Ringraziamo i molti studenti e borsisti che hanno lavorato su questo modello per migliorarne l&#39;efficacia, e per capire i suoi meccanismi. Un ringraziamento particolare va al dott del. Mary Johnson (University of Maryland-Baltimore), Nitza Goldenberg-Cohen (Schneiderman Childrens Hospital, Petah-Tikva, Israel), Charles Zhang (Einstein Medical College, Bronx, NY), e Valerie Touitou (Hopital Salpetrie, Parigi, Francia). Questo studio è stato finanziato in parte da RO1 EY015304 per SLB.

Questo protocollo è stato approvato dalla University of Maryland Istituzionale Cura animali e del Comitato di utilizzo (IACUC, Baltimore, MD, USA) 1. set-up sperimentale <ol><li> Fare una lente a contatto personalizzato progettato da un grado ottico chiaro circolare 7 mm di diametro plexiglass, di 3 mm di spessore. Tagliare le lenti circolari con un trapano a colonna. Utilizzare una punta da trapano standard per rendere la curva interna, e, infine, lucidare le curve esterne ed interne usando una lucidatrice di lenti a contatto di grana ultrafine (1000/3000). </li><li> Preparare 2,5 mM Rosa Bengala (RB) in pH 7,4 tampone fosfato salino (PBS) in anticipo, filtro sterilizzare con un filtro a siringa da 0,45 micron e conservare 1 ml aliquote a -20 ° C in un contenitore a tenuta di luce fino a 6 mesi. NOTA: L&#39;uso di animali maschi outbred albini, come Sprague Dawley riduce al minimo lo sforzo differenze di risposta dipendono, consente una maggiore facilità di induzione e riduce la variabilità che può verificarsi con estro cycling femminile. </li><li> Impostare la frequenza raddoppiata Neodimio-ittrio granato di alluminio (Fd-YAG) laser medico oftalmico, che genera una luce laser 532 nm. Montare il laser su una lampada a fessura oftalmica Haag-Streit utilizzando un adattatore laser oftalmico standard. Questo dispositivo disponibile in commercio consente la visualizzazione simultanea di applicazione punto dell&#39;occhio e laser dell&#39;animale. Il laser medico ha anche un fascio di puntamento per una corretta messa a fuoco e centratura utilizzando lo stesso formato di punto come l&#39;induzione laser. parametri di potenza laser sono i seguenti: <ol><li> Per ratto induzione rNAION: utilizzare 500 micron punto dimensioni / 50 mW di potenza del laser / 1.000 durata msec / 1.000 ms intervallo. </li><li> Per il mouse induzione rNAION: cambiare la dimensione del punto a 300 micron per disco ottico più piccolo e lasciare gli altri parametri lo stesso come impostazione di ratto. </li><li> Ordinariamente utilizzare un misuratore di potenza laser per assicurare la potenza del laser. </li></ol></li></ol> 2. Procedura sperimentale <ol><li> Accendere la potenza del lasere impostare il parametro del laser appropriata. Riscaldare il laser per almeno cinque minuti prima dell&#39;uso. </li><li> Pesare l&#39;animale per determinare la dose appropriata per ketamina / xylazina e tintura RB. Anestetizzare l&#39;animale mediante iniezione intraperitoneale di 1 ml / kg della miscela di 80 mg / ml di ketamina e 4 mg / ml xilazina. </li><li> Lasciare l&#39;animale in una gabbia riscaldata fino a completo anestetizzato. Verificare la presenza di alcuna risposta a stimoli avversi (coda o pizzicare la punta). Controllare animali per profondità dell&#39;anestesia ogni 10 min. </li><li> Dilata allievi dell&#39;animale con 1% tropicamide e anestetizzare la superficie dell&#39;occhio con 0,5% proparacaina. Se si utilizzano gli animali pigmentate, come Long Evans, a 2,5% gocce oculari neosynephrine aumenterà la dilatazione della pupilla. </li><li> Usare le forbici per tagliare i baffi vicino al muso sul lato per essere indotti a evitare di bloccare la vista. </li><li> Mettere una goccia di 1% di metilcellulosa o di altra goccia accoppiamento oftalmica all&#39;interno della lente a contatto