Name: an acute retinal model for evaluating blood retinal barrier breach and potential drugs for treatment
Uploaded: 2016-09-13T15:00:00.000+00:00
Duration: 9 min 33 s
Description: Watch this Scientific Journal Video about An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment at JoVE.com

Bringhurst Meats (Berlin, NJ) is acknowledged for their genuine help in providing the swine eyeballs.

Tutti i protocolli sono stati effettuati nel rispetto delle norme del Comitato di cura e l&#39;uso di animali istituzionale, se del caso. 1. Preparazione <ol><li> Preparare il supporto di stabilizzazione con il 75% di Dulbecco Modified Eagle Medium (DMEM), il 25% Hanks &#39;soluzione salina bilanciata (HBSS). Mescolare bene, aliquotare e conservare a -20 ° C fino al momento dell&#39;uso. </li><li> Preparare il tampone fosfato (PBS) e sterilizzare in autoclave. Conservare la soluzione a temperatura ambiente. Riscaldare la PBS a 37 ° C prima dell&#39;esperimento (5 ml per pozzetto). </li><li> Preparare 10%, 20% e 30% di saccarosio in PBS. Filtrare tutte le soluzioni individualmente oltre separati 0,22 micron filtri per la sterilizzazione. Salvare le soluzioni a 4 ° C. </li><li> Preparare la soluzione di istamina magazzino in PBS ad una concentrazione di 90 mm. Sterilizzare la soluzione per filtrazione su filtro 0,22 micron. Aliquotare e salvare la soluzione a -80 ° C. Evitare ripetuti cicli di congelamento e scongelamento. </li><li> Rendere fresca 4% paraformaldeide (PFA) in PBS prima dell&#39;esperimento. Posizionarlo sul ghiaccio. Attenzione: tenere PFA in una cappa aspirante e indossare equipaggiamento protettivo. </li><li> Scongelare il supporto di stabilizzazione (20 ml per retina). Aggiungere penicillina-streptomicina ad una concentrazione finale di 100 U / ml. parte calda del mezzo (15 ml per retina) a 37 ° C in incubatore prima dell&#39;esperimento. Lasciare il resto del mezzo su ghiaccio. </li><li> Posizionare il HBSS (10 ml per retina) sul ghiaccio. </li></ol> 2. retina Cultura Organotipica <ol><li> Trasferire i campioni ad una cappa coltura tissutale. Aprire l&#39;oculare tagliando intorno alla lente. Utilizzando un pennello, rimuovere delicatamente il vitreo senza tirare sulla retina. staccare delicatamente la retina dal bordo di taglio con una spazzola per esporre il disco ottico. Grave il nervo ottico con un rasoio e rilasciare la retina. </li><li> Trasferire la retina in una capsula di Petri e risciacquare con cura la retina una volta con HBSS freddo. Trasferire il campione in HBSS su ICE. Sezionare il maggior numero di retine fuori, se necessario ripetendo passo 2.1 e questo passo. </li><li> Tagliare la retina a metà simmetricamente con un rasoio. Delicatamente trasferire i campioni di un 6-bene con 3 ml di mezzi di stabilizzazione per pozzetto. Consentire equilibrazione per 30 min a 37 ° C in un incubatore con l&#39;atmosfera contenente il 5% di CO 2. </li><li> Preparare i seguenti reagenti durante l&#39;incubazione a Passo 2.3: Diluire la soluzione di istamina magazzino alla concentrazione desiderata (450 micron) in media caldo. Sciogliere LXA4 (immagazzinato sotto argon) in mezzo ad una concentrazione finale di 250 ng / ml. NOTA: Quando si misura l&#39;effetto di LXA4, una metà da una singola retina viene utilizzato solo per l&#39;istamina, mentre l&#39;altra metà è utilizzato per l&#39;istamina con LXA4. </li><li> Aspirare il mezzo di stabilizzazione con attenzione e aggiungere il mezzo preparato in ogni pozzetto (3 ml per pozzetto). Incubare i campioni a 37 ° C per 1 ora in un incubatore con l&#39;atmosfera contenente il 5% di CO 2. </li><li> Sciacquare ilcampioni di volta in caldo, PBS sterile. </li></ol> 3. Preparare i campioni per Immunocolorazione <ol><li> Aspirare PBS dalle piastre. Aggiungere 4% PFA (3 ml per pozzetto) e incubare per 15 min a temperatura ambiente. </li><li> aspirare rapidamente il PFA. Lavare i campioni una volta con PBS. Trasferire i campioni al 10% di saccarosio (5 ml per pozzetto) e incubare per 2 a 4 ore a 