y el ratón es monitoreado de cerca. </li></ol> 3. Cosecha de las retinas y analizar la supervivencia de CGR en el Día 11 El propósito de este procedimiento es la cosecha retinaeto analizar la supervivencia de células ganglionares retinianas. <ol><li> En el día 11 (día 7 después de ONC), el ratón es sacrificado por la infusión de CO 2 y la dislocación cervical. </li><li> Los ojos son enucleados con la ayuda de un par de pinzas, aplicando presión a la órbita. </li><li> Los ojos se fijaron en solución de paraformaldehído al 4% durante dos horas y se lavaron con PBS tres veces. Diseccionar la retina. El procedimiento de disección de la retina ya ha sido publicado previamente por Júpiter en 2010 por Gustmann, S. et al. 6, y por lo tanto no se describe aquí. </li><li> Las imágenes de las células ganglionares de la retina sobrevivientes son tomadas con un microscopio de fluorescencia en las regiones definidas de la retina, y la densidad de las células ganglionares de la retina se puede contar. Un resultado representativo de las células ganglionares de la retina de fluorescencia con etiqueta, con o sin lesión de aplastamiento del nervio óptico se muestra en la Figura 3. </li></ol> 4. Los resultados representativos: Para analizar la supervivencia de células ganglionares retinianas, siete días después de la lesión aplastamiento del nervio óptico, la retina se recogieron, se fija, aplanado y montaje. Imágenes de la retina fueron tomadas con un microscopio de fluorescencia. En comparación con el r normales sin lesionesetina (izquierda, Normal), el número de CGR viables (verde, retrógradamente marcados por el tinte FluoroGold neural marcador se inyecta en el colículo superior) es significativamente menor en las retinas con una lesión del nervio óptico aplastar (derecha, ONC) (Figura 3) . <img src="/files/ftp_upload/2685/2685fig1.jpg" alt="Figura 1" /> Figura 1. Línea de base retrógrada etiquetado de las células ganglionares de la retina Con el fin de contar las células viables ganglionares de la retina, en diferentes momentos después de la lesión aplastamiento del nervio óptico, las células ganglionares de la retina están etiquetados retrógrada mediante la inyección de un tinte trazador neural (color verde) en el colículo superior en el cerebro tres días antes del nervio óptico ( rojo) lesión por aplastamiento. Dado que los axones de las células ganglionares de la retina residen en el colículo superior, el tinte trazador se tomarán por la CGR retrógrada y proporciona un indicador de la CGR de vida. <img src="/files/ftp_upload/2685/2685fig2.jpg" alt="Figura 2" /> Figura 2. Nervio óptico lesión por aplastamiento Con el fin de analizar el estado de la supervivencia de células ganglionares retinianas, una lesión por aplastamiento se aplica en el nervio óptico (rojo) para causar un daño primario a los axones. Esto llevará a una muerte gradual de las células ganglionares de la retina. Tres días después de que el trazador de tinta (color verde) de la inyección, el ratón está anestesiado. La conjuntiva de un ojo se hace una incisión. Desviar los músculos orbitales y los puso a un lado. Exponer el nervio óptico en su salida del globo ocular. Aplique una lesión por aplastamiento del nervio óptico a unos 2 mm del globo ocular durante unos 3 segundos con un par de pinzas de acción transversal. <img src="/files/ftp_upload/2685/2685fig3.jpg" alt="Figura 3" /> Figura 3. Fluorescencia de células marcadas con ganglionares de la retina, con o sin lesión de aplastamiento del nervio óptico Para analizar la supervivencia de células ganglionares retinianas, siete días después de la lesión aplastamiento del nervio óptico, la retina se recogieron, se fija, aplanado y montaje. Imágenes de la retina fueron tomadas con un microscopio de fluorescencia. En comparación con la retina ileso normal (a la izquierda, Normal), el número de CGR viables (verde, retrógrada etiquetado por el FluoroGold neural trazador se inyecta un contraste en el colículo superior) fue significativamente menor en las retinas con una lesión del nervio óptico aplastar (ONC derecho, ) (Figura 3). Barra de escala: 50 micras

