Unsere Forschung wird durch die Interne Research Program des NIH, National Eye Institute unterstützt.

Alle Geräte und Reagenzien sind steril. Alle Tierversuche wurden von der Animal Care genehmigt und Verwenden Committee (ACUC) an der NEI / NIH (Tier Studienprotokoll NEI-570), und wurden nach den NIH-Richtlinien und Vorschriften durchgeführt. 1. Baseline Retrograde Markierung von retinalen Ganglienzellen (RGC) Am Tag 1 Der Zweck dieses Verfahrens ist es, die retinalen Ganglienzellen retrograd Etikett durch die Injektion eines neuronalen Markierungsfarbstoff in den Colliculus superior der Mäuse drei Tage vor dem Sehnerv Quetschverletzung. Der Farbstoff wird retrograd entnommen werden von den retinalen Ganglienzellen und bietet ein Marker für die Lebenden RGCs (Abbildung 1). Dieser Ansatz führt zu reproduzierbaren Kennzeichnung von lebensfähigen RGCs mit wenig Variation 1-5. <ol><li> Tief betäuben die Maus. Reinigen Sie das Haar am Oberkopf. Platzieren Sie die Maus in einem kleinen stereotaktischen Apparat. Protective Salbe ist an beiden Augen angewendet. Desinfizieren Sie die Kopfhaut jeweils dreimal mit einem Iodophor und einem 70% igem Alkohol zu schrubben. </li><li> Machen Sie einen Schnitt in der Haut am Schädel freizulegen, und halten Sie sie trocken und sauber mit 3% Wasserstoffperoxid. </li><li> Identifizieren und markieren Sie die Bregma. Bohren Sie ein Loch über dem Colliculus superior bei 2,9 mm hinter dem Bregma und 0,5 mm lateral der Mittellinie jeder Hemisphäre 7. Während des Bohrens, gelten Kochsalzlösung auf der Stelle, wo das Loch gebohrt, um sekundäre Wärme Verletzungen zu vermeiden ist. </li><li> Mit Hilfe eines stereotaktischen Messgerät und einem Hamilton Spritze die Fluorogold neuronalen Markierungsfarbstoff (1 ul von 3% Fluorochrom in Kochsalzlösung) sehr langsam in den Colliculus superior jeder Hemisphäre in einer Tiefe von 1,6 mm von der knöchernen Oberfläche des Gehirns. </li><li> Suture die Inzisionsstelle. Geben Sie eine subkutane Injektion von Buprenorphin zur Analgesie. </li><li> Bewegen Sie die Maus an einen warmen und trockenen Ort und zu überwachen, bis es in der Lage, eine aufrechte Haltung zu bewahren, und senden es an seinen eigenen Käfig ist. Für die ersten drei Tage nach der Kennzeichnung Verfahren, sind systemische Analgetika (zB Buprenorphin) und topische Antibiotika-Salbe zweimal täglich verabreicht und die Maus wird streng überwacht. </li></ol> 2. Optic Nerve Crush Injury An Tag 4 In diesem Verfahren werden wir ein Auge Verletzung des Sehnervs gelten für eine primäre Schädigung der Axone (Abb. 2), was zu einem allmählichen Absterben der retinalen Ganglienzellen führt zu verursachen. <ol><li> Drei Tage nach der Markierungsfarbstoff Anwendung ist die Maus tief narkotisiert. Mit einem Binokular ist der Bindehaut eines Auges an über die 4-Uhr-Position mit einem Paar Frühling Schere eingeschnitten. </li><li> Sanft lenken den orbitalen Muskeln und legte sie beiseite. Setzen Sie die weiße Sehnerv an seiner Austrittsstelle aus dem Auge Globus. Achten Sie besonders darauf, keine Blutgefäße beschädigen. </li><li> Mit Hilfe eines Paares von cross-action Zange, gelten verknallt Verletzung des Sehnervs bei etwa 2 mm vom Augapfel für ca. 3 Sekunden. Achten Sie besonders darauf nicht an die A. ophthalmica Schäden an Blutungen verursachen. </li><li> Nach Abschluss der Quetschverletzungen, wird der Einschnitt vernäht. </li><li> Bevor Sie die Maustaste wieder aus der Narkose, eine subkutane Injektion von Buprenorphin zur Analgesie. </li><li> Bewegen Sie die Maus an einen warmen und trockenen Ort und überwachen sie, bis sie in der Lage, eine aufrechte Haltung zu bewahren und senden es an seinen eigenen Käfig ist. Für die ersten drei Tage nach dem Quetschverletzung Verfahren, sind systemische Analgetika (zB Buprenorphin) und topische Antibiotika-Salbe zweimal täglich verabreicht und die Maus