4 ° C. </li><li> Trasferire i campioni ad un saccarosio 30% (5 ml per pozzetto) e incubare una notte a 4 ° C. Mescolare una parte del mezzo di congelamento commerciale con due parti di 20% di saccarosio per rendere il mezzo congelamento lavorare. Lasciare a 4 ° C per una notte o più a lungo fino a quando l&#39;aria bolle scompaiono. </li><li> Trasferire i campioni al mezzo di congelamento di lavoro e raggiungere l&#39;equilibrio per 5 min a temperatura ambiente. Tagliare ogni retina in rettangoli (circa 3 mm di 5 mm). </li><li> Trasferire accuratamente i campioni nei mezzi congelamento lavorare contenuta in un contenitore cilindrico (circa 1 cm Altezza Raggio x 2 cm) devzato da un foglio di alluminio. Assicurarsi che le fette retiniche verticale (orientata per fornire una sezione trasversale della retina upon sezionamento) e congelare in azoto liquido. Salvare i blocchi a -80 ° C fino al momento dell&#39;uso. </li><li> Sezione ogni blocco su un criostato in 14 micron fette spesse e montare le sezioni su vetrini. Conservare i vetrini a -80 ° C fino al momento dell&#39;uso. </li></ol> 4. immunocolorazione <ol><li> Lavare le sezioni con tampone fosfato 5% di saccarosio (SPB, 5% di saccarosio in PBS, pH 7,4) una volta. Bloccare le non specifici siti di legame incubando le sezioni in tampone di bloccaggio (5% SPB con 10% siero di capra normale e 0,5% Triton X-100) per 1 ora a temperatura ambiente. </li><li> Incubare sezioni (su diapositive) con l&#39;anticorpo primario appropriata (GFAP o MAP2, diluito 1: 500 in tampone di bloccaggio) per 1 ora. Aspirare la soluzione. Lavare tre volte in 5% SPB. Per la colorazione del endogena IgG, saltare questo passaggio. </li><li> Incubare le sezioni con la seconda corrispondenteanticorpi Dary (Cyanine3 / FITC coniugato asino anti-topo / anti-IgG di coniglio, diluito 1: 800 in tampone di bloccaggio) per 1 ora. </li><li> Lavare le sezioni accuratamente con 5% SPB tre volte. Preparare i campioni per la microscopia da loro montaggio in mezzo di montaggio contenente DAPI e copertura con lamelle. </li><li> Utilizzando un laser microscopio confocale, catturare immagini attraverso una lente 20X sotto parametri identici come l&#39;intensità del laser, valore di guadagno, tempo di sosta, ecc tra i gruppi. Impostare le lunghezze d&#39;onda di eccitazione / emissione per il canale blu (per rilevare DAPI) come 408/447 nm, per il canale rosso (per rilevare Cyanine3) come 561/785 nm e per il canale verde (per rilevare FITC) come 488/525 nm . </li></ol> Analisi 5. Immagine <ol><li> Quantificare la percentuale dei vasi sanguigni che perde in base ai seguenti criteri 12: Include solo vasi sanguigni con nuclei delle cellule endoteliali all&#39;interno del piano di sezione nel conteggio; prendere in considerazione una che perde vaso sanguigno se mostra aggardiente di immunostaining che circonda la nave. </li><li> Per determinare il livello di espressione, selezionare regioni di interesse e misurare il valore di grigio medio utilizzando il software di analisi di immagine. </li><li> Per misurare la larghezza del processo, scegliere un&#39;area della sezione in cui i processi sono distinti (strato esterno nucleare ONL, per esempio). Quindi scegliere un campo ottico in cui tali processi sono visibili e continuo. Impostare la barra di scala in base all&#39;impostazione microscopico, tracciare una linea attraverso il processo ed eseguire la misurazione. </li><li> Per analizzare la continuità del processo, selezionare regioni di interesse, applicare il filtro chiamato varianza che esalta le bordi dell&#39;immagine sostituendo ogni pixel con la sua varianza zona, regolare automaticamente il contrasto e la luminosità, contare le righe e normalizzare il conteggio dal la zona. </li><li> Calcola significatività statistica con t-test di Student a due code. *, P &lt;0,05; **, P &lt;0,01; ***, P &lt;0.001. Pmolto il risultato come media ± SEM. </li></ol>

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The authors have nothing to disclose.

