Questo lavoro è stato parzialmente supportato dal NIH RO1-EY019951 (AAF), Illinois Società per la Prevenzione della Cecità (JCC, AAF), i fondi non vincolati al Dipartimento di Oftalmologia di ricerca per prevenire la cecità (RPB), NY e RPB Medical Student Fellowship di Grant (CCM).

1. Preparazione della retina <ol><li> Enucleare l&#39;occhio mouse. Con una sola mano, aprire le palpebre in modo che l&#39;occhio è visibile. Con l&#39;altra mano, utilizzando una pinza curva (curva rivolta verso l&#39;alto), applicare una pressione su aspetti superiore e inferiore dell&#39;orbita fino sporge globo. Chiudere delicatamente forcipe nel lato posteriore dell&#39;occhio e sollevare in un movimento continuo di enucleare l&#39;occhio. </li><li> Fissare gli occhi con il 10% formalina tamponata neutra per almeno 24 ore. </li><li> Luogo occhio in soluzione PBS in una piccola scatola di Petri. </li><li> Sezionare retina attentamente al microscopio, facendo attenzione a non indurre grandi lacrime. Fai un primo taglio nella cornea con le forbici dissezione. Poi, mentre si tiene l&#39;area di taglio con pinze diritte, usare le forbici per tagliare lungo il bordo della cornea e la sclera rimozione della cornea. Utilizzando una pinza sottile curve e rette, togliere la sclera e coroide lontano dalla retina verso il nervo ottico. Questo passaggio richiede pazienza comeliberando rapidamente la retina può portare a rotture. Rimuovere la lente e di qualsiasi diaframma in eccesso e detriti dalla retina 16. </li></ol> 2. L&#39;acqua lava <ol><li> Posto retina in un piatto da 24 pozzetti e coprire con acqua (circa 500 pl, filtrata con un filtro almeno 45 micron). </li><li> Cambiare l&#39;acqua ogni 30 minuti-1 ora, almeno 4-5 volte. </li><li> Lasciare una notte in acqua con un leggero scuotimento a temperatura ambiente. </li></ol> Nota: Le fasi di lavaggio sono molto importanti in quanto aiutano separare gli strati della retina neurale dai vasi sanguigni. 3. Tripsina Digest <ol><li> Rimuovere l&#39;acqua ed retina incubare in una soluzione di 3% tripsina (Difco 1:250) in 0,1 M tampone Tris (pH 7,8) a 37 ° C con agitazione delicata per nessuna. Tripsina digest è completata quando il tessuto comincia a mostrare segni di disintegrazione (circa 1,5 ore). Nota: evitare di rapido scuotimentocome questo può danneggiare il sistema vascolare. </li><li> Con attenzione, pipetta fuori tripsina in un separato ben utilizzando un microscopio per evitare di toccare la retina. Questo eccesso tripsina può essere utilizzato in seguito, quando si manipolano i vasi sotto il microscopio, al passo 4.3. </li></ol> 4. Separare il sistema vascolare <ol><li> Aggiungere acqua filtrata alla retina. Quindi, tentare di rimuovere la membrana limitante interna con una pinza, usando le forbici