We wish to thank Peter Munro PhD for his assistance with optic nerve sectioning. This study was supported by the Medical Research Council (G0901303), and in part by the Dorothy Hodgkin Postgraduate Award/Medical Research Council, the Helen Hamlyn Trust, Fight for Sight, and Moorfields special trustess,.

Etica dichiarazione: Tutti gli esperimenti sugli animali sono stati condotti in conformità con la cura e l&#39;uso degli animali Comitato Istituzionale (IACUC), e sono stati approvati in accordo con le linee guida Regno Unito home office ( <a href="http://goo.gl/FLkirW" target="_blank">http://goo.gl/FLkirW</a> , ultimo accesso il 10 giugno , 2014) e la Dichiarazione ARVO per l&#39;uso di animali in Oftalmica e Vision Research ( <a href="http://goo.gl/4LFOjD" target="_blank">http://goo.gl/4LFOjD</a> , ultimo accesso il 10 giugno 2014). 1. ipertensione oculare Induzione <ol><li> Indurre glaucoma sperimentale elevando la pressione intraoculare (IOP) tramite iniezione unilaterale di microsfere paramagnetici nella camera anteriore di Brown Norvegia ratti, basata sul metodo di Samsel et al. 3 Altri ratti pigmentati possono essere adatti, anche se questi dovrebbero essere convalidata primo dall&#39;utente. </li><li> Casa 250-300 g femminileex-allevatore Brown Norvegia ratti in un ambiente di scarsa illuminazione costante (40-60 lux) per ridurre al minimo le fluttuazioni diurne in IOP 7, con l&#39;accesso a cibo e acqua ad libitum. </li><li> Prendere le misure IOP di base in animali svegli 8 prima anestesia e iniezione tallone, utilizzando un tonometro rimbalzo tarato per l&#39;uso negli occhi di ratto 9. IOP è preso come media di cinque letture. </li><li> Anaesthetize ratti con 37,5 mg / kg di ketamina, e 0,25 mg / kg medetomidina cloridrato consegnato per via intraperitoneale. Confermare profondità dell&#39;anestesia testando i riflessi del piede posteriore dell&#39;animale, prima dell&#39;applicazione di iodio povidone (vedi punto 1.5), e l&#39;iniezione tallone (vedi punto 1.8). Amministrare 0.5% proparacaina cloridrato per l&#39;analgesia. NOTA: Non dilatare la pupilla, in ogni fase. Questo aiuterà le perline per stabilirsi meglio nel dell&#39;angolo iridocorneale, e prevenire vincolante alla lente. Applicare pomata oculare contro l&#39;essiccamento della cornea su un-operato occhio controlaterale. </li><li> Wcenere l&#39;occhio operativa con 5% povidone iodio in acqua 10 5 min prima dell&#39;iniezione. </li><li> Dopo 5 minuti, stoppino lo iodio povidone off con garza sterile, e lavare l&#39;occhio con soluzione salina sterile allo 0,9%. Mantenere l&#39;occhio umido durante l&#39;anestesia con l&#39;applicazione regolare di soluzione salina sterile. </li><li> Posizionare un magnete toroidale intorno all&#39;occhio. </li><li> Iniettare 25 ml di una soluzione contenente 30 mg / ml di gamma-irradiazione sterilizzato 8 micron microsfere magnetiche in soluzione salina bilanciata di Hank (HBSS) nella camera anteriore, utilizzando un ago smussato 33 G. <ol><li> Per preparare perline, lavare da ri-sospensione, poi centrifugazione 3 volte a 10.000 xg per 5 minuti con 1 ml HBSS, prima di fare la 30 mg / ml soluzione definitiva. Mantenere condizioni di sterilità in tutto. </li><li> Per l&#39;iniezione, fare attenzione ad evitare di inserire l&#39;ago l&#39;iride, per ridurre al minimo il rischio di traumi iris. Ciò può essere evitato orientando l&#39;ago tangente alla superficie corneale, come paralleladell&#39;iride possibile. Questo aiuterà anche a ridurre al minimo la perdita di tallone dal sito di iniezione. NOTA: Iniettare perle a un ritmo rapido per garantire una distribuzione uniforme intorno all&#39;angolo iridocorneale, che è fondamentale per aumentare IOP. Inoltre, negozio perline, aghi e anelli magnetici separatamente in modo che le perle non formano i cluster, che li rende difficili da caricare nella siringa e iniettare, e l&#39;ago non diventa magnetizzato. </li></ol></li><li> Lasciare l&#39;ago in posizione per 1 minuto dopo l&#39;iniezione al fine di garantire che le microsfere sistemazione in angolo iridocorneale impedire il drenaggio acquosa dal trabecolato. Leggermente angolo l&#39;ago dopo le perline hanno inizialmente risolto per consentire alcune perdite di acquosa, per ridurre al minimo aumenti transitori IOP. Al termine dell&#39;intervento lavare l&#39;ago attraverso con il primo tampone fosfato salino (PBS), poi 70% di etanolo, seguita da acqua distillata, per garantire l&#39;uso continuato dell&#39;ago in procedure separate. In alternativa, si potrebbe utilizzareaghi monouso se la