In this report, we present a powerful ex vivo acute retinal model of BRB dysfunction using the swine retina. This model system does not require special instruments and can be easily adapted under most laboratory settings. However, to obtain a successful result, several steps require close attention. After obtaining the eyeballs from the source, they must be kept at 4 &#176;C or on ice and processed as soon as possible. When the effect of a treatment is being analyzed, two halves of the same retina must be used -...

Un crescente corpo di prove sostiene l&#39;esistenza di barriera retinica sangue (BRB) 1-5 e la sua somiglianza con la barriera emato-encefalica (BBB) ​​6,7. Compromesso della BBB è stata strettamente legata causalmente o come marcatore diagnostico per le malattie neurodegenerative croniche come il morbo di Alzheimer (AD) 8,9 e acuti condizioni come il delirio 10. intuizioni meccanicistici in queste patologie e scoperte per i potenziali bersagli farmacologici sono generalmente ostacolati dalla limitata accessibilità e network complessità del cervello. Alternative come l&#39;imaging in vivo 11, cervello organotipica cultura 12, colture cellulari primarie 13,14 e sistemi di co-coltura di 15 sono stati generati. Tuttavia, la maggior parte di questi modelli richiede strumenti speciali, lunghi periodi sperimentali o più marcatori per identificare le cellule. somiglianze funzionali e strutturali tra BBB e BRB nonché una correlazione tra la dysfunctions dei due sono stati sostenuto 16-19. Inoltre, un più facile accesso, tipi cellulari ben definiti e una struttura stratificata hanno consentito la retina ben caratterizzato come una finestra al cervello. Le identità strutturali e funzionali della BBB e BRB ancora essere confrontato nel dettaglio. Tuttavia, patologie retiniche, soprattutto la breccia BRB, sono stati strettamente associati con la progressione di varie malattie, compreso il diabete 18-19 e AD 21,22. Pertanto, è di interesse per stabilire un sistema disfunzione BRB non solo per delineare il meccanismo ma anche per schermo potenziali farmaci. In questo rapporto, un protocollo che consente BRB disfunzione utilizzando una semplice cultura della retina acuta è sviluppato e presentato. Aumento della permeabilità BBB e cambiamenti patologici AD-simili sono stati stabiliti in una cultura organotipica cervello incubate con l&#39;istamina, un mediatore pro-infiammatorio 12. Pertanto, nel sistema presentato, istamina era applied alla cultura della retina ex vivo per indurre disfunzioni BRB. Retine da diverse specie, come il Mus musculus e Bos taurus, sono stati testati. Grazie alla loro disponibilità commerciale e somiglianza con tessuti umani, bulbi oculari suina fresca sono stati utilizzati per fornire i dati qui riportati. Dopo incubazione con istamina e / o altri farmaci, le retine sono state preparate per la valutazione mediante immunocolorazione per diverse proteine ​​12, come immunoglobuline G (IgG), uno dei principali componenti del sangue; proteina silicea fibrillare gliale (GFAP), un indicatore ben noto per l&#39;attivazione gliale; e proteine ​​associate ai microtubuli 2 (MAP2), una specifica dei neuroni del citoscheletro proteina essenziale per l&#39;assemblaggio dei microtubuli. Inoltre, la struttura a strati della retina consente un&#39;analisi dettagliata dei processi di cellule Müller e cellule gangliari, quali i cambiamenti nella loro larghezza e continuità. Così diversi parametri aggiuntivi sono disponibili per valutare le conseguenze dellaBRB violazione in una fase iniziale e per valutare gli effetti di inversione di potenziali trattamenti pure. In questo protocollo, i potenziali effetti di inversione dei farmaci schermati vengono valutati da tre prospettive: la fuoriuscita di vasi sanguigni (BVS), l&#39;attivazione delle cellule gliali e il danno-risposta delle cellule neuronali. Diversi metodi di quantificazione sono utilizzati, per esempio, il livello di espressione dimostra intensità della immunocolorazione, misura della larghezza di un processo e la continuità dei processi neuronali mostrati da un filtro accessorio. Per meglio illustrare il metodo e per interpretare i risultati, Lipoxin A4 (LXA4), un composto endogeno sintetizzato in risposta al danno infiammatorio e attenuando la disfunzione endoteliale 23, è stata scelta a scopo dimostrativo.