su misura, e quindi applicare l&#39;obiettivo sulla tha gli occhi di ratto. </li><li> Posto l&#39;animale su una piattaforma regolato all&#39;altezza della lampada a fessura. Impostare testa dell&#39;animale con un angolo di 45 ° in modo che l&#39;occhio è perpendicolare alla lampada a fessura e laser. </li><li> Visualizza l&#39;occhio attraverso la lampada a fessura oftalmica. Assicurarsi che il fascio di puntamento è la dimensione giusta, così come messo a fuoco e centrato direttamente sul nervo ottico visualizzato. Fotografare il fondo della retina con una fotocamera digitale con alta ASA (1.200 - 2.000) di velocità montato su uno dei pezzi occhio della lampada a fessura con un adattatore su misura. NOTA: La capacità di vedere i vasi coroideali rivela la trasparenza della retina. Questo è un segnale importante per essere in grado di rilevare successivamente ischemia retinica, che possono confondere l&#39;interpretazione se si verifica. rNAION è ottico ischemia del nervo, che si traduce in perdita isolato RGC, mentre i risultati ischemia retinica in danni alla retina che colpisce tutte le cellule degli strati retinici interni. </li><li> Facoltativamente, ulteriormente immagine retina enervo ottico utilizzando una spettrale dominio a coerenza ottica tomografo (SD-8 ottobre) per valutare l&#39;occhio non-indotta. Scansione en face (Figura 3B) e 7 scansioni trasversali attraverso la retina (Figura 3C). L&#39;SD-OCT di imaging utilizza la stessa lente a contatto utilizzato per l&#39;induzione laser. </li><li> Iniettare 1 ml / kg per via endovenosa attraverso RB vena della coda, e attendere 30 secondi, quindi attivare la potenza del laser. Questa temporizzazione consente RB di distribuire uniformemente nella circolazione. Usiamo un impulso laser di 50 mW a una sec / impulso. energie maggiori (≥ 60 MW) possono danneggiare la retina o causare ischemia vascolare retinica. Attenzione: Assicurarsi che ognuno di noi ha un paio di occhiali filtranti sicurezza laser della lunghezza d&#39;onda di blocco appropriata per evitare che la luce laser diffusa entra l&#39;occhio del ricercatore. NOTA: La somministrazione laser deve essere rapidamente dopo l&#39;iniezione IV RB poiché il colorante viene eliminato rapidamente dalla circolazione. Più a lungo l&#39;induzione laser, il più grave l&#39;ottica ischemia del nervo. Generalmente, gli animali sono date 7 - 12 impulsi sec in rapida successione. Il contatto della luce laser con il colorante circola dà vasi nervo ottico bella bagliore dorato che può essere visto attraverso la lampada a fessura (Figura 4B). Questo dimostra il colorante è stato iniettato per via sistemica e distribuita nel flusso sanguigno. Se la luce è debole, o del tutto assenti (Figura 4A), il colorante non è stato iniettato per via endovenosa. In questo caso, non danno una seconda iniezione immediatamente, poiché l&#39;animale dovrà recuperare per almeno due giorni prima reiniezione. </li><li> Subito dopo induzione, rimuovere le lenti a contatto. Coprire entrambi gli occhi con antibiotico oftalmico tripla (Neosporin / polimixina / bacitracina) pomata con desametasone, e mettere l&#39;animale in un riscaldamento pad 37 ° C in una sola gabbia alloggiata sotto stretta osservazione fino al pieno recupero. <ol><li> Pulire la lente a contatto con acqua distillata wAter e asciugare con un panno non abrasivo per un utilizzo futuro. </li><li> Due giorni dopo l&#39;induzione valutare l&#39;edema nervo ottico sia da fotografia a colori del fondo oculare e SD OCT-analisi 8. NOTA: Confrontando il grado di edema nervo ottico fornisce una stima della gravità della ischemia nervo ottico. In due giorni, il margine del disco nervo ottico è offuscata, e le vene della retina sono leggermente dilatati, rispetto con l&#39;occhio (non-indotta) controlaterale. Eseguire elettroretinografia (ERG) e flash visivo potenziali evocati (VEP 11) a due e quattro settimane inviare ad induzione per l&#39;analisi elettrofisiologia. </li></ol></li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonarteritic+anterior+ischemic+optic+neuropathy+(NAION)+and+its+experimental+models.">Nonarteritic anterior ischemic optic neuropathy (NAION) and its experimental models.