combinazione calibro e la siringa corretta è disponibile. Optional: aghi possono essere affilano con una beveller per prolungare il loro utilizzo. </li><li> A questo punto, se necessario, rimuovere il magnete, e usarlo per disegnare perle in aree di copertura incompleta. </li><li> Lasciare il magnete in posizione intorno all&#39;occhio per altri 10 min dopo l&#39;iniezione per assicurare perline stabilirsi bene nel dell&#39;angolo iridocorneale. </li><li> Anestesia Reverse con 0,25 mg / kg atipemezole cloridrato. </li><li> Amministrare cloramfenicolo, o altro pomata antibiotica, per esempio gentamicina o terramycin topica per prevenire l&#39;infezione, e lo 0,5% proparacaina cloridrato per l&#39;analgesia. Lasciare gli animali a recuperare su una stuoia di calore fino a ritrovare il movimento, poi il trasferimento ad una scatola caldo e fornitura con alimentazione supplementare, come un integratore alimentare o dieta regolare inumidito fino al recupero è completo. Analgesia sistemica o locale deve essere somministrato agli animali che presentano segni di dolore 24 ore dopo l&#39;intervento chirurgico. Se These sintomi persistono nonostante il trattamento, gli animali devono essere abbattuti in modo umano. </li><li> Utilizzare l&#39;occhio controlaterale come controllo non operato. </li><li> Prendere le misure IOP ogni 2-3 giorni dopo la somministrazione tallone, e ogni 2-3 giorni successivi con un tonometro rimbalzo tarato per l&#39;uso negli occhi di ratto 9. </li><li> I criteri per l&#39;inclusione occhi a studi possono essere: se 1) la IOP è elevata al di sopra della pressione di controllo controlaterale di 5 mmHg, e 2) non superiore a 60 mmHg. </li><li> Occhi dove la pressione torna a basale (di solito da 1 settimana dopo l&#39;iniezione) non devono essere inclusi negli studi, tuttavia è possibile re-iniettare perline in occhi che non riescono a sviluppare l&#39;alta pressione intraoculare, se desiderato. </li><li> Euthanize animali di CO 2 asfissia alla fine dell&#39;esperimento. </li><li> Sezionare occhi e nervi ottici, e fissare nel 4% paraformaldeide (PFA) durante la notte per ulteriori analisi istologica. </li></ol> 2. Valutare danni alla retina Neuron Uso TUNEL STaining <ol><li> Per quantificare cellule apoptotiche in tutto il monte retinasuse test deossinucleotidil terminale transferasi-mediata dUTP nick-etichettatura (TUNEL), secondo le istruzioni del produttore. </li><li> Sezionare la retina dalla oculare, lavare per 3 x 5 min in 0,3% Triton X-100 in tampone fosfato (PBS-T). </li><li> Permeabilize il tessuto in 3% T-PBS per 2 ore. </li><li> Pre-equilibrare retine in Equilibration tampone per 10 minuti, prima di incubazione in soluzione di reazione TUNEL per 1 ora a 37 ° C. </li><li> Lavare il tessuto da 3 x 5 min a 0,3% T-PBS, sciacquare in 0.3% T-PBS contenente 5 micron DAPI, e TV a montare in supporti di montaggio. </li><li> Per quantificare TUNEL nuclei positivi utilizzano un microscopio confocale a prendere 10 micron z-stack attraverso lo strato di cellule gangliari a 20X di ingrandimento. Prendere 3 immagini su ciascuno dei 4 petali, in siti vicino al nervo ottico, in media periferia, e alla periferia lontano della retina, dando un totale di 12 immagini per tutta mount, il campionamento di circa 7.000 cellule. Criteri morfologici discriminati cellule non neuronali (endoteliale e gliali) dalle cellule neuronali. </li><li> Selezionare le aree per l&#39;imaging utilizzando solo il canale DAPI, e gli investigatori maschera di gruppi di trattamento. </li></ol> 3. Valutare danno del nervo ottico Utilizzando Toluidine Blu colorazione <ol><li> Fissare nervi ottici durante la notte in soluzione di Karnovsky a 4 ° C. </li><li> Trattare campioni per 2 ore a 1% (w / v) di osmio tetrossido e poi disidratato in 100% di etanolo. </li><li> Incubare nervi ottici in ossido di propilene per 30 min, e posizionare in una miscela 50:50 di ossido di propilene: overnight araldite. </li><li> Cambiare questa soluzione a 100% araldite, seguita da incubazione per una notte a 60 ° C. </li><li> Cut semisottile sezioni (0,75 millimetri di spessore) e macchia con l&#39;1% di toluidina blu / di borace (TB) in etanolo al 50% prima di esame al microscopio ottico. </li></ol> 4. Analisi statistica <ol><li> Eseguire ana statistichelisa utilizzando un apposito software di statistiche. Un ANOVA a due vie con test post-hoc di Newman-Keul può essere utilizzata per calcolare la significatività statistica per IOP cambia nel tempo. </li><li> Un valore p inferiore a 0,05 può essere considerato significativo. </li></ol>