<table><tbody><tr><td>DMEM</td><td>Life Technologies&amp;nbsp;</td><td>11965-092</td><td></td></tr><tr><td>HBSS</td><td>Life Technologies&amp;nbsp;</td><td>14170-112</td><td></td></tr><tr><td>Sucrose</td><td>J.T.Baker</td><td>4072-05</td><td></td></tr><tr><td>Histamine&amp;nbsp;</td><td>Sigma</td><td>H7125-1G</td><td></td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>PFA</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Freezing Media&amp;nbsp;</td><td>Triangle Biomedical Sciences</td><td>TFM-5</td><td></td></tr><tr><td>Normal Goat Serum&amp;nbsp;</td><td>Rockland</td><td>D104-00-0050</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>GFAP Antibody</td><td>Millipore</td><td>AB5804</td><td></td></tr><tr><td>MAP2 Antibody</td><td>EMD Millipore</td><td>MAB3418</td><td></td></tr><tr><td>FITC conjugated Donkey anti-rabbit IgG</td><td>Jackson ImmunoResearch Laboratories, Inc.</td><td>711-095-152</td><td></td></tr><tr><td>Cy3 conjugated Donkey anti-mouse IgG</td><td>Jackson ImmunoResearch Laboratories, Inc.</td><td>715-165-150</td><td></td></tr><tr><td>mounting medium containing DAPI</td><td>Vector Laboratories, Inc.</td><td>H-1200</td><td></td></tr><tr><td>Laser Confocal Microscope</td><td>Nikon</td><td>Eclipse Ti microscope</td><td></td></tr><tr><td>ImageJ</td><td>National Institutes of Health</td><td>1.45s</td><td></td></tr></tbody></table>

an acute retinal model for evaluating blood retinal barrier breach and potential drugs for treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

Vi presentiamo un basso costo, sistema di time-efficiente e facile da usare per valutare i potenziali trattamenti che potrebbero proteggere contro la violazione BRB indotta da istamina. IgG è vincolato all&#39;interno dei vasi in retina di controllo (Figura 1A), ma le perdite fuori dei vasi sanguigni su esposizione istamina (Figura 1B), a conferma che il modello è stata stabilita. LXA4 è st...

Watch this Scientific Journal Video about An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment at JoVE.com

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A basso costo, il sistema facile da usare e potente è stabilito per valutare i potenziali trattamenti che potrebbero migliorare barriera emato-retinica violazione indotta da istamina. perdita dei vasi sanguigni, l&#39;attivazione delle cellule Müller e la continuità dei processi neuronali sono utilizzati per valutare la risposta al danno e la sua inversione con un potenziale farmaco, Lipoxin A4.

Un modello acuta della retina per la valutazione sanguigni della retina barriera breccia e potenziali farmaci per il trattamento

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

an-acute-retinal-model-for-evaluating-blood-retinal-barrier-breach

Research

JoVE Journal

Medicine

7.1K Views.  Rowan University.  A basso costo, il sistema facile da usare e potente è stabilito per valutare i potenziali trattamenti che potrebbero migliorare barriera emato-retinica violazione indotta da istamina. perdita dei vasi sanguigni, l'attivazione delle cellule Müller e la continuità dei processi neuronali sono utilizzati per valutare la risposta al danno e la sua inversione con un potenziale farmaco, Lipoxin A4. 

Video: An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

Modelli sperimentali per lo Studio di Fisiologia dell&#39;epitelio pigmentato retinico e fisiopatologia

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

Ricerca

Neuroscienze

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

Modello Rat di emato-encefalica Turbativa Barriera per consentire mirato Therapeutics neurovascolare

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

Medicina

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

Utilizzando virus adeno-associati come strumento per studiare le barriere della retina nella malattia

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Generazione della degenerazione retinica per studiare le strategie di riparazione cellulare in Xenopus

Generating Retinal Injury Models in Xenopus Tadpoles

Retinal neurodegenerative diseases are the leading causes of blindness. Among numerous therapeutic strategies being explored, stimulating self-repair recently emerged as particularly appealing. A cellular source of interest for retinal repair is the M&#252;ller glial cell, which harbors stem cell potential and an extraordinary regenerative capacity in anamniotes. This potential is, however, very limited in mammals. Studying the molecular mechanisms underlying retinal regeneration in animal models with regenerative capabilities should provide insights into how to unlock the latent ability of mammalian M&#252;ller cells to regenerate the retina. This is a key step for the development of therapeutic strategies in regenerative medicine. To this aim, we developed several retinal injury paradigms in Xenopus: a mechanical retinal injury, a transgenic line allowing for nitroreductase-mediated photoreceptor conditional ablation, a retinitis pigmentosa model based on CRISPR/Cas9-mediated rhodopsin knockout, and a cytotoxic model driven by intraocular CoCl2 injections. Highlighting their advantages and disadvantages, we describe here this series of protocols that generate various degenerative conditions and allow the study of retinal regeneration in Xenopus.