se necessario. Agitare con lieve agitazione per 5 minuti. Attentamente, Pipetta fuori l&#39;acqua sotto il microscopio per evitare di danneggiare la retina. </li><li> Ripetere lavaggi con acqua (per 5 minuti ciascuno) per aiutare a liberare la rete di vasi dal tessuto retinico aderente. Lavaggi con acqua vengono completati quando vi è poca o nessuna detriti rimanendo in acqua dopo il lavaggio. </li><li> Manipolazioni attente al microscopio di dissezione sono ora necessari per liberare ulteriormente la rete di vasi. Qui di seguito sono diverse tecniche utili, che possono essere usati in sequenza o individually per ottenere il risultato desiderato in base alle preferenze tecnica: <ol class="lalpha"><li> Usando una pipetta 200 ml, pipettare con cura l&#39;acqua su e giù accanto alle navi, che soffia acqua a vasi, causando agitazione. </li><li> Utilizzando 200 microlitri pipetta, Pipetta la tripsina su e giù per le pareti cappotto della pipetta. Poi, Pipettare attentamente l&#39;intera rete nave su e giù una volta per aiutare a rompere il tessuto non vascolare. Nota: centraggio della punta della pipetta al nervo ottico, piuttosto che i bordi della rete vascolare può impedire al sistema vascolare di attaccarsi alla punta. </li><li> Sollevare delicatamente vascolare con una pinza sottile di acqua per rimuovere i componenti non-vascolari. </li><li> Se i passaggi precedenti non funzionano, aggiungere tripsina alla retina per aiutare abbattere il tessuto e incubare a 37 ° C per alcuni minuti. Quindi, rimuovere tripsina e ripetere i passaggi 4,1-4,3. </li></ol></li></ol> &lt;strong&gt; Nota: Assicurati di rivestire tutti gli strumenti che possono entrare in contatto con le navi in tripsina (ad esempio la punta di pipetta, pinze). Ciò contribuirà a evitare l&#39;adesione del sistema vascolare per l&#39;attrezzatura. 5. Visualizzare la vascolarizzazione <ol><li> Mettere una goccia d&#39;acqua su un vetrino pulito. Usando una pipetta 200 microlitri o pinze, trasferire i vasi digeriti in acqua. Assicurarsi che il piano di montaggio vascolare viene spiegato. La tensione superficiale dell&#39;acqua aiuta a spiegare la rete vascolare ora diafana. Se il tessuto non si dipana, aggiungere ulteriore acqua e scuotere delicatamente il vetrino per facilitare dispiegarsi. </li><li> Rimuovere l&#39;acqua in eccesso molto lentamente da pipettaggio o utilizzando un tovagliolo di carta. </li><li> Lasciare vetrini si asciughino completamente. </li><li> Colorare i vetrini tramite protocollo appropriato sia con PAS / ematossilina o H &amp; E macchia. Nota: PAS / ematossilina è utile per identificare parete del vaso e nuclei cellulari; H &amp; E è utile per demonstration di RBC pure. </li><li> Disidratarsi e montare (Permount mezzo di montaggio). </li></ol>