<ol>
	<li>Yoles, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GM1+reduces+injury-induced+metabolic+deficits+and+degeneration+in+the+rat+optic+nerve.">GM1 reduces injury-induced metabolic deficits and degeneration in the rat optic nerve.</a> Investigative ophthalmology &#38; visual science. 33, 3586-3591 (1992).</li><li>Fisher, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccination+for+neuroprotection+in+the+mouse+optic+nerve:+implications+for+optic+neuropathies.">Vaccination for neuroprotection in the mouse optic nerve: implications for optic neuropathies.</a> J Neurosci. 21, 136-142 (2001).</li><li>Levkovitch-Verbin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RGC+death+in+mice+after+optic+nerve+crush+injury:+oxidative+stress+and+neuroprotection.">RGC death in mice after optic nerve crush injury: oxidative stress and neuroprotection.</a> Investigative ophthalmology &#38; visual science. 41, 4169-4174 (2000).</li><li>Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-B+inhibits+apoptosis+via+VEGFR-1-mediated+suppression+of+the+expression+of+BH3-only+protein+genes+in+mice+and+rats.">VEGF-B inhibits apoptosis via VEGFR-1-mediated suppression of the expression of BH3-only protein genes in mice and rats.</a> The Journal of clinical investigation. 118, 913-923 (2008).</li><li>Tang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+effect+of+PDGF-CC+rescues+neurons+from+apoptosis+in+both+brain+and+retina+by+regulating+GSK3beta+phosphorylation.">Survival effect of PDGF-CC rescues neurons from apoptosis in both brain and retina by regulating GSK3beta phosphorylation.</a> The Journal of experimental medicine. 207, 867-880 (2010).</li><li>Gustmann, S., Dunker, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo-like+organotypic+murine+retinal+wholemount+culture.">In vivo-like organotypic murine retinal wholemount culture.</a> J Vis Exp. , (2010).</li><li>Franklin, K., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (1997).</li></ol>

El aplastamiento del nervio óptico modelo murino lesión es útil para estudiar el proceso de la muerte de las CGR y la supervivencia. Este modelo también se utiliza a menudo para investigar los efectos de diferentes reactivos y los genes de la apoptosis y la supervivencia de las CGR. Una de las ventajas de este modelo es que tiene un alto grado de reproducibilidad con variaciones mínimas. Sin embargo, se requiere un cuidado especial en varias etapas en este modelo. En primer lugar, la ho...

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<td>Stereotaxic apparatus</td>
<td>&nbsp;</td>
<td>ASI Instruments</td>
<td>SAS-4100</td>
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<td>Dissecting microscope</td>
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<td>World Precision Instruments</td>
<td>PZMIV-BS </td>
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<td>Scalpel handle</td>
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<td>Fine Science Tools </td>
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<td>Scalpel Blades #15</td>
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<td>Microdrill</td>
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<td>Fine Science Tools </td>
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<td>Fine Science Tools </td>
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<td>Sigma</td>
<td>459844</td>
<td>70%</td>
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<td>H2O2</td>
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<td>Sigma</td>
<td>H3410</td>
<td>3%</td>
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an optic nerve crush injury murine model to study retinal ganglion cell survival

La lesión del nervio óptico puede conducir a la degeneración axonal, seguido de una muerte gradual de las células ganglionares de la retina (CGR), que se traduce en la pérdida de visión irreversible. Ejemplos de tales enfermedades en humanos son la neuropatía óptica traumática y la degeneración del nervio óptico en el glaucoma. Se caracteriza por los cambios típicos en la cabeza del nervio óptico, degeneración progresiva del nervio óptico y la pérdida de células ganglionares de la retina, si no se controla, lo que lleva a la pérdida de visión y ceguera. El aplastamiento del nervio óptico (ONC) lesión modelo de ratón es un modelo importante para la enfermedad experimental neuropatía óptica traumática, glaucoma, etc En este modelo, la lesión por aplastamiento del nervio óptico conduce a la gradual ganglionares de la retina apoptosis de las células. Este modelo de la enfermedad puede ser usado para estudiar los procesos generales y los mecanismos de muerte neuronal y la supervivencia, que es esencial para el desarrollo de medidas terapéuticas. Además, los enfoques farmacológicos y moleculares pueden ser utilizados en este modelo para identificar y comprobar el potencial de reactivos terapéuticos para tratar diferentes tipos de neuropatía óptica. Aquí, le ofrecemos una demostración paso a paso (I) el etiquetado de referencia retrógrada de las células ganglionares de la retina (CGR) en el día 1, (II) lesión aplastamiento del nervio óptico en el día 4, (III) la cosecha de la retina y analizar la supervivencia de las CGR en el día 11, y (IV) son el resultado Representante.

