Notre recherche est soutenu par le Programme de recherche intra-muros des NIH, l&#39;Institut National Eye.

Tous les équipements et les réactifs utilisés sont stériles. Toutes les expériences sur les animaux ont été approuvés par le soin des animaux et utilisation comité (ACUC) à l&#39;IEN / NIH (protocole de l&#39;étude des animaux IEN-570), et ont été réalisées selon les directives du NIH et des règlements. 1. Étiquetage base rétrograde des cellules ganglionnaires rétiniennes (CGR), au Jour 1 Le but de cette procédure est de marquer les cellules ganglionnaires de la rétine rétrograde par injection d&#39;un colorant traceur neuronal dans le colliculus supérieur de la souris trois jours avant la lésion par écrasement du nerf optique. Le colorant sera rétrograde prises par les cellules ganglionnaires de la rétine et fournit un marqueur de la CGR de vie (figure 1). Cette approche donne étiquetage reproductible de CGR viable avec peu de variation 1-5. <ol><li> Profondément anesthésier la souris. Nettoyez les cheveux sur le dessus de la tête. Placez la souris dans un petit appareil de stéréotaxie. Oinment protecteur est appliqué sur les deux yeux. Désinfecter la peau de la tête à trois reprises chacun utilisant un iodophore et un gommage alcool à 70%. </li><li> Faire une incision dans la peau d&#39;exposer le crâne, et le garder propre et sec à l&#39;aide de peroxyde d&#39;hydrogène à 3%. </li><li> Identifier et marquer le bregma. Percez un trou au-dessus du colliculus supérieur à 2,9 mm derrière le bregma et 0,5 mm latéralement à la ligne médiane de chaque hémisphère 7. Pendant le forage, appliquer une solution saline à l&#39;endroit où le trou est creusé pour prévenir les blessures de chaleur secondaire. </li><li> L&#39;utilisation d&#39;un appareil de mesure stéréotaxique et d&#39;une seringue Hamilton, injecter le colorant traceur FluoroGold neurones (1 pi de 3% dans une solution saline fluorochrome) très lentement dans le colliculus supérieur de chaque hémisphère à une profondeur de 1,6 mm de la surface osseuse du cerveau. </li><li> Suture du site d&#39;incision. Donner une injection sous-cutanée de la buprénorphine pour l&#39;analgésie. </li><li> Déplacez la souris à un endroit chaud et sec et de le surveiller jusqu&#39;à ce qu&#39;il soit capable de maintenir une posture droite, et le retourner dans sa cage à la maison. Pour les trois premiers jours après la procédure de labellisation, les analgésiques systémiques (par exemple, buprénorphine) et pommade antibiotique sont donnés deux fois par jour et de la souris est étroitement surveillée. </li></ol> 2. Optic blessures écrasement du nerf au jour 4 Dans cette procédure, nous allons appliquer une lésion par écrasement du nerf optique de causer un dommage aux primaires des axones (figure 2), ce qui conduira à une mort progressive des cellules ganglionnaires de la rétine. <ol><li> Trois jours après l&#39;application colorant traceur, la souris est profondément anesthésié. En utilisant un microscope à dissection, la conjonctive d&#39;un œil est incisé au sujet de la position 04 heures en utilisant une paire de ciseaux à ressort. </li><li> Doucement les muscles détourner l&#39;orbite et les mettre de côté. Exposer le nerf optique blanche à sa sortie du globe oculaire. Prenez grand soin de ne pas endommager les vaisseaux sanguins. </li><li> Avec l&#39;aide d&#39;une paire de pinces à action croisée, appliquer une lésion par écrasement du nerf optique, à environ 2 mm de l&#39;œil pendant environ 3 secondes. Prenez grand soin de ne pas endommager l&#39;artère ophtalmique pour provoquer des saignements. </li><li> Après achèvement de la lésion par écrasement, l&#39;incision est suturée. </li><li> Avant la souris se remet de l&#39;anesthésie, donner une injection sous-cutanée de la buprénorphine pour l&#39;analgésie. </li><li> Déplacez la souris à un endroit chaud et sec et de le surveiller, jusqu&#39;à ce qu&#39;il soit capable de maintenir une posture droite et le retourner dans sa cage à la maison. Pour les trois premiers jours après la procédure de blessure par écrasement, les analgésiques systémiques (par