La nostra ricerca è sostenuta dal programma di ricerca intramurale del NIH, National Eye Institute.

Tutte le attrezzature e reagenti utilizzati sono sterili. Tutti gli esperimenti sugli animali sono stati approvati dalla cura degli animali e utilizzo Comitato (ACUC) al NEI / NIH (protocollo di studio su animali NEI-570), e sono stati eseguiti secondo le linee guida NIH e regolamenti. 1. Baseline marcatura retrograda di cellule gangliari della retina (RGCs) al giorno 1 Lo scopo di questa procedura è quello di etichettare le cellule gangliari della retina retrogrado, iniettando un colorante tracciante neurale nel collicolo superiore dei topi tre giorni prima della lesione schiacciamento del nervo ottico. Il colorante sarà retrogrado ripreso dalle cellule gangliari della retina e fornisce un indicatore per la RGCs vita (Figura 1). Questo approccio produce etichettatura riproducibile di RGCs praticabile con piccole variazioni 1-5. <ol><li> Profondamente anestetizzare il mouse. Pulire i capelli sulla sommità della testa. Posizionare il mouse in un piccolo apparecchio stereotassico. Oinment protettiva viene applicata in entrambi gli occhi. Disinfettare la cute testa tre volte utilizzando un iodofori e una macchia di alcool al 70%. </li><li> Fare un&#39;incisione nella pelle per esporre il cranio, e tenerlo asciutto e pulito con acqua ossigenata al 3%. </li><li> Identificare e contrassegnare il bregma. Praticare un foro al di sopra del collicolo superiore al 2,9 mm dietro il bregma e 0,5 mm lateralmente alla linea mediana di ciascun emisfero 7. Durante la perforazione, applicare soluzione salina per il sito in cui si è esercitato il buco per evitare lesioni secondarie di calore. </li><li> Utilizzando un dispositivo stereotassico di misura e una siringa Hamilton, iniettare il FluoroGold neurale tracciante colorante (1 ml di Fluorocromo 3% in soluzione salina) molto lentamente nel collicolo superiore di ogni emisfero, ad una profondità di 1,6 mm dalla superficie ossea del cervello. </li><li> Sutura del sito di incisione. Dare un iniezione sottocutanea di buprenorfina per l&#39;analgesia. </li><li> Muovi il mouse in una zona calda e secca e monitorare fino a quando non è in grado di mantenere una postura eretta, e tornare alla sua gabbia casa. Per i primi tre giorni dopo la procedura di etichettatura, analgesici sistemici (es. buprenorfina) e di attualità pomata antibiotica sono dati due volte al giorno e il mouse è strettamente monitorato. </li></ol> 2. Lesioni del nervo ottico Crush Al 4 ° giorno In questa procedura, si applica una lesione da schiacciamento del nervo ottico per causare un danno primario al assoni (Figura 2), che porterà a una morte graduale delle cellule gangliari retiniche. <ol><li> Tre giorni dopo l&#39;applicazione tracciante colorante, il mouse è profondamente anestetizzati. Utilizzando un microscopio da dissezione, la congiuntiva di un occhio è incisa a circa la posizione 04:00 con un paio di forbici primavera. </li><li> Delicatamente deviare i muscoli orbitali e metterle da parte. Esporre il nervo ottico bianco alla sua uscita dal globo oculare. Fate attenzione a non danneggiare alcuna vasi sanguigni. </li><li> Con l&#39;aiuto di un paio di cross-azione forcipe, applicare una lesione da schiacciamento del nervo ottico a circa 2 mm dal bulbo oculare per circa 3 secondi. Fare molta attenzione a non danneggiare l&#39;arteria oftalmica per causare sanguinamento. </li><li> Dopo il completamento della lesione da schiacciamento, l&#39;incisione viene suturata. </li><li> Prima che il topo recupera da anestesia, dare una iniezione sottocutanea di buprenorfina per l&#39;analgesia. </li><li> Muovi il mouse in una zona calda e secca e monitorarlo, fino a quando non è in grado di mantenere una postura eretta e tornare alla sua gabbia casa. Per i primi tre giorni dopo la procedura di lesione da schiacciamento, analgesici sistemici (es. buprenorfina) e di attualità pomata antibiotica sono dati due volte al giorno e il mouse è strettamente monitorato. </li></ol> 3. Raccogliere i retina e