<ol>
	<li>Kuwabara, T., Cogan, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+retinal+vascular+patterns.+I.+Normal+architecture.">Studies of retinal vascular patterns. I. Normal architecture.</a> Arch. Ophthalmol. 64, 904-911 (1960).</li><li>Kuwabara, T., Cogan, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+vascular+patterns.+VII.+Acellular+change.">Retinal vascular patterns. VII. Acellular change.</a> Invest. Ophthalmol. 4, 1049-1064 (1965).</li><li>Bell, W. R., Green, W. R., Goldberg, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathologic+and+trypsin+digestion+studies+of+the+retina+in+incontinentia+pigmenti.">Histopathologic and trypsin digestion studies of the retina in incontinentia pigmenti.</a> Ophthalmology. 115, 893-897 (2008).</li><li>Danis, R. P., Wallow, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HRP/trypsin+technique+for+studies+of+the+retinal+vasculature.">HRP/trypsin technique for studies of the retinal vasculature.</a> Invest. Ophthalmol. Vis. Sci. 27, 434-437 (1986).</li><li>Kim, S. H., Chu, Y. K., Kwon, O. W., McCune, S. A., Davidorf, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+studies+of+the+retina+in+a+new+diabetic+model;+SHR/N:Mcc-cp+rat.">Morphologic studies of the retina in a new diabetic model; SHR/N:Mcc-cp rat.</a> Yonsei Med. J. 39, 453-462 (1998).</li><li>Cuthbertson, R. A., Mandel, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+mouse+retina.+Endothelial+cell-pericyte+ratio+and+capillary+distribution.">Anatomy of the mouse retina. Endothelial cell-pericyte ratio and capillary distribution.</a> Invest. Ophthalmol. Vis. Sci. 27, 1659-1664 (1986).</li><li>Laver, N. M., Robison, W. G., Pfeffer, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+procedures+for+isolating+intact+retinal+vascular+beds+from+diabetic+humans+and+animal+models.">Novel procedures for isolating intact retinal vascular beds from diabetic humans and animal models.</a> Invest. Ophthalmol. Vis. Sci. 34, 2097-2104 (1993).</li><li>Midena, E., 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=Studies+on+the+retina+of+the+diabetic+db/db+mouse.+I.+Endothelial+cell-pericyte+ratio.">Studies on the retina of the diabetic db/db mouse. I. Endothelial cell-pericyte ratio.</a> Ophthalmic Res. 21, 106-111 (1989).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+30.">Chapter 30.</a> Diabetic Retinopathy. , (2005).</li><li>Rosenblatt, B., Benson, W. E., Yannoff, M., Duker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+6.19+Diabetic+Retinopathy.">Chapter 6.19 Diabetic Retinopathy.</a> Ophthalmology. , (2008).</li><li>Friedenwald, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+some+problems+of+retinal+vascular+disease.">A new approach to some problems of retinal vascular disease.</a> Am. J. Ophthalmol. 32, 487-498 (1949).</li><li>Podesta, F., 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=Bax+is+increased+in+the+retina+of+diabetic+subjects+and+is+associated+with+pericyte+apoptosis+in+vivo+and+in+vitro.">Bax is increased in the retina of diabetic subjects and is associated with pericyte apoptosis in vivo and in vitro.</a> Am. J. Pathol. 156 (10), 1025-1032 (2000).</li><li>Dagher, Z., 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=Studies+of+rat+and+human+retinas+predict+a+role+for+the+polyol+pathway+in+human+diabetic+retinopathy.">Studies of rat and human retinas predict a role for the polyol pathway in human diabetic retinopathy.</a> Diabetes. 53, 2404-2411 (2004).</li><li>Puro, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinovascular+physiology+and+pathophysiology:+new+experimental+approach/new+insights.">Retinovascular physiology and pathophysiology: new experimental approach/new insights.</a> Prog. Retin. Eye Res. 31, 258-270 (2012).</li><li>Roy, S., Nasser, S., Yee, M., Graves, D. T., Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+long-term+siRNA+strategy+regulates+fibronectin+overexpression+and+improves+vascular+lesions+in+retinas+of+diabetic+rats.">A long-term siRNA strategy regulates fibronectin overexpression and improves vascular lesions in retinas of diabetic rats.</a> Mol. Vis. 17, 3166-3174 (2011).</li><li>Li, Q., 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=Diabetic+eNOS-knockout+mice+develop+accelerated+retinopathy.">Diabetic eNOS-knockout mice develop accelerated retinopathy.</a> Invest. Ophthalmol. Vis. Sci. 51, 5240-5246 (2010).</li><li>Connor, K. M., 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=Quantification+of+oxygen-induced+retinopathy+in+the+mouse:+a+model+of+vessel+loss,+vessel+regrowth+and+pathological+angiogenesis.">Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis.</a> Nature Protocols. 4, 1565-1573 (2009).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione.