<ol>
	<li>Quigley, H. A., Broman, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+number+of+people+with+glaucoma+worldwide+in+2010+and+2020.">The number of people with glaucoma worldwide in 2010 and 2020.</a> Br J Ophthalmol. 90 (3), 262-267 (2006).</li><li>Weinreb, R. N., Aung, T., Medeiros, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathophysiology+and+treatment+of+glaucoma:+a+review.">The pathophysiology and treatment of glaucoma: a review.</a> JAMA. 311 (18), 1901-1911 (2014).</li><li>Samsel, P. A., Kisiswa, L., Erichsen, J. T., Cross, S. D., Morgan, J. 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+novel+method+for+the+induction+of+experimental+glaucoma+using+magnetic+microspheres.">A novel method for the induction of experimental glaucoma using magnetic microspheres.</a> Invest Ophthalmol Vis Sci. 52 (3), 1671-1675 (2011).</li><li>Libby, R. T., 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=Inherited+glaucoma+in+DBA/2J+mice:+pertinent+disease+features+for+studying+the+neurodegeneration.">Inherited glaucoma in DBA/2J mice: pertinent disease features for studying the neurodegeneration.</a> Vis Neurosci. 22 (5), 637-648 (1017).</li><li>Morrison, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+intraocular+pressure+and+optic+nerve+injury+models+in+the+rat.">Elevated intraocular pressure and optic nerve injury models in the rat.</a> J Glaucoma. 14 (4), 315-317 (2005).</li><li>Levkovitch-Verbin, H., 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=Translimbal+laser+photocoagulation+to+the+trabecular+meshwork+as+a+model+of+glaucoma+in+rats.">Translimbal laser photocoagulation to the trabecular meshwork as a model of glaucoma in rats.</a> Invest Ophthalmol Vis Sci. 43 (2), 402-410 (2002).</li><li>Moore, C. G., Johnson, E. C., Morrison, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythm+of+intraocular+pressure+in+the+rat.">Circadian rhythm of intraocular pressure in the rat.</a> Curr Eye Res. 15 (2), 185-191 (1996).</li><li>Morrison, J. C., Jia, L., Cepurna, W., Guo, Y., Johnson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+and+sensitivity+of+the+TonoLab+rebound+tonometer+in+awake+Brown+Norway+rats.">Reliability and sensitivity of the TonoLab rebound tonometer in awake Brown Norway rats.</a> Invest Ophthalmol Vis Sci. 50 (6), 2802-2808 (2009).</li><li>Wang, W. H., Millar, J. C., Pang, I. H., Wax, M. B., Clark, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+measurement+of+rodent+intraocular+pressure+with+a+rebound+tonometer.">Noninvasive measurement of rodent intraocular pressure with a rebound tonometer.</a> Invest Ophthalmol Vis Sci. 46 (12), 4617-4621 (2005).</li><li>Isenberg, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ocular+application+of+povidone-iodine.">The ocular application of povidone-iodine.</a> Community Eye Health. 16 (46), 30-31 (2003).</li><li>Morrison, J. C., 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+rat+model+of+chronic+pressure-induced+optic+nerve+damage.">A rat model of chronic pressure-induced optic nerve damage.</a> Exp Eye Res. 64 (1), 85-96 (1997).</li><li>Foxton, R. H., 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=VEGF-A+is+necessary+and+sufficient+for+retinal+neuroprotection+in+models+of+experimental+glaucoma.">VEGF-A is necessary and sufficient for retinal neuroprotection in models of experimental glaucoma.</a> Am J Pathol. 182 (4), 1379-1390 (2013).</li></ol>

David T. Shima is an employee of Hofmann-La Roche, Switzerland. The remaining authors declare they have no competing financial interests.