exemple, buprénorphine) et pommade antibiotique sont donnés deux fois par jour et de la souris est étroitement surveillée. </li></ol> 3. Récolte les rétines et analyser la survie RGC au 11e jour Le but de cette procédure est de récolter le retinaeto d&#39;analyser la rétine survie des cellules ganglionnaires. <ol><li> Au jour 11 (7 jours après l&#39;ONC), la souris est euthanasié par le CO 2 de perfusion et dislocation cervicale. </li><li> Les yeux sont énucléés avec l&#39;aide d&#39;une pince en exerçant une pression à l&#39;orbite. </li><li> Les yeux sont fixés dans une solution de paraformaldéhyde 4% pendant deux heures et lavées dans du PBS à trois reprises. Disséquer la rétine. La procédure de dissection rétine a déjà été publiée auparavant par Jove en 2010 par Gustmann, S. et al. 6, et donc ne sera pas décrite ici. </li><li> Images des cellules ganglionnaires de la rétine survivants sont prises en utilisant un microscope à fluorescence dans des régions définies de la rétine, et la densité des cellules ganglionnaires de la rétine peut être compté. Un résultat représentatif des cellules marquées par fluorescence ganglionnaires de la rétine, avec ou sans lésion du nerf optique est écraser le montre la figure 3. </li></ol> 4. Les résultats représentatifs: Pour analyser la rétine survie des cellules ganglionnaires, sept jours après une lésion du nerf optique, écraser, les rétines ont été récoltés, fixe, aplaties et montées. Images rétiniennes ont été prises avec un microscope à fluorescence. Par rapport à la normale r indemneetina (gauche, normal), le nombre de CGR viables (vert, marqués de façon rétrograde par le colorant FluoroGold neurones traceur injecté dans le colliculus supérieur) est significativement plus faible dans les rétines avec une blessure par écrasement du nerf optique (à droite, ONC) (Figure 3) . <img src="/files/ftp_upload/2685/2685fig1.jpg" alt="Figure 1" /> Figure 1. L&#39;étiquetage de base rétrograde de cellules ganglionnaires de la rétine Pour compter les cellules viables ganglionnaires de la rétine à différents moments après une lésion nerveuse écraser optique, les cellules ganglionnaires de la rétine sont étiquetés rétrograde par injection d&#39;un colorant traceur neuronal (couleur verte) dans le colliculus supérieur dans les trois jours avant le cerveau du nerf optique ( rouge) écrasent blessure. Depuis les axones des cellules ganglionnaires de la rétine résider dans le colliculus supérieur, le colorant sera repris par le CGR rétrograde et fournit un marqueur de la CGR vie. <img src="/files/ftp_upload/2685/2685fig2.jpg" alt="Figure 2" /> Figure 2. Optic blessure par écrasement du nerf Afin d&#39;analyser l&#39;état de la survie des cellules ganglionnaires de la rétine, une blessure par écrasement est appliquée au nerf optique (rouge) de causer un dommage aux primaires des axones. Cela conduira à une mort progressive des cellules ganglionnaires de la rétine. Trois jours après le traceur colorant (vert) d&#39;injection, la souris est anesthésiée. La conjonctive d&#39;un œil est incisé. Détourner les muscles orbitaux et les mettre de côté. Exposez le nerf optique à sa sortie du globe oculaire. Appliquer une lésion par écrasement du nerf optique, à environ 2 mm de l&#39;œil pendant environ 3 secondes à l&#39;aide d&#39;une paire de pinces à action croisée. <img src="/files/ftp_upload/2685/2685fig3.jpg" alt="Figure 3" /> Figure 3. Marqué par fluorescence des cellules ganglionnaires de la rétine, avec ou sans lésion du nerf optique, écraser Pour analyser la rétine survie des cellules ganglionnaires, sept jours après une lésion du nerf optique, écraser, les rétines ont été récoltés, fixe, aplaties et montées. Images rétiniennes ont été prises avec un microscope à fluorescence. Par rapport à la rétine indemne normal (à gauche, normal), le nombre de CGR viables (vert, marqués de façon rétrograde par le colorant FluoroGold neurones traceur injecté dans le colliculus supérieur) a été significativement plus faible dans les rétines avec une blessure par écrasement du nerf optique (à droite, ONC ) (figure 3). Barre d&#39;échelle: 50 um