Tripsina digest è un metodo standard per valutare la vascolarizzazione della retina. Purtroppo è tecnicamente impegnativo e può causare elevato tasso di perdita di campione, se non eseguite correttamente. Inoltre, la procedura è particolarmente difficile nei topi, che può limitare l&#39;applicazione di questa tecnica nei comunemente utilizzati modelli animali genetici di malattie oculari. Questo documento fornisce una guida su come eseguire la procedura efficace e coerente con gli occhi del mouse. <p class="jov...

Visualizzare la vascolarizzazione della retina è un approccio estremamente importante per sezionare i meccanismi delle varie malattie oculari come la retinopatia diabetica. Essa permette di valutare le prime anomalie vascolari, tra cui microaneurismi, degenerazione dei capillari, e perdita pericyte 8,9. Ad oggi, sono state sviluppate diverse tecniche per analizzare la vascolarizzazione della retina. Perfusione di vari coloranti è stato utilizzato per evidenziare i vasi, ma tutti hanno condiviso limitazioni simili. Iniezione evidenzia raramente l&#39;intero sistema vascolare della retina a meno dato ad alta pressione, che rischia di rompersi e danneggiare le navi 1. Immunostaining dell&#39;endotelio vascolare con fluoroforo marcato G isolectin B4 (Alexa Fluor 594 coniugato; I21413, Invitrogen; diluizione 1:100) e monta piatti della retina può evidenziare l&#39;architettura complessiva dei vasi, ma senza la visualizzazione dettagliata di capillari, membrane basali e periciti . È stato osservato dallaFriedenwald che le navi possono essere evidenziate dalla colorazione piatta-monti della retina con acido periodico di Schiff (PAS) o ematossilina ed eosina (H &amp; E) colorazione 10. Tuttavia, la colorazione era non-specifico ai vasi ed evidenziato il tessuto non vascolare pure, rendendo difficile distinguere i vasi. Nel 1960, Cogan e Kuwabara sviluppato la tecnica tripsina digest che ha reso più facile visualizzare la vascolarizzazione della retina da digerire componenti non vascolari della retina 1. Da quel momento, la tripsina digest è diventato il metodo gold standard nell&#39;analisi della vascolarizzazione della retina 2-5. Tuttavia, è importante notare che altre tecniche alternative per isolare il sistema vascolare sono stati descritti. L&#39;uso di lisi osmotica è stato usato per isolare il sistema vascolare e consentire studi biochimici del tessuto 11,12, ma la procedura non è stata utilizzata come metodo primario per studio anatomico. Il metodo di stampa dei tessuti è statautilizzato per isolare ampi segmenti microcircolo e permette la capacità di studiare l&#39;architettura elettrotoniche del sistema vascolare 13. In teoria, questa tecnica potrebbe essere utilizzata anche per studiare i cambiamenti anatomici, come la qualità dei vasi è elevata. Tuttavia, è solo in grado di isolare segmenti della intera rete vascolare. Sebbene questi metodi non possono sostituire tripsina digest, è importante notare che essi hanno diversi vantaggi e le carenze e sono complementari a questo riguardo. Il metodo digest tripsina è tecnicamente impegnativo e difficile da eseguire in modo coerente 6,7. Inoltre, è stato notato che la tripsina digest è particolarmente difficile in un modello di topo, in particolare quando la si vuole preservare l&#39;architettura complessiva vascolare della retina 6,7. Sfide includono (1) sopra-digestione della retina che causa la perdita sia del sistema vascolare così come il tessuto non vascolare, (2) sotto-digestione, richiedendodissezione estesa meccanico che porterebbe al danneggiamento del letto recipiente, (3) scarsa separazione del tessuto non vascolare dalla vascolatura conseguente colorazione aspecifica. Diversi manoscritti hanno evidenziato queste sfide, ma nessuno ha fornito un protocollo dettagliato e coerente per superarli 14,15. Questo manoscritto introdurrà una metodologia step-by-step dettaglio la tecnica per eseguire la tripsina digest su topo e ratto retine con consigli specifici su come gestire i passaggi particolarmente difficili. Una panoramica schematica è mostrata in Figura 1.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>10% neutral buffered formalin</td>
			<td>Fischer Scientific</td>
			<td>SF100-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin (1:250)</td>
			<td>Amresco</td>
			<td>0458-50G</td>
			<td>Make 3% trypsin in 0.1 M Tris</td>
		</tr>
		<tr>
			<td>TRIZMA base</td>
			<td>Sigma</td>
			<td>T1503-1KG</td>
			<td>Create 0.1 M Tris Buffer (pH 7.8 @ 37 &deg;C)</td>
		</tr>
		<tr>
			<td>TRIZMA</td>
			<td>Sigma</td>
			<td>T3253-500G</td>
		</tr>
		<tr>
			<td>Steritop sterile vacuum bottle</td>
			<td>Millipore</td>
			<td>SCGPS05RE</td>
			<td>Create filtered water</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Any microscope that allows good visualization of the retina is adequate. (10x is sufficient with working distance of 24 mm)</td>
		</tr>
		<tr>
			<td>Straight scissors</td>
			<td>Fine Science Tools</td>
			<td>15024-10</td>
			<td>Cutting edge: 8 mm; Tip diameter: 0.2 mm; 10 cm</td>
		</tr>
		<tr>
			<td>Straight Forceps (Inox)</td>
			<td>Dumont #5</td>
			<td>11252-20</td>
			<td>Biologie tip; 0.05 x 0.02 mm; 11 cm</td>
		</tr>
		<tr>
			<td>Curved Forcepts (Dumostar)</td>
			<td>Dumont #7</td>
			<td>11297-10</td>
			<td>Biologie tip; 0.07 x 0.04 mm; 11.5 cm</td>
		</tr>
		<tr>
			<td>Dubnoff Metabolic Shaker Incubator</td>
			<td>Precision Scientific</td>
			<td>BDG59442</td>
			<td>Shaker optional</td>
		</tr>
		<tr>
			<td>24-well dish</td>
			<td>Falcon</td>
			<td>353047</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>VWR</td>
			<td>82027-132</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