Qui mostriamo un metodo per indurre elevata IOP nel ratto, iniettando microsfere magnetiche nella camera anteriore dell&#39;occhio. Questo metodo è semplice da portare a termine, e richiede poca perizia chirurgica, o ore di pratica e raffinatezza. Inoltre, il procedimento è efficace; raramente richiede più di una singola iniezione di perline di indurre una forte, robusto aumento della pressione (circa il 10% tasso di reiniezione). Questo può fornire un vantaggio rispetto ai metodi inducibili esistenti, come la vena episceral tecnicamente impegnativo sclerosi 11 modelli, o laser protocollo fotocoagulazione 6, che può richiedere più procedure da mantenere sollevate IOP. Affinché il metodo per avere successo tuttavia, ci sono alcuni piccoli passi critici che devono essere prese. In primo luogo, è utile utilizzare un magnete a forma toroidale per disegnare perline in angolazione iridocorneale. Questo passo è una modifica del protocollo originale, where le perline sono state iniettate nella camera anteriore, e poi spostati a mano libera intorno all&#39;occhio 3. Utilizzando un magnete toroidale significa che microsfere dovrebbero risolvere in modo uniforme in tutto l&#39;angolo, richiedendo il minimo redistribuzione manuale. In secondo luogo, il tasso di iniezione deve essere veloce - troppo lento e le perline si accumula prevalentemente su un lato dell&#39;angolo, con conseguente copertura incompleta e potenzialmente nessun aumento di pressione. In generale però, il metodo è abbastanza semplice che l&#39;utente potrebbe facilmente apportare modifiche al protocollo, ad esempio variando la dimensione o volume delle particelle di microsfere, forse per tentare di alterare il grado di elevazione IOP. Tuttavia, uno svantaggio potenziale del metodo è che si ha poco controllo sulla misura della ipertensione, che in circa 5-10% dei casi osservati salito sopra 60 mmHg. Aumenti eccessivi di IOP può essere molto distruttivo per tessuto retinico, e può rendere lo studio dei meccanismi e biologia di morte cellulare sfidare. Tuttavia, il metodo produce una patologia neuronale consistente, sia nella retina e nervo ottico, che può essere manipolato farmacologicamente 12. Questo può rendere il modello interessante per lo sviluppo di nuove terapie per il trattamento del glaucoma. Inoltre, poiché le perle sono diretti in angolo iridocorneale, questo lascia l&#39;asse visivo gratuito per l&#39;imaging dal vivo del disco retina o ottico. Prevediamo che questo modello sarà adattato e utilizzato per applicazioni future in altre specie, tra cui mouse.

Glaucoma primario è una malattia degli occhi devastante che colpisce circa 60,5 milioni di persone in tutto il mondo 1, che può portare alla perdita della vista vita che alterano e cecità 2. La ricerca sui meccanismi della malattia, e lo sviluppo di nuove terapie per il glaucoma, dipendono buoni modelli della malattia che ricapitolano alcune delle caratteristiche della patologia. Presentiamo qui un modello di ratto glaucoma basato sul metodo di Samsel et al. 3 L&#39;obiettivo generale di questa tecnica è quello di aumentare la pressione intraoculare (IOP) nell&#39;occhio iniettando microsfere magnetiche nella camera anteriore, e utilizzando un anello magnetico, diretta li in angolo iridocorneale. Questo impedisce deflusso acquosa, che aumenta IOP, con conseguente danno neuronale e perdita di cellule. Il protocollo è stato sviluppato per tentare di fornire un semplice modello inducibile del glaucoma. Questo protocollo può avere alcuni vantaggirispetto alle tecniche esistenti. Modelli genetici come topi DBA / 2J sono disponibili, che non richiedono procedure di avvio; tuttavia questi possono avere un esordio imprevedibile di progressione della malattia 4. Al contrario, i modelli inducibili, molti dei quali si basano su chirurgicamente elevando IOP nei roditori, hanno il vantaggio che l&#39;iniziazione può essere controllata dall&#39;utilizzatore. Alcuni di questi metodi possono avere inconvenienti della loro tuttavia, compreso tecnicamente impegnativo 5, e possono richiedere più procedure per mantenere elevata IOP 6. Al contrario, il metodo inducibile descritto in questo manoscritto è una tecnica semplice, efficace e riproducibile che produce stabili, robusti aumenti di pressione, con minima necessità di reiniezione. Inoltre, esso non comporta attrezzature costose, e richiede solo abilità chirurgiche di base per eseguire. Questo protocollo può essere appropriato per i lettori che stanno cercando di creare una inducibile tecnicamente meno impegnativaModello glaucoma nel loro laboratorio.