</a> Prog Retin Eye Res. 30, 167-187 (2011).</li><li>Nicholson, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PGJ2+Provides+Prolonged+CNS+Stroke+Protection+by+Reducing+White+Matter+Edema.">PGJ2 Provides Prolonged CNS Stroke Protection by Reducing White Matter Edema.</a> PLoS One. 7 (12), (2012).</li><li>Goldenberg-Cohen, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendrocyte+Dysfunction+Following+Induction+of+Experimental+Anterior+Optic+Nerve+Ischemia.">Oligodendrocyte Dysfunction Following Induction of Experimental Anterior Optic Nerve Ischemia.</a> Invest Ophthalmol Vis Sci. 46, 2716-2725 (2005).</li><li>Bernstein, S. L., Guo, Y., Kelman, S. E., Flower, R. W., Johnson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+cellular+responses+in+a+novel+rodent+model+of+anterior+ischemic+optic+neuropathy.">Functional and cellular responses in a novel rodent model of anterior ischemic optic neuropathy.</a> Invest Ophthalmol Vis Sci. 44, 4153-4162 (2003).</li><li>Berger, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral-Domain+Optical+Coherence+Tomography+of+the+Rodent+Eye:+Highlighting+Layers+of+the+Outer+Retina+Using+Signal+Averaging+and+Comparison+with+Histology.">Spectral-Domain Optical Coherence Tomography of the Rodent Eye: Highlighting Layers of the Outer Retina Using Signal Averaging and Comparison with Histology.</a> PLoS One. 9 (5), (2014).</li><li>Huang, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effects+of+systemic+treatment+with+methylprednisolone+in+a+rodent+model+of+non-arteritic+anterior+ischemic+optic+neuropathy+(rNAION).">Protective effects of systemic treatment with methylprednisolone in a rodent model of non-arteritic anterior ischemic optic neuropathy (rNAION).</a> Exp Eye Res. 131, 69-76 (2015).</li><li>Mantopoulos, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Experimental+Model+of+Optic+Nerve+Head+Injury.">An Experimental Model of Optic Nerve Head Injury.</a> Invest Ophthalmol Vis Sci. 57, 6222 (2014).</li><li>You, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+Evoked+Potential+Recording+in+a+Rat+Model+of+Experimental+Optic+Nerve+Demyelination.">Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination.</a> J. Vis. Exp. (101), e52934 (2015).</li><li>Templeton, J. P., Geisert, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+approach+to+optic+nerve+crush+in+the+mouse.">A practical approach to optic nerve crush in the mouse.</a> Mol Vis. 18, 2147-2152 (2012).</li><li>Magharious, M. M., D'Onofrio, P. M., Koeberle, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optic+Nerve+Transection:+A+Model+of+Adult+Neuron+Apoptosis+in+the+Central+Nervous+System.">Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System.</a> J Vis Exp. (51), e2241 (2011).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Rodent+Model+of+Posterior+Ischemic+Optic+Neuropathy.">A Novel Rodent Model of Posterior Ischemic Optic Neuropathy.</a> JAMA Ophthalmol. 131 (2), 194-204 (2013).</li><li>Huang, T. L., Chang, C. H., Chang, S. W., Lin, K. H., Tsai, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+Intravitreal+Injections+of+Antivascular+Endothelial+Growth+Factor+Agents+in+a+Rat+Model+of+Anterior+Ischemic+Optic+Neuropathy.">Efficacy of Intravitreal Injections of Antivascular Endothelial Growth Factor Agents in a Rat Model of Anterior Ischemic Optic Neuropathy.</a> IOVS. 56, 2290-2296 (2015).</li></ol>