wird streng überwacht. </li></ol> 3. Ernte der Netzhaut und Analysieren RGC Überleben an Tag 11 Der Zweck dieses Verfahrens ist die Ernte der retinaeto analysieren retinalen Ganglienzellen Überleben. <ol><li> Am Tag 11 (Tag 7 nach ONC), wird die Maus durch CO 2-Infusion und Genickbruch getötet. </li><li> Die Augen sind mit Hilfe einer Pinzette durch Druck auf die Bahn entkernte. </li><li> Die Augen sind in 4% Paraformaldehyd-Lösung für 2 Stunden fixiert und gewaschen in PBS dreimal. Präparieren Sie die Netzhaut. Das Verfahren der Netzhaut Dissektion wurde bereits zuvor von JoVE im Jahr 2010 veröffentlicht durch Gustmann, S. et al. 6, und wird daher hier nicht beschrieben werden. </li><li> Bilder der überlebenden retinalen Ganglienzellen getroffen werden mit Hilfe eines Fluoreszenz-Mikroskops in definierten Regionen der Netzhaut und des retinalen Ganglienzellen Dichte gezählt werden können. Ein repräsentatives Ergebnis der Fluoreszenz-markierten retinalen Ganglienzellen mit oder ohne Sehnerv Quetschverletzung ist in Abbildung 3 dargestellt. </li></ol> 4. Repräsentative Ergebnisse: Zur Analyse retinalen Ganglienzellen Überleben, sieben Tage nach der Sehnerv Quetschverletzung wurden die Netzhäute geerntet, fixiert, abgeflacht und montiert. Netzhautbilder wurden mit einem Fluoreszenz-Mikroskop. Verglichen mit dem unverletzten normalen retina (links, Normal), ist die Anzahl der lebensfähigen RGCs (grün, retrograd durch die Fluorogold neuronale Tracer Farbstoff in den Colliculus superior injiziert beschriftet) signifikant niedriger in der Netzhaut mit einer Sehnerv Quetschverletzung (rechts, ONC) (Abbildung 3) . <img src="/files/ftp_upload/2685/2685fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Baseline retrograde Markierung von retinalen Ganglienzellen Um die lebensfähigen retinalen Ganglienzellen zu verschiedenen Zeitpunkten nach der Sehnerv Quetschverletzung zählen, sind die retinalen Ganglienzellen retrograd durch die Injektion eines neuronalen Markierungsfarbstoff (grüne Farbe) in den Colliculus superior im Gehirn drei Tage vor dem Sehnerv markierten ( rot) Quetschverletzung. Da die Axone der retinalen Ganglienzellen in den Colliculus superior befinden, wird der Tracer-Farbstoff durch die RGCs retrograd entnommen werden und stellt einen Marker für die Lebenden RGCs. <img src="/files/ftp_upload/2685/2685fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Sehnerv Quetschverletzung Um den Status der retinalen Ganglienzellen das Überleben der Zelle zu analysieren, ist eine Quetschverletzung des Sehnervs (rot), die auf eine primäre Schädigung der Axone bewirken. Dies führt zu einer allmählichen Absterben der retinalen Ganglienzellen führen. Drei Tage nach der Tracer-Farbstoff (grün) Injektion, ist die Maus narkotisiert. Die Bindehaut eines Auges ist eingeschnitten. Auslenken Orbital Muskeln und legte sie beiseite. Setzen Sie den Sehnerv an seiner Austrittsstelle aus dem Auge Globus. Tragen Sie einen Quetschverletzung des Sehnervs bei etwa 2 mm vom Augapfel für etwa 3 Sekunden unter Verwendung eines Paares von Cross-Aktion Pinzette. <img src="/files/ftp_upload/2685/2685fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Fluoreszenz-markierten retinalen Ganglienzellen mit oder ohne Sehnerv Quetschverletzung Zur Analyse retinalen Ganglienzellen Überleben, sieben Tage nach der Sehnerv Quetschverletzung wurden die Netzhäute geerntet, fixiert, abgeflacht und montiert. Netzhautbilder wurden mit einem Fluoreszenz-Mikroskop. Verglichen mit dem unverletzten normalen Netzhaut (links, Normal), wurde die Anzahl der lebensfähigen RGCs (grün, retrograd durch die Fluorogold neuronale Tracer Farbstoff in den Colliculus superior injiziert beschriftet) signifikant niedriger in der Netzhaut mit einer Sehnerv Quetschverletzung (rechts, ONC ) (Abbildung 3). Maßstabsbalken: 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>