<table border="0" cellpadding="0" cellspacing="0" width="784">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">Name of Material/ Equipment</td>
			<td style="width:175px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:324px;">250-300g female Brown Norway ex-breeder rats</td>
			<td style="width:175px;">Harlan UK</td>
			<td style="width:119px;">203</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Tonolab Rebound Tonometer</td>
			<td style="width:175px;">Tiolat</td>
			<td style="width:119px;">TV02</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:324px;">Ketaset (Ketamine)</td>
			<td style="width:175px;">Fort Dodge Animal health</td>
			<td>BN1000118</td>
			<td>37.5 mg/kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Domitor (medetomidine hydrochloride)</td>
			<td style="width:175px;">Orion Pharma</td>
			<td style="width:119px;">140-999</td>
			<td>0.25 mg/kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Povidone iodine</td>
			<td style="width:175px;">Ecolab</td>
			<td>BN4369LE10</td>
			<td>5% in H2O</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Minim&#39;s Saline Solution</td>
			<td style="width:175px;">Bausch and Lomb</td>
			<td>PL00033/5017</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Toroidal magnet</td>
			<td style="width:175px;">Supermagnete</td>
			<td>R-10-07-03-N</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Magnetic Microspheres</td>
			<td style="width:175px;">Bangs Laboratories</td>
			<td>UMC4N/9692</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">HBSS</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">14025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">33-guage bevelled needle</td>
			<td style="width:175px;">Hamilton</td>
			<td>7747-01</td>
			<td>Custom needle</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Luer tip syringe</td>
			<td style="width:175px;">Hamilton</td>
			<td>80601</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Antisedan (atipemezole hydrochloride )</td>
			<td style="width:175px;">Orion Pharma</td>
			<td style="width:119px;">141-003</td>
			<td>0.25 mg/kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Chloramphenicol ointment</td>
			<td style="width:175px;">Medicom</td>
			<td>18956-0005</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">TUNEL staining kit</td>
			<td style="width:175px;">Promega</td>
			<td style="width:119px;">G3250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Triton X-100</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">T8787</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">DAPI</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9542</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Vectashield Mounting Media</td>
			<td style="width:175px;">Vector Labs</td>
			<td style="width:119px;">H-1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

experimental glaucoma induced by ocular injection of magnetic microspheres

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present here a protocol for elevating intraocular pressure (IOP) in the rat, by injecting magnetic microspheres into the anterior chamber of the eye. The use of magnetic particles allows the user to manipulate the beads into the iridocorneal angle, thus providing a very effective blockade of fluid outflow from the trabecular meshwork. This leads to long-lasting IOP rises, and eventually neuronal death in the ganglion cell layer (GCL) as well as optic nerve pathology, as seen in patients with the disease. This method is simple to perform, as it does not require machinery, specialist surgical skills, or many hours of practice to perfect. Furthermore, the pressure elevations are very robust, and reinjection of the magnetic microspheres is not usually required unlike in some other models using plastic beads. Additionally, we believe this method is suitable for adaptation for the mouse eye.