analizzare sopravvivenza RGC Al Giorno 11 Lo scopo di questa procedura è quello di raccogliere le retinaeto analizzare la sopravvivenza delle cellule gangliari della retina. <ol><li> Al giorno 11 (giorno 7 dopo l&#39;ONC), il mouse è eutanasia da CO 2 infusione e dislocazione cervicale. </li><li> Gli occhi sono enucleato con l&#39;aiuto di una pinza facendo pressione per l&#39;orbita. </li><li> Gli occhi sono fissi in soluzione di paraformaldeide al 4% per due ore e lavate in PBS per tre volte. Sezionare la retina. La procedura di dissezione retina è già stato pubblicato precedentemente da Giove nel 2010 da Gustmann, S. et al. 6, e quindi non sarà descritta qui. </li><li> Le immagini delle cellule sopravvissute gangliari della retina sono effettuate utilizzando un microscopio a fluorescenza in regioni definite della retina, e la densità delle cellule gangliari della retina può essere contato. Un risultato rappresentante della fluorescenza marcata con cellule gangliari della retina, con o senza lesioni schiacciamento del nervo ottico è mostrato nella Figura 3. </li></ol> 4. Rappresentante dei risultati: Per analizzare la sopravvivenza delle cellule gangliari della retina, sette giorni dopo l&#39;infortunio schiacciamento del nervo ottico, la retina sono state raccolte, fisso, appiattita e montato. Immagini retiniche sono state scattate con un microscopio a fluorescenza. Rispetto al normale r illesoetina (a sinistra, Normale), il numero di RGCs vitali (verde, retrograda etichettato dal colorante FluoroGold neurale tracciante iniettato nel collicolo superiore) è significativamente più bassa nel retina con una ferita schiacciamento del nervo ottico (a destra, ONC) (Figura 3) . <img src="/files/ftp_upload/2685/2685fig1.jpg" alt="Figura 1" /> Figura 1. Baseline marcatura retrograda di cellule gangliari della retina Al fine di contare le cellule vitali gangliari della retina in diversi momenti dopo l&#39;infortunio schiacciamento del nervo ottico, le cellule gangliari della retina sono etichettati retrogrado iniettando una neurale tracciante colorante (colore verde) nel collicolo superiore del cervello tre giorni prima che il nervo ottico ( rosso) schiacciare lesioni. Dal momento che gli assoni delle cellule gangliari retiniche risiedono nel collicolo superiore, il colorante tracciante sarà ripresa dalla RGCs retrogrado e fornisce un indicatore per la RGCs vivere. <img src="/files/ftp_upload/2685/2685fig2.jpg" alt="Figura 2" /> Figura 2. Lesione da schiacciamento del nervo ottico Al fine di analizzare lo stato di sopravvivenza delle cellule gangliari della retina, una lesione da schiacciamento viene applicato al nervo ottico (rosso) per causare un danno primario al assoni. Questo porterà a una morte graduale delle cellule gangliari retiniche. Tre giorni dopo il colorante tracciante (verde) di iniezione, il mouse è anestetizzato. La congiuntiva di un occhio è incisa. Deviare i muscoli orbitali e metterle da parte. Esporre il nervo ottico alla sua uscita dal globo oculare. Applicare una lesione da schiacciamento del nervo ottico a circa 2 mm dal bulbo oculare per circa 3 secondi con un paio di cross-azione pinze. <img src="/files/ftp_upload/2685/2685fig3.jpg" alt="Figura 3" /> Figura 3. Fluorescenza etichettati cellule gangliari della retina, con o senza lesioni schiacciamento del nervo ottico Per analizzare la sopravvivenza delle cellule gangliari della retina, sette giorni dopo l&#39;infortunio schiacciamento del nervo ottico, la retina sono state raccolte, fisso, appiattita e montato. Immagini retiniche sono state scattate con un microscopio a fluorescenza. Rispetto alla retina indenne normale (a sinistra, Normale), il numero di RGCs vitali (verde, retrograda etichettato dalla neurali FluoroGold colorante tracciante iniettato nel collicolo superiore) è stata significativamente più bassa nel retina con una ferita schiacciamento del nervo ottico (a destra, ONC ) (Figura 3). Scala grafica: 50 micron

<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>

Nessun conflitto di interessi dichiarati.