trypsin digest protocol to analyze the retinal vasculature of a mouse model

Tripsina digest è il metodo gold standard per analizzare la retina vascolare 1-5. Permette la visualizzazione di tutta la rete di vasi complessi tridimensionali sanguigni e capillari retinici creando una bidimensionale TV a monte dei canali vascolari interconnessi dopo digestione dei componenti non vascolari della retina. Questo permette di studiare le varie alterazioni patologiche vascolari, come microaneurismi, degenerazione capillare e abnorme endoteliali ai rapporti di periciti. Tuttavia, il metodo è tecnicamente impegnativo, soprattutto nei topi, che sono diventati il modello animale più ampiamente disponibile per studiare la retina a causa della facilità di manipolazioni genetiche 6,7. Nell&#39;occhio topo, è particolarmente difficile da rimuovere completamente i componenti non vascolari mantenendo l&#39;architettura complessiva dei vasi sanguigni della retina. Ad oggi, vi è una carenza di letteratura che descrive la tecnica tripsina digest in dettaglio in il mouse. Questo manoscritto fornisce una metodologia dettagliata passo-passo della tripsina digest nel topo retina, fornendo anche suggerimenti sulla risoluzione dei passaggi difficili.

Il prodotto finale di una procedura di successo è una TV a monte di tutta la rete del mouse vascolarizzazione della retina, con l&#39;architettura mantenuto, colorate sia con PAS / ematossilina o H &amp; E, come mostrato nelle figure 2-4. Chiara differenziazione delle cellule endoteliali e periciti può essere visto come mostrato in Figura 3. Nella retina, i nuclei delle cellule endoteliali sono ovali o allungata e si trova interamente all&#39;interno della parete del vaso. Nuclei peri...

Watch this Scientific Journal Video about Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model at JoVE.com

Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Tripsina digest è uno dei metodi più comunemente utilizzati per analizzare vascolarizzazione retinica. Questo manoscritto descrive il metodo in dettaglio, comprese le alterazioni fondamentali per ottimizzare la tecnica e rimuovere il tessuto non vascolare preservando l&#39;architettura generale dei vasi.

Tripsina Digest protocollo per analizzare la vascolarizzazione della retina di un modello murino

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex ...

trypsin-digest-protocol-to-analyze-retinal-vasculature-mouse

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Neuroscience

18.2K Views.  Northwestern University Feinberg School of Medicine. Tripsina digest è uno dei metodi più comunemente utilizzati per analizzare vascolarizzazione retinica. Questo manoscritto descrive il metodo in dettaglio, comprese le alterazioni fondamentali per ottimizzare la tecnica e rimuovere il tessuto non vascolare preservando l'architettura generale dei vasi.

Video: Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Bloccare Registrazioni patch da neuroni della retina del mouse in un Dark-adattato Preparazione Slice

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

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Neuroscienze

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

Dissezione di un occhio mouse per un monte intero di dell&#39;epitelio pigmentato retinico

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

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 ...