Iniezione di perline magnetiche nel angolo iridocorneale costantemente indotto un aumento prolungato e robusto in pressione (figura 1), che era facilmente osservabile al primo punto temporale, 3 giorni dopo l&#39;iniezione. Inoltre, l&#39;aumento della pressione è stata mantenuta per tutta la durata dell&#39;esperimento, e sebbene il nostro corso tempo finito a 18 giorni dopo l&#39;iniezione, altri hanno riportato che la pressione persiste a lungo termine 3. La media IOP media sulla lunghezza della esperimento di controllo, occhi non-bead-iniettati era 19,7 ± 0,3 mmHg, rispetto a 40,5 ± 2,8 mmHg per occhi branello-iniettato (P &lt;0.001). Inoltre, picco IOP è aumentato da 22.8 ± 0.3 mmHg a 49,9 ± 2,3 mmHg. Per determinare se l&#39;elevazione della PIO porta alla morte delle cellule gangliari retiniche, abbiamo effettuato TUNEL sulla retina, e istologia su sezioni trasversali del nervo ottico (Figura 2). In retina<html> abbiamo osservato un aumento della colorazione TUNEL (Figura 2A) in occhi tallone-iniettata con elevata pressione intraoculare. Il numero di nuclei apoptotici aumentato di circa 15 volte, da 1,6 ± 0,5 cellule nei controlli controlaterali, a 24,5 ± 0,5 cellule retine ipertesi (Figura 2B; P &lt;0.05). Inoltre, negli occhi in cui biglie magnetiche sono state iniettate ma pressione non è aumentato (probabilmente a causa del blocco incompleta dell&#39;angolo iridocorneale), il numero di cellule TUNEL-positivi non erano significativamente differenti da quelli dei controlli uninjected (P&gt; 0,05). Questo suggerisce che la morte cellulare era legato a pressione aumenta, non causa tossicità diretta delle microsfere magnetiche. Infine, abbiamo studiato la patologia del nervo ottico nel modello glaucoma, e visto accumulo di blu di toluidina in molti degli assoni, indicando la degenerazione di questi processi cellulari (Figura 2C). ure 1 &quot;src =&quot; / files / ftp_upload / 52400 / 52400fig1highres.jpg &quot;/&gt; Figura 1. aumento della pressione intraoculare con microsfere magnetiche. Iniezione di microsfere magnetiche nella camera anteriore indotto un robusto, significativo aumento della pressione intraoculare (IOP) in confronto al controllo controlaterale, occhi non iniettati. Unità asse Y = millimetri di mercurio (mmHg). Dati = media ± SEM. * = P &lt;0.001; N = 12. Questo dato è stato modificato da Foxton et al, Am.. J. Pathol 182 (4):. 1379-1390. <img alt="Figura 2" src="/files/ftp_upload/52400/52400fig2highres.jpg" /> Figura 2. Elevazione della IOP tramite iniezione di microsfere magnetiche in angolo iridocorneale, indotta morte neuronale nello strato di cellule gangliari (GCL). (A) Immagini rappresentative della retine di controllo (a sinistra) e glaucoma (a destra) gli occhi macchiati di nuclei apoptotici da TUNEL (verde;frecce bianche) e DAPI (blu), che indica il numero di nuclei apoptotici aumentato rosa IOP. (B) Quantificazione delle cellule positive TUNEL in GCL, mostrando che gli occhi con elevata IOP (al centro), avevano cellule significativamente più apoptotici rispetto a (a sinistra). Al contrario, negli occhi perline-iniettata in cui la pressione non ha aumento (a destra), è stato osservato alcun aumento significativo TUNEL colorazione. Dati = media ± SEM. * = P &lt;0.05; N = 7 - 8 (C) Immagini rappresentative della colorazione ottico nervo, dimostrando un aumento toluidina blu accumulo (frecce nere) in assoni danneggiati da glaucoma (a destra), ma non il controllo occhi (a sinistra).. Bar scale = 50 micron. Questa cifra è stata modificata da Foxton et al., Am. J. Pathol 182 (4):. 1379-1390.

Watch this Scientific Journal Video about Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres at JoVE.com

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises.

Experimental Glaucoma indotta da iniezione oculare di microsfere magnetiche

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present ...

experimental-glaucoma-induced-ocular-injection-magnetic

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JoVE Journal

Medicine

16.9K Views.  University College London Institute of Ophthalmology. We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises.

Video: Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

Generazione di microsfere di alginato per applicazioni biomediche

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

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Medicina

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

Iniezione sottoretinico di vettori di terapia genica e cellule staminali nel Eye mouse

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

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 ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Distacco della retina Modello in Roditori da iniezione sottoretinico di ialuronato di sodio

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events of myocardial injuries, various mouse models of myocarditis have been widely used. This study involved experimental autoimmune myocarditis (EAM) induced with cardiac myosin heavy chain (Myhc)-&#945; 334-352 in A/J mice; the affected animals develop lymphocytic myocarditis but with no apparent clinical signs. In this model, the utility of magnetic resonance microscopy (MRM) as a non-invasive modality to determine the cardiac structural and functional changes in animals immunized with Myhc-&#945; 334-352 is shown. EAM and healthy mice were imaged using a 9.4 T (400 MHz) 89 mm vertical core bore scanner equipped with a 4 cm millipede radio-frequency imaging probe and 100 G/cm triple axis gradients. Cardiac images were acquired from anesthetized animals using a gradient-echo-based cine pulse sequence, and the animals were monitored by respiration and pulse oximetry. The analysis revealed an increase in the thickness of the ventricular wall in EAM mice, with a corresponding decrease in the interior diameter of ventricles, when compared with healthy mice. The data suggest that morphological and functional changes in the inflamed hearts can be non-invasively monitored by MRM in live animals. In conclusion, MRM offers an advantage of assessing the progression and regression of myocardial injuries in diseases caused by infectious agents, as well as response to therapies.