L&#39;ottica schiacciare modello murino lesione del nervo è utile per studiare il processo di morte RGC e sopravvivenza. Questo modello è anche spesso usato per studiare gli effetti di reagenti diversi e geni RGC apoptosi e la sopravvivenza. Un vantaggio di questo modello è che ha un alto grado di riproducibilità con minime varianti. Tuttavia, particolare attenzione è richiesta in più fasi in questo modello. In primo luogo, al momento della lesione da schiacciamento del nervo ottico, ?...

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

Lesione al nervo ottico può portare alla degenerazione assonale, seguita da una morte graduale delle cellule gangliari della retina (RGCs), che si traduce in perdita di visione irreversibile. Esempi di questo tipo di malattie umane sono traumatiche neuropatia ottica e la degenerazione del nervo ottico nel glaucoma. E &#39;caratterizzato da cambiamenti tipici della testa del nervo ottico, progressiva degenerazione del nervo ottico, e la perdita delle cellule gangliari della retina, se non controllata, porta alla perdita della vista e cecità. Schiacciare il nervo ottico (ONC) del modello lesioni mouse è un importante modello sperimentale di malattia traumatica neuropatia ottica, glaucoma, ecc In questo modello, la lesione da schiacciamento del nervo ottico conduce all&#39;apoptosi delle cellule gangliari della retina graduale. Questo modello di malattia può essere usato per studiare i processi generali ed ai meccanismi di morte neuronale e la sopravvivenza, che è essenziale per lo sviluppo di misure terapeutiche. Inoltre, gli approcci farmacologici e molecolari può essere utilizzato in questo modello per individuare e testare i reagenti potenziale terapeutico per il trattamento di diversi tipi di neuropatia ottica. Qui, mettiamo a disposizione una dimostrazione passo passo di (I) l&#39;etichettatura di base retrograda di cellule gangliari della retina (RGCs) al giorno 1, (II) lesione da schiacciamento del nervo ottico al giorno 4, (III), raccogliere la retina e analizzare la sopravvivenza RGC al giorno 11, e (IV) risultato Rappresentante.

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

Questo protocollo mostra come etichetta retrogrado cellule gangliari della retina, e come fare successivamente un lesione da schiacciamento del nervo ottico al fine di analizzare la sopravvivenza delle cellule gangliari della retina e l&#39;apoptosi. Si tratta di un modello di malattia sperimentale per diversi tipi di neuropatia ottica, compreso il glaucoma.

Una lesione del nervo ottico Modello Crush murini per studiare la sopravvivenza delle cellule gangliari della retina

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 Views.  NIH. Questo protocollo mostra come etichetta retrogrado cellule gangliari della retina, e come fare successivamente un lesione da schiacciamento del nervo ottico al fine di analizzare la sopravvivenza delle cellule gangliari della retina e l'apoptosi. Si tratta di un modello di malattia sperimentale per diversi tipi di neuropatia ottica, compreso il 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.

Resezione del nervo ottico: un modello di apoptosi Neuron adulti nel Sistema Nervoso Centrale

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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Neuroscienze

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 preparazione isolati retina per registrare Risposta Luce da Geneticamente etichetta cellule gangliari della retina

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.

Registrazione potenziale evocato visivo in un modello murino di sperimentale del nervo ottico demielinizzazione

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.

Sequenza RNA a singola cellula di sottogruppi definiti delle cellule del Ganglio retinico

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.

Radice dorsale Ganglion iniezione e dorsale radice Lesioni Crush come un modello per sensoriale 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 ...

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.

Transection parziale del nervo ottico in ratti: un modello stabilito con un nuovo approccio operativo per valutare la degenerazione secondaria delle cellule ganglionari retiniche

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.

Ganglio simpatico cervicale indotta da etanolo blocco applicazioni per promuovere la rigenerazione del nervo alveolare inferiore canini utilizzando un nervo artificiale

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.

Analisi kinematica 3D per la valutazione funzionale nel modello di ratto di lesioni da schiacciamento del nervo sciatico

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.

Valutazione multifattoriale del comportamento motorio nei ratti dopo la lesione unilaterale da schiacciamento del nervo sciatico

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 Metodi per valutare la funzione e la struttura delle cellule gangliari retiniche e del nervo ottico in animali di grandi dimensioni

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 ...