Gli autori non hanno nulla da rivelare.

Mentre ci sono un certo numero di modelli di danno del nervo ottico (nervo ottico crush 12, ottico resezione del nervo 13, e PION 14), il modello rNAION è umano, adattabile a entrambi i ratti e topi. Assomiglia più da vicino le condizioni cliniche umana di NAION. Questa condizione include progressiva anteriore del nervo ottico edema, una sindrome ottico scompartimento del nervo anteriore, focale ischemia assonale, isolato retina cellule gangliari danno assonale e la perdita di più di ...

Nonarteritic neuropatia ottica ischemica anteriore (NAION) è una lesione ischemica focale della porzione anteriore del nervo ottico (ON) 1. NAION è la causa più comune di improvvisa fibre nervose perdita della vista ottico in soggetti di età superiore a 50 2. Il meccanismo è ritenuta essere una sindrome vano che provoca edema intraneurale, e provoca la compressione dei capillari forniscono gli assoni nel nervo ottico 3. Dal momento che l&#39;ON è in realtà un tratto del sistema nervoso centrale (CNS), la NAION roditore modello (rNAION) può essere utilizzato per studiare i meccanismi e le risposte di isolati colpi della sostanza bianca del sistema nervoso centrale. Il modello rNAION può quindi essere utile in sezionare molti problemi connessi con danni relativi corsa di materia bianca. Può essere usato per valutare diverse strategie neuroprotettive e agenti in bianco ictus materia. Una delle caratteristiche più interessanti del modello è che èun indolore, procedura non invasiva. La potenza del laser può essere regolata per produrre vari gradi di danno ischemico. Un&#39;altra caratteristica è che si basa su laser indotta radicali superossido danneggiare l&#39;endotelio capillare, producendo una disfunzione capillare progressiva. È questa disfunzione e progressiva edema che si ritiene essere notevolmente simile al meccanismo che causa NAION. La ricerca ha dimostrato che non provoca la coagulazione capillare diretta, ma agisce attraverso almeno due meccanismi: superossido morte indotta e stripping di alcuni capillare cellule endoteliali 4, e NFkB (fattore nucleare kappa-light-chain-enhancer di cellule B attivate ) associato regolazione infiammatorio in endotelio restante, con un aumento del trasporto di liquidi attraverso le membrane cellulari nell&#39;interstizio 5. La chiusura dei capillari del nervo ottico e compressione causate da interstiziale risultato accumulo di liquido in ottica ischemia testa del nervo. Un quadro schematico è mostrato inFigura 2. Il modello rNAION può essere utilizzato in entrambe le specie di ratti e topi 6,7, e può essere variata nel livello della sua gravità, da una lesione lieve a una distruzione completa, ma indolore del nervo ottico e la retina, tale come occlusione dell&#39;arteria centrale della retina (CRAO).

<table><tbody><tr><td>50 mW 532 nm laser</td><td>Iridex</td><td></td><td>Standard Ophthalmic Laser</td></tr><tr><td>0 - 100 mW 532 nm laser</td><td>Laserglow technologies</td><td></td><td>Substitute for iridex</td></tr><tr><td>Laser slit lamp adapter</td><td>Iridex</td><td></td><td>SMA coupled adapter for laser output</td></tr><tr><td>Coherent Fieldmate laser meter with thermopile sensor</td><td>Coherent</td><td></td><td>others also appropriate</td></tr><tr><td>Ophthalmic Examing Slit lamp biomicroscope</td><td>Various</td><td></td><td>Haag-Streit is the best; cheaper versions available on ebay</td></tr><tr><td>Rose Bengal</td><td>Sigma</td><td>330000-1G</td><td>Photoinducing agent</td></tr><tr><td>Fundus Contact lens or glass cover slip</td><td>custom/Cantor and Nissel (UK)</td><td></td><td>Custom designed planoconvex plastic lens for eye exam and induction</td></tr><tr><td>Tropicamide 1%</td><td></td><td></td><td></td></tr><tr><td>Tamiya polishing compound</td><td>Tamiya, INC</td><td>87068</td><td>polishing contact lens</td></tr><tr><td>2.5% Hypromellose (Goniovisc)/1% Methycellulose</td><td>HUB Pharmaceuticals</td><td></td><td>contact lens coupling agent</td></tr><tr><td>2.5% Neosynephrine Ophthalmic drops</td><td>Alcon labs</td><td></td><td>pupil dilating agent</td></tr><tr><td>Tropicamide 1%</td><td>Alcon labs</td><td></td><td>pupil dilating agent</td></tr><tr><td>0.5% Proparacaine</td><td>Alcon labs</td><td></td><td>topical Anesthetic</td></tr><tr><td>30 G fused needle insulin syringe</td><td>Various</td><td>Various</td><td>for intravenous injection of rose bengal</td></tr><tr><td>Ophthamic Antibiotic ointment with dexamethasone added (Triple antibiotic ointment)</td><td>Various</td><td>Various</td><td>Apply after induciton to minimize corneal scarring</td></tr><tr><td>Heidelberg Corporation Spectral domain-Optical Coherence Tomograph</td><td>Heidelberg Corporation</td><td></td><td>For Optical coherence measurements baseline and post-induction; not essential for induction</td></tr></tbody></table>