<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>

Le modèle du nerf optique écraser blessures murin est utile d&#39;étudier le processus de la mort et la survie du CJR. Ce modèle est également souvent utilisé pour étudier les effets de différents réactifs et les gènes de l&#39;apoptose et la survie du CJR. Un avantage de ce modèle est qu&#39;il a un degré élevé de reproductibilité avec des variations minimes. Cependant, une attention particulière est requise en plusieurs étapes dans ce modèle. Premièrement, lors de la lé...

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<td>Stereotaxic apparatus</td>
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<td>ASI Instruments</td>
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an optic nerve crush injury murine model to study retinal ganglion cell survival

Blessure au nerf optique peuvent conduire à une dégénérescence axonale, suivie par une mort progressive des cellules ganglionnaires de la rétine (CGR), qui se traduit par une perte de vision irréversible. Des exemples de telles maladies à l&#39;homme comprennent traumatiques neuropathie optique et une dégénérescence du nerf optique dans le glaucome. Elle est caractérisée par des modifications typiques de la tête du nerf optique, progressive dégénérescence du nerf optique, et la perte de cellules ganglionnaires de la rétine, si incontrôlée, conduisant à une perte de vision et de cécité. L&#39;écrasement du nerf optique (ONC) modèle de lésion de la souris est un modèle expérimental pour la maladie importante neuropathie optique traumatique, glaucome, etc Dans ce modèle, la lésion par écrasement du nerf optique conduit à l&#39;apoptose des cellules rétiniennes ganglionnaires progressive. Ce modèle de la maladie peut être utilisée pour étudier les processus généraux et les mécanismes de mort neuronale et de la survie, ce qui est essentiel pour le développement de mesures thérapeutiques. En outre, les approches pharmacologiques et moléculaires peuvent être utilisés dans ce modèle afin d&#39;identifier et de tester le potentiel de réactifs thérapeutiques pour traiter différents types de neuropathie optique. Ici, nous offrons une démonstration étape par étape de (I) l&#39;étiquetage de base rétrograde de cellules ganglionnaires de la rétine (CGR) au jour 1, (ii) des blessures écrasement du nerf optique au jour 4, (III) Récolte des rétines et analyser la survie au jour du CJR 11, et le résultat Représentant (IV).

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

Ce protocole montre comment rétrograde marquer les cellules ganglionnaires de la rétine, et la façon de faire ultérieurement une lésion du nerf optique écraser dans le but d&#39;analyser la rétine survie des cellules ganglionnaires et l&#39;apoptose. Il s&#39;agit d&#39;un modèle de maladie expérimentale pour différents types de neuropathie optique, notamment un glaucome.

Un modèle Optic Nerve Crush blessures murins pour étudier la rétine de survie des cellules ganglionnaires

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

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Neuroscience

30.2K vues.  NIH. Ce protocole montre comment rétrograde marquer les cellules ganglionnaires de la rétine, et la façon de faire ultérieurement une lésion du nerf optique écraser dans le but d'analyser la rétine survie des cellules ganglionnaires et l'apoptose. Il s'agit d'un modèle de maladie expérimentale pour différents types de neuropathie optique, notamment un glaucome.

Vidéo: 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.

Section du nerf optique: un modèle de l&#39;apoptose neuronale des adultes dans le système nerveux 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 ...

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Neurosciences

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.

Une préparation isolée rétinienne pour enregistrer la réponse de Lumière génétiquement marqués cellules ganglionnaires rétiniennes

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.

Enregistrement Potentiel évoqué visuel dans un modèle de rat of Experimental Optic Nerve démyélinisation

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.

L&#39;ARN monocellulaire-séquentiel des sous-ensembles définis des cellules ganglionnaires rétiniennes

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.

Racine Dorsale Ganglion Injection et Racine Dorsale Crush blessures comme modèle pour la régénération sensorielle Axon

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.

Résection partielle du nerf optique chez le rat : un modèle mis en place avec une nouvelle approche du dispositif d’évaluation secondaire dégénérescence des cellules ganglionnaires rétiniennes

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.

Applications de blocs induite par l’éthanol du Ganglion sympathique Cervical pour promouvoir la régénération Canine nerf alvéolaire inférieur à l’aide d’un nerf artificiel

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.

Analyse cinématique 3D pour l’évaluation fonctionnelle dans le modèle de rat de la blessure d’écrasement de nerf sciatique

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.

Évaluation multifactorielle du comportement moteur chez les rats après une blessure unilatérale d’écrasement du nerf sciatique

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 Méthodes d’évaluation de la fonction et de la structure des cellules ganglionnaires rétiniennes et du nerf optique chez les grands animaux

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 ...