This study demonstrates the successful establishment of magnetic resonance microscopy imaging as a non-invasive tool to assess the cardiac abnormalities in mice affected with autoimmune myocarditis. The data indicate that the technique can be used to monitor the disease-progression in live animals.

La valutazione non invasiva della Cardiac Anomalie in Experimental Autoimmune miocardite da Risonanza Magnetica Imaging Microscopy nel mouse

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events ...

A Magnetic Microbead Occlusion Model to Induce Ocular Hypertension-Dependent Glaucoma in Mice

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial neurodegenerative disease. With the advent of numerous transgenic mouse lines, there is increasing interest in inducible murine models of ocular hypertension. Here, we present an occlusion model of glaucoma based on the injection of magnetic microbeads into the anterior chamber of the eye using a modified microneedle with a facetted bevel. The magnetic microbeads are attracted to the iridocorneal angle using a handheld magnet to block the drainage of aqueous humour from the anterior chamber. This disruption in aqueous dynamics results in a steady elevation of intraocular pressure, which subsequently leads to the loss of retinal ganglion cells, as observed in human glaucoma patients. The microbead occlusion model presented in this manuscript is simple compared to other inducible models of glaucoma and also highly effective and reproducible. Importantly, the modifications presented here minimize common issues that often arise in occlusion models. First, the use of a bevelled glass microneedle prevents backflow of microbeads and ensures that minimal damage occurs to the cornea during the injection, thus reducing injury-related effects. Second, the use of magnetic microbeads ensures the ability to attract most beads to the iridocorneal angle, effectively reducing the number of beads floating in the anterior chamber avoiding contact with other structures (e.g., iris, lens). Lastly, the use of a handheld magnet allows flexibility when handling the small mouse eye to efficiently direct the magnetic microbeads and ensure that there is little reflux of the microbeads from the eye when the microneedle is withdrawn. In summary, the microbead occlusion mouse model presented here is a powerful investigative tool to study neurodegenerative changes that occur during the onset and progression of glaucoma.

Here, we present a protocol to induce ocular hypertension in the murine eye that results in the loss of retinal ganglion cells as observed in glaucoma. Magnetic microbeads are injected into the anterior chamber and attracted to the iridocorneal angle using a magnet to block the outflow of aqueous humour.

Un magnetica microperla occlusione modello per indurre ipertensione oculare-Dependent Glaucoma nei topi

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial ...

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to microperimetry (MY) in eyes affected by dry Age-related Macular Degeneration (AMD) over time through the production and secretion of growth factors (GFs) on surrounding tissue. Patients were randomly assigned to each study group. All patients were diagnosed with dry AMD and with BCVA equal to or greater than 1 logarithm of the minimum angle of resolution (logMAR). A suprachoroidal autologous graft by Limoli Retinal Restoration Technique (LRRT) was carried out on group A, which included 11 eyes from 11 patients. The technique was performed by implanting adipocytes, adipose-derived stem cells obtained from the stromal vascular fraction, and platelets from platelet-rich plasma in the suprachoroidal space. Conversely, group B, including 14 eyes of 14 patients, was used as a control group. For each patient, diagnosis was verified by confocal scanning laser ophthalmoscope and spectral domain-optical coherence tomography (SD-OCT). In group A, BCVA improved by 0.581 to 0.504 at 90 days and to 0.376 logMAR at 180 days (+32.20%) postoperatively. Furthermore, MY test increased by 11.44 dB to 12.59 dB at 180 days. The different cell types grafted behind the choroid were able to ensure constant GF secretion in the choroidal flow. Consequently, the results indicate that visual acuity (VA) in the grafted group can increase more than in the control group after six months.

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

The goal of this study is to assess whether the suprachoroidal graft of adipose-derived stem cells included in the stromal vascular fraction and platelets derived from platelet-rich plasma by the Limoli Retinal Restoration Technique can improve visual acuity and retinal sensitivity responses in eyes affected by dry age-related macular degeneration.

La terapia rigenerativa da Suprachoroidal cella autoinnesto nella degenerazione maculare senile secca: Report preliminare In Vivo

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to ...