Autore in primo piano: Esplorazione dei meccanismi di rigenerazione retinica nella rana Xenopus 

We have developed several protocols to induce retinal damage or retinal degeneration in Xenopus laevis tadpoles. These models offer the possibility of studying retinal regeneration mechanisms.

Generazione di modelli di lesioni retiniche nei girini di Xenopus

Retinal neurodegenerative diseases are the leading causes of blindness. Among numerous therapeutic strategies being explored, stimulating self-repair ...

Biologia dello sviluppo

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, ...

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To properly leverage this greater translatability, there is a need to devise reproducible methods of drawing quantitative data from these images. Optical coherence tomography (OCT) imaging can resolve retinal histology at micrometer resolution and reveal functional differences in vascular blood flow. Here, we delineate noninvasive vascular readouts that we use to characterize pathological damage post vascular insult in an optimized mouse model of retinal vein occlusion (RVO). These readouts include live imaging analysis of retinal morphology, disorganization of retinal inner layers (DRIL) measure of capillary ischemia, and fluorescein angiography measures of retinal edema and vascular density. These techniques correspond directly to those used to examine patients with retinal disease in the clinic. Standardizing these methods enables direct and reproducible comparison of animal models with clinical phenotypes of ophthalmic disease, increasing the translational power of vascular injury models.

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO).

In vivo Letture di lesioni vascolari nella retina del topo per promuovere la riproducibilità

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

Valutazione fisiopatologica retinica in un modello di ratto

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and subsequent vision loss. The inner blood-retinal barrier (iBRB) is mainly composed of retinal vascular endothelium with low permeability under physiological conditions. This feature of low permeability is tightly regulated and maintained by low rates of paracellular transport between adjacent retinal microvascular endothelial cells, as well as transcellular transport (transcytosis) through them. The assessment of retinal transcellular barrier permeability may provide fundamental insights into iBRB integrity in health and disease. In this study, we describe an endothelial cell (EC) transcytosis assay, as an in vitro model for evaluating iBRB permeability, using human retinal microvascular endothelial cells (HRMECs). This assay assesses the ability of HRMECs to transport transferrin and horseradish peroxidase (HRP) in receptor- and caveolae-mediated transcellular transport processes, respectively. Fully confluent HRMECs cultured on porous membrane were incubated with fluorescent-tagged transferrin (clathrin-dependent transcytosis) or HRP (caveolae-mediated transcytosis) to measure the levels of transferrin or HRP transferred to the bottom chamber, indicative of transcytosis levels across the EC monolayer. Wnt signaling, a known pathway regulating iBRB, was modulated to demonstrate the caveolae-mediated HRP-based transcytosis assay method. The EC transcytosis assay described here may provide a useful tool for investigating the molecular regulators of EC permeability and iBRB integrity in vascular pathologies and for screening drug delivery systems.

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

This protocol illustrates an in vitro endothelial cell transcytosis assay as a model to evaluate inner blood-retinal barrier permeability by measuring the ability of human retinal microvascular endothelial cells to transport horseradish peroxidase across cells in caveolae-mediated transcellular transport processes.

Saggio di transcitosi delle cellule endoteliali come modello in vitro per valutare la permeabilità della barriera emato-retinica interna

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and ...

Un modello acuta della retina per la valutazione sanguigni della retina barriera breccia e potenziali farmaci per il trattamento

Riepilogo

Esplora Altri Video

Capitoli in questo video

Modelli sperimentali per lo Studio di Fisiologia dell'epitelio pigmentato retinico e fisiopatologia

Modello Rat di emato-encefalica Turbativa Barriera per consentire mirato Therapeutics neurovascolare

Utilizzando virus adeno-associati come strumento per studiare le barriere della retina nella malattia

Generazione di modelli di lesioni retiniche nei girini di Xenopus

Generazione di modelli di lesioni retiniche nei girini di Xenopus

Generazione di modelli di lesioni retiniche nei girini di Xenopus

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

In vivo Letture di lesioni vascolari nella retina del topo per promuovere la riproducibilità

Valutazione fisiopatologica retinica in un modello di ratto

Saggio di transcitosi delle cellule endoteliali come modello in vitro per valutare la permeabilità della barriera emato-retinica interna