Watch this Scientific Journal Video about An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival at JoVE.com

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Este protocolo indica cómo etiquetar retrógradamente las células ganglionares de la retina, y cómo hacer posteriormente una lesión del nervio óptico aplastar con el fin de analizar la supervivencia de células ganglionares retinianas y apoptosis. Se trata de un modelo de enfermedad experimental para los diferentes tipos de neuropatía óptica, incluyendo el glaucoma.

Un aplastamiento del nervio óptico Lesiones modelo murino para estudiar la supervivencia de células ganglionares retinianas

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

an-optic-nerve-crush-injury-murine-model-to-study-retinal-ganglion

Research

JoVE Journal

Neuroscience

30.2K vistas.  NIH. Este protocolo indica cómo etiquetar retrógradamente las células ganglionares de la retina, y cómo hacer posteriormente una lesión del nervio óptico aplastar con el fin de analizar la supervivencia de células ganglionares retinianas y apoptosis. Se trata de un modelo de enfermedad experimental para los diferentes tipos de neuropatía óptica, incluyendo el glaucoma.

Video: An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

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. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. 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 time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

La transección del nervio óptico: un modelo de apoptosis neuronal de adultos en el sistema nervioso central

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

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

Una preparación aislada de retina para registrar la respuesta de la luz a partir de semillas etiquetadas las células ganglionares retinianas

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

Grabación de potencial evocado visual en un modelo de rata de Experimental del Nervio Óptico Desmielinización

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

RNA-Seq de una sola célula de subconjuntos definidos de células de ganglio retiniano

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Inyección de raíz dorsal ganglio de raíz dorsal y Lesión por aplastamiento como modelo para la regeneración del axón sensorial

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection is considered a useful and reproducible model. Compared with other optic nerve injury models used commonly for assessing secondary degeneration, e.g. complete optic nerve transection and optic nerve crush models, the partial optic nerve transection model is superior as it distinguishes primary from secondary degeneration in situ. Therefore, it serves as an excellent tool for evaluating secondary degeneration. This study describes a novel operative approach of partial optic nerve transection by directly accessing the area of the retrobulbar optic nerve through the orbital lateral wall of the eyeball. Moreover, we present a newly designed, low cost surgical instrument to assist with transection. As demonstrated by the representative results in distinguishing the boundary of primary and secondary injury areas, the new approach and instrument ensures high efficiency and stability of the model by providing adequate space for surgical operation. This in turn makes it easy to separate the meningeal sheath and ophthalmic vessels from the optic nerve before transection. An additional benefit is that this space-saving operative approach improves the investigators&#39; ability to administer drugs, carriers, or selective RGC tracers to the stump of the partially transected optic nerve, allowing the exploration of mechanisms behind secondary injury in RGCs, in a new way.

Secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. This study describes an innovative operative approach for partial optic nerve transection. The use of this space-saving operative approach extends the model's application range, and allows exploration of secondary injury mechanisms in RGCs in a new way.

Transección parcial del nervio óptico en ratas: un modelo con un nuevo enfoque operativo para evaluar la degeneración secundaria de las células ganglionares de la retina

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection ...