the rodent model of nonarteritic anterior ischemic optic neuropathy (rnaion)

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their lifetime. NAION results in optic nerve edema, selective loss of the retinal ganglion cell neurons (RGCs) and atrophy of the optic nerve. A rodent model of NAION that expresses most NAION features and sequelae has been developed, which is applicable to both rats and mice. This model utilizes a focal laser application of 532 nm wavelength to illuminate a photoactive dye, Rose Bengal (RB), to cause capillary damage and leakage at the targeted anterior optic nerve (the laminar region). After rNAION induction, there is an early optic nerve ischemia, optic nerve edema, and intraneural inflammation, followed by selective RGC and axonal loss. Since the optic nerve is a CNS white matter tract, the rNAION model is applicable to mechanistic studies of selective white matter ischemia, as well as neuroprotective analyses and short and long-term mechanisms of glial and neuronal response to ischemia.

<html> La visualizzazione lente a contatto abilitato centrale retina (Figura 1). Il punto laser focale illumina il disco ottico sul retro della retina (Figura 2). Il normale retina non-indotta è mostrato ripreso dalla lampada a fessura bio-microscopio (figura 3A) e SD-OCT (Figura 3B e 3C). Durante l&#39;induzione laser, quando nessun colorante è presente nella circolazione, luce laser non comporta recipiente e fluoresce...

Watch this Scientific Journal Video about The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy rNAION at JoVE.com

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy rNAION

The following report describes how to replicate the rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION), using the appropriate dye, contact lens and laser parameters. We also reveal the appropriate steps for evaluating the rNAION lesion in vivo.

Il modello di roditore di nonarteritic Neuropatia ottica ischemica anteriore (rNAION)

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their ...

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Research

JoVE Journal

Medicine

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy (rNAION)

8.9K Views.  University of Maryland-Baltimore School of Medicine. The following report describes how to replicate the rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION), using the appropriate dye, contact lens and laser parameters. We also reveal the appropriate steps for evaluating the rNAION lesion in vivo.

Video: The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy rNAION

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

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including 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 ...

Ricerca

Neuroscienze

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

Un modello di topo indotta da laser di cronica ipertensione oculare per caratterizzare difetti visivi

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

Medicina

Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history have&#160;remained poorly understood. Recently, we developed a reliable, reproducible animal model of PION and tested the treatment effect of some neurotrophic factors in this model1. The purpose of this video is to demonstrate our photochemically induced model of posterior ischemic optic neuropathy, and to evaluate its effects with retrograde labeling of retinal ganglion cells. Following surgical exposure of the posterior optic nerve, a photosensitizing dye, erythrosin B, is intravenously injected and a laser beam is focused onto the optic nerve surface. Photochemical interaction of erythrosin B and the laser during irradiation damages the vascular endothelium, prompting microvascular occlusion mediated by platelet thrombosis and edematous compression. The resulting ischemic injury yields a gradual but pronounced retinal ganglion cell dieback, owing to a loss of axonal input - a remote, injury-induced and clinically relevant outcome. Thus, this model provides a novel platform to study the pathophysiologic course of PION,&#160;and can be further optimized for testing therapeutic approaches for optic neuropathies as well as other CNS ischemic diseases.

The goal of this protocol is to photochemically induce ischemic injury to the posterior optic nerve in rat. This model is critical to studies of the pathophysiology of posterior ischemic optic neuropathy, and therapeutic approaches for this and other optic neuropathies, as well as of other CNS ischemic diseases.