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

Le analisi morfometriche delle sezioni della retina

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain proper, that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis. The tissue harvested is suitable for biochemical and physiological analysis, and the MAV has been shown to be sensitive to damage produced by amphetamine and hyperthermia 1,2. As well, the major and minor cerebral vasculatures harvested in MAV are of potentially high interest when investigating concussive types of head trauma. The MAV dissected in this presentation consists of the pial and some of the arachnoid membrane (less dura) of the meninges and the major and minor cerebral surface vasculature. The choroid plexus dissected is the structure that resides in the lateral ventricles as described by Oldfield and McKinley3,4,5,6. The methods used for harvesting these two tissues also facilitate the harvesting of regional cortical tissue devoid of meninges and larger cerebral surface vasculature, and is compatible with harvesting other brain tissues such as striatum, hypothalamus, hippocampus, etc. The dissection of the two tissues takes from 5 to 10 min total. The gene expression levels for the dissected MAV and choroid plexus, as shown and described in this presentation can be found at GSE23093 (MAV) and GSE29733 (choroid plexus) at the NCBI GEO repository. This data has been, and is being, used to help further understand the functioning of the MAV and choroid plexus and how neurotoxic events such as severe hyperthermia and AMPH adversely affect their function.

This video presentation shows a method of harvesting the two most important highly vascular structures that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis.

Una descrizione visiva della dissezione del sistema vascolare di superficie cerebrale e Meningi associate, e del plesso coroide da cervello di ratto

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

Implementazione morsetto dinamico con conduttanze sinaptiche e artificiale nel topo cellule gangliari della retina

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

Video Imaging e mappe spazio-temporale per analizzare la motilità gastrointestinale nei topi

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

Protocol for Isolating the Mouse Circle of Willis

The cerebral arterial circle (circulus arteriosus cerebri) or circle of Willis (CoW) is a circulatory anastomosis surrounding the optic chiasma and hypothalamus that supplies blood to the brain and surrounding structures. It has been implicated in several cerebrovascular disorders, including cerebral amyloid angiopathy (CAA)-associated vasculopathies, intracranial atherosclerosis and intracranial aneurysms. Studies of the molecular mechanisms underlying these diseases for the identification of novel drug targets for their prevention require animal models. Some of these models may be transgenic, whereas others will involve isolation of the cerebro-vasculature, including the CoW.The method described here is suitable for CoW isolation in any mouse lineage and has considerable potential for screening (expression of genes, protein production, posttranslational protein modifications, secretome analysis, etc.) studies on the large vessels of the mouse cerebro-vasculature. It can also be used for ex vivo studies, by adapting the organ bath system developed for isolated mouse olfactory arteries.

We describe here a reproducible protocol for isolating the mouse circle of Willis.

Protocollo per isolare il circolo di Willis mouse

The cerebral arterial circle (circulus arteriosus cerebri) or circle of Willis (CoW) is a circulatory anastomosis surrounding the optic chiasma and ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

Preparazione delle sezioni ricrello della retina

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a detailed method pointing out critical steps is provided with recommendations for optimization to achieve consistently successful occlusion rates across different genetically modified mouse strains. The RVO mouse model consists primarily of the intravenous administration of a photosensitizer dye followed by laser photocoagulation using a retinal imaging microscope attached to an ophthalmic guided laser. Three variables were identified as determinants of occlusion consistency. By adjusting the wait time after rose bengal administration and balancing the baseline and experimental laser output, the variability across experiments can be limited and a higher success rate of occlusions achieved. This method can be used to study retinal diseases that are characterized by retinal edema and hypoxic-ischemic injury. Additionally, as this model induces vascular injury, it can also be applied to study the neurovasculature, neuronal death, and inflammation.

Here, we describe an optimized protocol for retinal vein occlusion using rose bengal and a laser-guided retinal imaging microscope system with recommendations to maximize its reproducibility in genetically modified strains.

Ottimizzazione del modello murino di occlusione venosa retinica per limitare la variabilità

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a ...