Der Sehnerv Quetschverletzung Mausmodell ist nützlich, um den Prozess der RGC Tod und Überleben zu untersuchen. Dieses Modell ist auch oft verwendet, um die Auswirkungen der verschiedenen Reagenzien und Gene auf RGC Apoptose und Überleben zu untersuchen. Ein Vorteil dieses Modells ist, dass es ein hohes Maß an Reproduzierbarkeit bei minimalen Variationen hat. Allerdings ist besondere Sorgfalt in mehreren Schritten in diesem Modell erforderlich. Erstens, wenn was den Sehnerv Quetschverlet...

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an optic nerve crush injury murine model to study retinal ganglion cell survival

Eine Verletzung des Sehnervs kann die axonale Degeneration führen, gefolgt von einer allmählichen Tod von retinalen Ganglienzellen (RGC), die zu einer irreversiblen Verlust der Sehkraft führt. Beispiele für solche Krankheiten in menschlichen gehören traumatische Optikusneuropathie und Sehnerv-Degeneration bei Glaukom. Es ist durch die typischen Veränderungen im Sehnervenkopf, progressive Degeneration des Sehnervs und der Verlust von retinalen Ganglienzellen, wenn unkontrolliert, was zu Sehverlust und Erblindung aus. Der Sehnerv crush (ONC) Verletzungen Mausmodell ist eine wichtige experimentelle Krankheit Modell für traumatische Optikusneuropathie, Glaukom, etc. In diesem Modell das Gedränge Verletzung des Sehnervs führt zur allmählichen retinalen Ganglienzellen Apoptose. Diese Krankheit Modell kann verwendet werden, um die allgemeinen Prozesse und Mechanismen der neuronalen Tod und Überleben, die wesentlich für die Entwicklung von therapeutischen Maßnahmen ist zu studieren. Darüber hinaus können pharmakologische und molekularbiologische Ansätze in diesem Modell verwendet werden, um zu identifizieren und zu testen potenzielle therapeutische Reagenzien für verschiedene Arten von Optikusneuropathie zu behandeln. Hier bieten wir Ihnen eine Schritt für Schritt Demonstration von (I) Baseline retrograde Markierung von retinalen Ganglienzellen (RGC) am Tag 1, (II) Sehnerv Quetschverletzung am Tag 4, (III) Ernte der Netzhaut und zu analysieren RGC Überleben am Tag 11, und (IV) repräsentatives Ergebnis.

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

Dieses Protokoll zeigt, wie man retrograd Label retinalen Ganglienzellen, und wie man später machen ein Sehnerv Quetschverletzung um retinalen Ganglienzellen Überleben und Apoptose zu analysieren. Es ist eine experimentelle Krankheit Modell für verschiedene Arten von Optikusneuropathie, einschließlich Glaukom.

Ein Optic Nerve Crush Injury Mausmodell zur retinalen Ganglienzellen Survival Study

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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JoVE Journal

Neuroscience

30.2K Aufrufe.  NIH. Dieses Protokoll zeigt, wie man retrograd Label retinalen Ganglienzellen, und wie man später machen ein Sehnerv Quetschverletzung um retinalen Ganglienzellen Überleben und Apoptose zu analysieren. Es ist eine experimentelle Krankheit Modell für verschiedene Arten von Optikusneuropathie, einschließlich Glaukom.

Serie: 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.

Optic Nerve Durchtrennung: Ein Modell des Adult Neuron Apoptose in das zentrale Nervensystem

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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Neurowissenschaften

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.

Eine isolierte Retinal Vorbereitung auf leichte Ansprache aus genetisch markierten retinalen Ganglienzellen Rekord

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.

Visuell evozierten Potential-Aufnahme in einem Rattenmodell für Experimentelle Optic Nerve Demyelinisierung

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.

Einzelzell-RNA-Seq von definierten Subsets von Retinal-Ganglion-Zellen

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.

Dorsalwurzelganglion Injection und dorsale Wurzel Quetschverletzungen als Modell für Sensory Axonregeneration

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.

Partielle Sehnerv Durchtrennung bei Ratten: ein Modell gegründet mit einem neuen operativen Ansatz zur sekundären Degeneration der retinalen Ganglienzellen bewerten

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.

Ethanol-induzierte zervikalen sympathischen Ganglion Block-Anwendungen für die Förderung der Eckzahn minderwertig alveoläre Nervenregeneration mit einer künstlichen Nerv

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.

3D Kinematische Analyse zur funktionellen Bewertung im Rattenmodell von Ischias-Nerven-Crush-Verletzungen

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.

Multifaktorielle Beurteilung des motorischen Verhaltens bei Ratten nach einseitiger Ischiasnervenquetschverletzung

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.

Im lebenden Organismus Methoden zur Beurteilung der Funktion und Struktur retinaler Ganglienzellen und Sehnerven bei Großtieren

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 ...