An Experimental Model of Diet-Induced Metabolic Syndrome in Rabbit: Methodological Considerations, Development, and Assessment

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased prevalence due to the rise of sedentary lifestyles and unhealthy eating habits. Thanks to animal models, basic research can investigate the mechanisms underlying pathological processes such as MetS. Here, we describe the methods used to develop an experimental rabbit model of diet-induced MetS and its assessment. After a period of acclimation, animals are fed a high-fat (10% hydrogenated coconut oil and 5% lard), high-sucrose (15% sucrose dissolved in water) diet for 28 weeks. During this period, several experimental procedures were performed to evaluate the different components of MetS: morphological and blood pressure measurements, glucose tolerance determination, and the analysis of several plasma markers. At the end of the experimental period, animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia with low HDL, high LDL, and an increase of triglyceride (TG) levels, thus reproducing the main components of human MetS. This chronic model allows new perspectives for understanding the underlying mechanisms in the progression of the disease, the detection of preclinical and clinical markers that allow the identification of patients at risk, or even the testing of new therapeutic approaches for the treatment of this complex pathology.

We describe methods to develop an experimental model of diet-induced metabolic syndrome (MetS) in rabbits using a high-fat, high-sucrose diet. Animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia, thus reproducing the main components of human MetS. This chronic model will allow acquisition of knowledge underlying mechanisms of disease progression.

Un modello sperimentale di sindrome metabolica indotta da dieta in coniglio: considerazioni metodologiche, sviluppo e valutazione

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased ...

A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known risk factor for glaucoma. This protocol demonstrates a step-by-step guide on this technique in Long Evans rats and C57BL/6 mice. Under general anesthesia, a "purse-string" suture is applied on the conjunctiva, around the equator and behind the limbus of the eye. The fellow eye serves as an untreated control. Over the duration of our study, which was a period of 8 weeks for rats and 12 weeks for mice, IOP remained elevated, as measured regularly by rebound tonometry in conscious animals without topical anesthesia. In both species, the sutured eyes showed electroretinogram features consistent with preferential inner retinal dysfunction. Optical coherence tomography showed selective thinning of the retinal nerve fiber layer. Histology of the rat retina in cross-section found reduced cell density in the ganglion cell layer, but no change in other cellular layers. Staining of flat-mounted mouse retinae with a ganglion cell specific marker (RBPMS) confirmed ganglion cell loss. The circumlimbal suture is a simple, minimally invasive and cost-effective way to induce ocular hypertension that leads to ganglion cell injury in both rats and mice.

Chronic ocular hypertension is induced by applying a circumlimbal suture in rats and mice, leading to functional and structural deterioration of the retinal ganglion cells consistent with glaucoma.

Un modello del Glaucoma indotto da sutura Circumlimbal nei ratti e nei topi

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known ...

A Rabbit Model of Aqueous-Deficient Dry Eye Disease Induced by Concanavalin A Injection into the Lacrimal Glands: Application to Drug Efficacy Studies

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for quality of life and health care costs. The lack of informative animal models that recapitulate its key features impedes the search for new therapeutic agents for DED. Available DED animal models have limited reproducibility and efficacy. A model is presented here in which DED is induced by injecting the mitogen concanavalin A (Con A) into the orbital lacrimal glands of rabbits. Innovative aspects of this model are the use of ultrasound (US) guidance to ensure optimal and reproducible injection of Con A into the inferior lacrimal gland; injection of Con A into all orbital lacrimal glands that limits compensatory production of tears; and use of periodic repeat injections of Con A that prolong the state of DED at will. DED and its response to test agents are monitored with a panel of parameters that assess tear production, the stability of the tear film, and the status of the corneal and conjunctival mucosa. They include tear osmolarity, tear break-up time, Schirmer&#39;s tear test, rose bengal staining, and tear lactoferrin levels. The induction of DED and the monitoring of its parameters are described in detail. This model is simple, robust, reproducible, and informative. This animal model is suitable for the study of tear physiology and of the pathophysiology of DED as well as for the assessment of the efficacy and safety of candidate agents for the treatment of DED.

This article describes the development of a method to induce acute or chronic dry eye disease in rabbits by injecting concanavalin A to all portions of the orbital lacrimal gland system. This method, superior to those already reported, generates a reproducible, stable model of dry eye suitable for the study of pharmacological agents.

Un modello di coniglio di malattia dell'occhio secco acquosa-decisa indotta da Concanavalin Un'iniezione nelle ghiandole lacrimali: applicazione agli studi sull'efficacia dei farmaci

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for ...