Rat Modello di fotochimica-Induced posteriore Neuropatia ottica ischemica

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history ...

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

Un modello di topo per Laser-indotta della coroide neovascolarizzazione

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the brain for visual perception. Damage to RGCs and their axons leads to vision loss and/or blindness. Although the specific cause of glaucoma is unknown, the primary risk factor for the disease is an elevated intraocular pressure. Glaucoma-inducing procedures in animal models are a valuable tool to researchers studying the mechanism of RGC death. Such information can lead to the development of effective neuroprotective treatments that could aid in the prevention of vision loss. The protocol in this paper describes a method of inducing glaucoma - like conditions in an in vivo rat model where 50 &#181;l of 2 M hypertonic saline is injected into the episcleral venous plexus. Blanching of the vessels indicates successful injection. This procedure causes loss of RGCs to simulate glaucoma. One month following injection, animals are sacrificed and eyes are removed. Next, the cornea, lens, and vitreous are removed to make an eyecup. The retina is then peeled from the back of the eye and pinned onto sylgard dishes using cactus needles. At this point, neurons in the retina can be stained for analysis. Results from this lab show that approximately 25% of RGCs are lost within one month of the procedure when compared to internal controls. This procedure allows for quantitative analysis of retinal ganglion cell death in an in vivo rat glaucoma model.

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is characterized by damage to retinal ganglion cells. Inducing glaucoma in animal models can provide insight into the study of this disease. Here, we outline a procedure that induces loss of RGCs in an in vivo rat model and demonstrates the preparation of whole-mount retinas for analysis.

Glaucoma inducono Procedura in un<em&gt; In Vivo</em&gt; Modello di ratto e tutto il montaggio Retina Preparazione

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the ...

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions. Rodent models of I/R injury are providing significant insights into mechanisms and treatment strategies for human I/R injury, especially with regard to neurodegenerative damage in the retinal neurovascular unit. Presented here is a protocol for inducing retinal I/R injury in mice through elevation of intraocular pressure (IOP). In this protocol, the ocular anterior chamber is cannulated with a needle, through which flows the drip of an elevated saline reservoir. Using this drip to raise IOP above systolic arterial blood pressure, a practitioner temporarily halts inner retinal blood flow (ischemia). When circulation is reinstated (reperfusion) by removal of the cannula, severe cellular damage ensues, resulting ultimately in retinal neurodegeneration. Recent studies demonstrate inflammation, vascular permeability, and capillary degeneration as additional elements of this model. Compared to alternative retinal I/R methodologies, such as retinal arterial ligation, retinal I/R injury by elevated IOP offers advantages in its&#160;anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit.

Un modello murino di retina ischemia-riperfusione lesioni a causa di aumento della pressione intraoculare

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

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 ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

Modello di retinopatia indotta dall'ossigeno per le malattie retiniche ischemiche nei roditori

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

Cauterizzazione a cerchio completo del plesso vascolare limbare per il glaucoma indotto chirurgicamente nei roditori

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. To investigate pathological mechanisms of retinal ischemia, relevant experimental models need to be developed. Anatomically, a main retinal blood supplying vessel is the ophthalmic artery (OpA) and OpA originates from the internal carotid artery of the common carotid artery (CCA). Thus, disruption of CCA could effectively cause retinal ischemia. Here, we established a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion (tBCCAO) to tie the right CCA with 6-0 silk sutures and to occlude the left CCA transiently for 2 seconds via a clamp, and showed that tBCCAO could induce acute retinal ischemia leading to retinal dysfunction. The current method reduces reliance on surgical instruments by only using surgical needles and a clamp, shortens occlusion time to minimize unexpected animal death, which is often seen in mouse models of middle cerebral artery occlusion, and maintains reproducibility of common retinal ischemic findings. The model can be utilized to investigate the pathophysiology of ischemic retinopathies in mice and further can be used for in vivo drug screening.

Here, we describe a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion using simple sutures and a clamp. This model can be useful for understanding the pathological mechanisms of retinal ischemia caused by cardiovascular abnormalities.

Un modello murino di lesione retinica ischemica indotta dall'occlusione dell'arteria carotide comune transiente

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. ...