Ethanol-Induced Cervical Sympathetic Ganglion Block Applications for Promoting Canine Inferior Alveolar Nerve Regeneration Using an Artificial Nerve

Polyglycolic acid collagen (PGA-C) tubes are bio-absorbable nerve tubes filled with collagen of multi-chamber structure, which consist of thin collagen films. Favorable clinical outcomes have been achieved when using these tubes for the treatment of damaged inferior alveolar nerve (IAN). A critical factor for the successful nerve regeneration using PGA-C tubes is blood supply to the surrounding tissue. Cervical sympathetic ganglion block (CSGB) creates a sympathetic blockade in the head and neck region thus increasing blood flow in the area. To ensure an adequate effect, the blockade must be administered with local anesthetics one to two times a day for several consecutive weeks; this poses a challenge when creating animal models for investigating this technique. To address this limitation, we developed an ethanol-induced CSGB in a canine model of long-term increase in blood flow in the orofacial region. We examined whether IAN regeneration via PGA-C tube implantation can be enhanced by this model. Fourteen Beagles were each implanted with a PGA-C tube across a 10-mm gap in the left IAN. The IAN is located within the mandibular canal surrounded by bone, therefore we chose piezoelectric surgery, consisting of ultrasonic waves, for bone processing, in order to minimize the risk of nerve and vessel injury. A good surgical outcome was obtained with this approach. A week after surgery, seven of these dogs were subjected to left CSGB by injection of ethanol. Ethanol-induced CSGB resulted in improved nerve regeneration, suggesting that the increased blood flow effectively promotes nerve regeneration in IAN defects. This canine model can contribute to further research on the long-term effects of CSGB.

We evaluated the effect of cervical sympathetic ganglion block on nerve repair using artificial nerve conduits. Male beagle dogs were each implanted with an artificial nerve across a 10-mm gap in the left inferior alveolar nerve; left cervical sympathetic ganglion was blocked by injecting 99.5% ethanol via lateral thoracotomy.

Aplicaciones en bloque del ganglio simpático Cervical inducido por etanol para promover la regeneración del nervio Alveolar Inferior canino utilizando un nervio Artificial

Polyglycolic acid collagen (PGA-C) tubes are bio-absorbable nerve tubes filled with collagen of multi-chamber structure, which consist of thin collagen ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Análisis cinemático 3D para la evaluación funcional en el modelo de rata de lesión de aplastamiento de nervios ciáticos

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Multifactorial Assessment of Motor Behavior in Rats after Unilateral Sciatic Nerve Crush Injury

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In addition, in the research field of movement disorders, sciatic crush injury has been employed to trigger a dystonia-like phenotype in genetically predisposed DYT-TOR1A rodent models of dystonia. To achieve consistent, reproducible and comparable results after a sciatic nerve crush injury, a standardized method for inducing the nerve crush is essential, in addition to a standardized phenotypical characterization. Attention must be paid not only to the specific assortment of behavioral tests, but also to the technical requirements, the correct execution and consecutive data analysis. This protocol describes in detail how to perform a sciatic nerve crush injury and provides a behavioral test battery for the assessment of motor deficits in rats that includes the open field test, the CatWalk XT gait analysis, the beam walking task, and the ladder rung walking task.

We provide a protocol for the assessment of motor behavior via a behavioral test battery in rats after sciatic nerve crush injury.

Evaluación Multifactorial Del Comportamiento Motor En Ratas Después De La Lesión Unilateral Por Aplastamiento Del Nervio Ciático

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In ...

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury are essential for translating novel therapeutic strategies from rodent models to clinical application due to their closer similarities to humans in size and anatomy. Here we describe some in vivo methods to evaluate the function and structure of the retinal ganglion cells (RGCs) and optic nerve (ON) in large animals, including visual evoked potential (VEP), pattern electroretinogram (PERG) and optical coherence tomography (OCT). Both goat and non-human primate were employed in this study. By presenting these in vivo methods step by step, we hope to increase experimental reproducibility among different labs and facilitate the usage of large animal models of optic neuropathies.

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

Here we demostrate several in vivo tests (flash visual evoked potential, pattern electroretinogram and optic coherence tomography) in goat and rhesus macaque to understand the structure and function of the optic nerve and its neurons.

In vivo Métodos para evaluar la función y estructura de las células ganglionares de la retina y el nervio óptico en animales grandes

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury ...