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

Alle Protokolle wurden in Übereinstimmung mit den Richtlinien der Institutional Animal Care und Use Committee gegebenenfalls durchgeführt. 1. Vorbereitung <ol><li> Bereiten Sie die Stabilisierungsmedien mit 75% Dulbecco modifiziertem Eagle Medium (DMEM), 25% Hanks &#39;Balanced Salt Solution (HBSS). Gut mischen, aliquotieren und bei -20 ° C bis zur Verwendung. </li><li> Bereiten Sie die Phosphat-gepufferte Saline (PBS) und durch Autoklavieren sterilisieren. Bewahren Sie die Lösung bei Raumtemperatur. Erwärmen Sie den PBS auf 37 ° C vor dem Experiment (5 ml pro Vertiefung). </li><li> Herstellung von 10%, 20% und 30% Saccharose in PBS. Filter alle Lösungen einzeln über getrennte 0,22 um Filter für die Sterilisation. Speichern Sie die Lösungen bei 4 ° C. </li><li> Bereiten Sie die Histamin-Stammlösung in PBS auf eine Konzentration von 90 mM. Sterilisieren der Lösung durch Filtration über ein 0,22-um-Filter. Aliquot und die Lösung bei -80 ° C zu speichern. Vermeiden Sie mehrere Gefrier-und-Auftau-Zyklen. </li><li> Machen Sie frisch 4% Paraformaldehyd (PFA) in PBS vor dem Experiment. Legen Sie sie auf Eis. Vorsicht: Halten Sie PFA in einem Abzug und tragen Sie geeignete Schutzausrüstung. </li><li> Auftauen Stabilisierungsmedium (20 ml pro Retina). Hinzufügen, Penicillin-Streptomycin in einer Endkonzentration von 100 U / ml. Warmen Teil des Mediums (15 ml pro Retina) bis 37 ° C im Inkubator vor dem Experiment. Lassen Sie den Rest des Mediums auf dem Eis. </li><li> Legen Sie die HBSS (10 ml pro Retina) auf Eis. </li></ol> 2. Retinal Organotypischen Kultur <ol><li> Übertragen Sie die Proben auf eine Gewebekultur Kapuze. Öffnen Sie die Augenmuschel von rund um das Objektiv zu schneiden. Mit einer Bürste, entfernen Sie den Glaskörper ohne auf der Netzhaut zu ziehen. Vorsichtig lösen mit einem Pinsel auf die Netzhaut von der Schnittkante der Papille zu belichten. Schwere der Sehnerv mit einer Rasierklinge und die Netzhaut lösen. </li><li> Übertragen Sie die Netzhaut in eine Petrischale und sorgfältig auf die Netzhaut einmal mit kaltem HBSS spülen. Die Probe wird in HBSS auf ice. nach Bedarf Präparieren wie viele Netzhäuten aus Schritt 2.1 und dieser Schritt wird wiederholt. </li><li> Schneiden Sie die Netzhaut in zwei Hälften symmetrisch mit einem Rasiermesser. Sanft übertragen Proben auf eine 6-Well-Platte mit 3 ml Stabilisierung Medien pro Vertiefung. Erlauben Äquilibrierung für 30 min bei 37 ° C in einem Inkubator mit der Atmosphäre , die 5% CO 2. </li><li> Bereiten Sie die folgenden Reagenzien während der Inkubation bei Schritt 2.3: Verdünnen Sie die Histamin-Stammlösung auf die gewünschte Konzentration (450 &amp; mgr; M) in warmem Medium. Auflösen LXA4 (gespeichert unter Argon) in das Medium bis zu einer Endkonzentration von 250 ng / ml. HINWEIS: Wenn die Wirkung von LXA 4 zu messen, die eine Hälfte von einer einzigen Netzhaut für Histamin allein verwendet wird, während die andere Hälfte für Histamin mit LXA 4 verwendet wird. </li><li> Saugen Sie das Stabilisierungsmedium sorgfältig und fügen Sie das fertige Medium zu jeder Vertiefung (3 ml pro Vertiefung). Inkubieren der Proben bei 37 ° C für 1 h in einem Inkubator mit der Atmosphäre , die 5% CO 2. </li><li> Spülen Sie dieProben einmal in warmen, sterilen PBS. </li></ol> 3. Bereiten Sie die Proben für Immunostaining <ol><li> Absaugen PBS von den Platten. In 4% PFA (3 ml pro Vertiefung) und Inkubation für 15 min bei Raumtemperatur. </li><li> Schnell die PFA absaugen. Spülen Sie die Proben einmal mit PBS. Übertragen Sie die Proben auf 10% Sucrose (5 ml pro Vertiefung) und Inkubation für 2 bis 4 Stunden bei 4 ° C. </li><li> Übertragen Sie die Proben auf ein 30% Sucrose (5 ml pro Vertiefung) und Inkubation über Nacht bei 4 ° C. Mischen Sie einen Teil des kommerziellen Einfriermedium mit zwei Teilen von 20% Saccharose das Arbeits Einfriermedium zu machen. Lassen Sie es bei 4 ° C über Nacht oder länger, bis die Luftblasen verschwinden. </li><li> Übertragen Sie die Proben auf das Arbeits Einfriermedium und ermöglichen Äquilibrierung während 5 min bei Raumtemperatur. Schneiden Sie jede Netzhaut in Rechtecke (ca. 3 mm bis 5 mm). </li><li> übertragen Sie sorgfältig die Proben in die Arbeits Einfrieren Medien in einem zylindrischen Behälter enthalten sind (etwa 1 cm Radius x 2 cm Höhe) devISED aus Aluminiumfolie. Stellen Sie sicher, dass die Netzhaut Scheiben vertikal sind (orientiert einen Querschnitt der Netzhaut zu schaffen, auf sectioning) und frieren sie in flüssigem Stickstoff. Speichern Sie die Blöcke bei -80 ° C bis zur Verwendung. </li><li> Abschnitt jeder Block auf einem Kryostaten in 14 &amp; mgr; m dicke Scheiben schneiden und montieren Sie die Abschnitte auf Glasplatten. Lagern Sie die Folien bei -80 ° C bis zur Verwendung. </li></ol> 4. Immunostaining <ol><li> Waschen der Schnitte mit 5% Sucrose Phosphatpuffer (SPB, 5% Saccharose in PBS, pH 7,4) einmal. Blockieren die nicht-spezifischen Bindungsstellen durch Inkubation der Abschnitte in Blockierungspuffer (5% SPB mit 10% normalem Ziegenserum und 0,5% Triton X-100) 1 Stunde bei Raumtemperatur. </li><li> Inkubieren Sie die Abschnitte (auf Folien) mit dem entsprechenden primären Antikörper (GFAP oder MAP2, verdünnt auf 1: 500 in Blocking-Puffer) für 1 Stunde. Absaugen die Lösung. Waschen dreimal in 5% SPB. Für die endogene IgG-Färbung, diesen Schritt überspringen. </li><li> Inkubieren Sie die Abschnitte mit dem entsprechenden SECON1 h: dary Antikörper (800 in Blockierungspuffer bei 1 Cyanine3 / FITC konjugiertem Esel-anti-Maus / anti-Kaninchen-IgG, verdünnt). </li><li> Waschen Sie die Abschnitte gründlich mit 5% SPB dreimal. Bereiten Sie die Proben für die Mikroskopie von ihnen Montagemedium, das DAPI in Montage und Abdecken mit Deckgläsern. </li><li> Mit einem Laser konfokalen Mikroskop, die Aufnahmen über ein 20fach - Objektiv unter identischen Parametern wie Laserintensität, Gain - Wert, Verweilzeit usw. in Gruppen. Festlegen der Anregungs- / Emissionswellenlängen für den blauen Kanal (zu detektieren DAPI) als 408/447 nm, für den roten Kanal (zu detektieren Cyanine3) als 561/785 nm und für den grünen Kanal (zu erkennen FITC) als 488/525 nm . </li></ol> 5. Bildanalyse <ol><li> Quantifizieren Prozentsatz von undichten Blutgefäße nach den folgenden Kriterien 12: Geben Sie nur die Blutgefäße mit endothelialen Zellkerne innerhalb der Ebene des Abschnitts in der Zählung; ein Blutgefäß undicht prüfen, ob es ag zeigtradient der Immunfärbung des Behälters umgibt. </li><li> Um das Expressionsniveau bestimmen, wählen Sie Bereiche von Interesse und messen Sie den mittleren Grauwert unter Verwendung der Bildanalyse-Software. </li><li> Um die Breite des Prozesses zu messen, einen Bereich des Abschnitts zu wählen, wo die Prozesse unterscheiden (die äußere Kernschicht, ONL, zum Beispiel). Dann wählen Sie ein optisches Feld, wo solche Prozesse sichtbar und kontinuierlich sind. Stellen Sie den Maßstab entsprechend der mikroskopischen Einstellung, eine Linie über den Prozess ziehen und die Messung durchzuführen. </li><li> Um die Kontinuität des Prozesses, wählen Sie Bereiche von Interesse zu analysieren, wenden Sie den Filter genannt Varianz, die die Kanten im Bild verbessert, indem jedes Pixel mit seiner Nachbarschaft Varianz zu ersetzen, passen sich automatisch den Kontrast und die Helligkeit, die Linien zu zählen und die Zählung durch die Normalisierung Bereich. </li><li> Berechnen statistische Bedeutungen unter Verwendung eines two-tailed t - Test nach Student. * P &lt;0,05; ** P &lt;0,01; *** P &lt;0,001. Plot das Ergebnis als Mittelwert ± 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 -...

Eine wachsende Zahl von Hinweisen unterstützt die Existenz von Blut Retina - Schranke (BRB) 1-5 und seine Ähnlichkeit mit dem Blut - Hirn - Schranke (BBB) ​​6,7. Kompromiss der BBB wurde ursächlich oder als diagnostischer Marker für chronische neurodegenerative Erkrankungen, wie Alzheimer-Krankheit (AD) 8,9 und akuter Erkrankungen wie Delirium 10 fest verbunden. Mechanistische Einblicke in diese Pathologien und Entdeckungen für potentielle Arzneimittelziele werden durch die begrenzte Zugänglichkeit und Netzwerk Kompliziertheit des Gehirns im Allgemeinen behindert. Alternativen , wie beispielsweise in vivo - Bildgebung 11, Gehirn organotypische Kultur 12, primären Zellkulturen 13,14 und Co-Kultursysteme 15 erzeugt worden sind . Allerdings sind die meisten dieser Modelle erfordern spezielle Instrumente, lange Versuchszeiten oder mehrere Marker-Zellen zu identifizieren. Funktionelle und strukturelle Ähnlichkeiten zwischen BBB und BRB sowie eine Korrelation zwischen der dysfunctions der beiden wurden 16-19 argumentiert. Zusätzlich leichteren Zugang, gut definierte Zelltypen und eine Schichtstruktur haben, die gut charakterisierten Netzhaut als ein Fenster zum Gehirn erlaubt. Die strukturellen und funktionellen Identität der BBB und BRB bleiben im Detail verglichen werden. Jedoch retinale Pathologien, insbesondere die BRB Verletzung, wurden ebenfalls mit dem Fortschreiten von verschiedenen Krankheiten eng in Verbindung gebracht worden, einschließlich Diabetes 18-19 und AD 21,22. Somit ist es von Interesse, eine BRB Dysfunktion System zu etablieren, nicht nur den Mechanismus zu beschreiben, sondern auch potentielle Arzneimittel zu screenen. In diesem Bericht wird ein Protokoll ermöglicht BRB Dysfunktion eine einfache akute retinale Kultur unter Verwendung entwickelt und präsentiert. Erhöht BBB Permeabilität und AD-ähnliche pathologische Veränderungen wurden in einem Gehirn organotypischen Kultur etabliert mit Histamin inkubiert, einem pro-inflammatorischen Mediators 12. Daher wird in dem dargestellten System war Histamin applied zur ex - vivo - Retinal Kultur BRB Dysfunktion zu induzieren. Retinas von mehreren Arten, wie Mus musculus und Bos Taurus, getestet wurden. Aufgrund ihrer kommerziellen Verfügbarkeit und Ähnlichkeit mit menschlichem Gewebe, frisch wurden Schweine Augäpfel verwendet, um die Daten hier berichtet zu liefern. Nach Inkubation mit Histamin und / oder andere Medikamente wurden die Retinas zur Auswertung verarbeitet durch Immunfärbung für mehrere Proteine ​​12, wie Immunoglobulin G (IgG), eine der Hauptkomponenten des Blutes; glial fibrillary acidic protein (GFAP), ein bekannter Marker für Gliazellen Aktivierung; und Mikrotubuli-assoziierten Protein 2 (MAP2), ein Neuron-spezifischen Zytoskelettprotein essentiell für Mikrotubuluszusammenbau. Darüber hinaus ermöglicht die geschichtete Struktur der Netzhaut, eine detaillierte Analyse der Prozesse der Müller-Zellen und Ganglionzellen, wie beispielsweise Veränderungen in ihrer Breite und Kontinuität. So einige zusätzliche Parameter zur Verfügung, die Folgen der zu bewertenBRB Verletzung in einem frühen Stadium und auch die Umkehrung Auswirkungen möglicher Behandlungen zu bewerten. In diesem Protokoll werden mögliche Umkehrung Wirkungen gescreent Drogen aus drei Perspektiven bewertet: das Austreten von Blutgefäßen (BVs), die Aktivierung von Gliazellen und beschädigungs Reaktion von neuronalen Zellen. Mehrere Quantifizierungsverfahren werden verwendet, zum Beispiel die Expressionsniveaus von einem Verbesserungsfilter dargestellt durch Intensität der Immunfärbung Breitenmessung eines Prozesses und die Kontinuität der neuronalen Prozesse gezeigt. Zum besseren Verständnis der Methode veranschaulichen und zu helfen , Ergebnisse zu interpretieren, Lipoxin A4 (LXA 4) eine Verbindung synthetisiert endogen als Reaktion auf entzündliche Verletzungen und mildernde endotheliale Dysfunktion 23, wurde zu Demonstrationszwecken gewählt.

<table border="0" cellpadding="0" cellspacing="0" width="984">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">DMEM</td>
			<td style="width:269px;">Life Technologies&#160;</td>
			<td style="width:119px;">11965-092</td>
			<td style="width:305px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">HBSS</td>
			<td style="width:269px;">Life Technologies&#160;</td>
			<td style="width:119px;">14170-112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Sucrose</td>
			<td style="width:269px;">J.T.Baker</td>
			<td style="width:119px;">4072-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Histamine&#160;</td>
			<td style="width:269px;">Sigma</td>
			<td style="width:119px;">H7125-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Penicillin-Streptomycin&#160;</td>
			<td style="width:269px;">Invitrogen</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:291px;">PFA</td>
			<td style="width:269px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Freezing Media&#160;</td>
			<td style="width:269px;">Triangle Biomedical Sciences</td>
			<td style="width:119px;">TFM-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Normal Goat Serum&#160;</td>
			<td style="width:269px;">Rockland</td>
			<td style="width:119px;">D104-00-0050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Triton X-100</td>
			<td style="width:269px;">Sigma</td>
			<td style="width:119px;">T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">GFAP Antibody</td>
			<td style="width:269px;">Millipore</td>
			<td style="width:119px;">AB5804</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">MAP2 Antibody</td>
			<td style="width:269px;">EMD Millipore</td>
			<td style="width:119px;">MAB3418</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">FITC conjugated Donkey anti-rabbit IgG</td>
			<td style="width:269px;">Jackson ImmunoResearch Laboratories, Inc.</td>
			<td style="width:119px;">711-095-152</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Cy3 conjugated Donkey anti-mouse IgG</td>
			<td style="width:269px;">Jackson ImmunoResearch Laboratories, Inc.</td>
			<td style="width:119px;">715-165-150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">mounting medium containing DAPI</td>
			<td style="width:269px;">Vector Laboratories, Inc.</td>
			<td style="width:119px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Laser Confocal Microscope</td>
			<td style="width:269px;">Nikon</td>
			<td style="width:119px;">Eclipse Ti microscope</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">ImageJ</td>
			<td style="width:269px;">National Institutes of Health</td>
			<td style="width:119px;">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.

Wir stellen eine kostengünstige, zeitsparende und einfach zu bedienendes System, mögliche Behandlungen zu bewerten, die gegen die BRB Verletzung durch Histamin induzierte schützen könnte. IgG wird in den Gefäßen in Kontrollnetzhaut (Abbildung 1A) beschränkt, sondern tritt aus der Blutgefäße auf Histamin - Exposition (Abbildung 1B), was bestätigt , dass das Modell erfolgreich etabliert. <p class="jove_content" fo:keep-together.within-page="1...

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

Eine kostengünstige, einfach zu bedienende und leistungsfähige System etabliert mögliche Behandlungen zu bewerten, die Blut Retina-Schranke Verletzung durch Histamin induzierte verbessern könnte. Blutgefäße Leckage, Müller-Zell-Aktivierung und die Kontinuität der neuronalen Prozesse werden genutzt, um die Schadensantwort und deren Umkehrung mit einem potenziellen Medikament, Lipoxin A4 zu beurteilen.

Eine akute Retinal Modell zur Bewertung von Blut Retinal Barrier Breach und potenzielle Medikamente für die Behandlung

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 Aufrufe.  Rowan University.  Eine kostengünstige, einfach zu bedienende und leistungsfähige System etabliert mögliche Behandlungen zu bewerten, die Blut Retina-Schranke Verletzung durch Histamin induzierte verbessern könnte. Blutgefäße Leckage, Müller-Zell-Aktivierung und die Kontinuität der neuronalen Prozesse werden genutzt, um die Schadensantwort und deren Umkehrung mit einem potenziellen Medikament, Lipoxin A4 zu beurteilen. 

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

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

Visualisierung und Analyse des Blutflusses und der Sauerstoffverbrauch in hepatischen Mikrozirkulation: Anwendung auf eine akute Hepatitis-Modell

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

Forschung

Medizin

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Sauerstoff-Glukose Deprivation und Reoxygenierung als<em&gt; In-vitro-</em&gt; Ischämie-Reperfusion Injury Modell zum Studium der Blut-Hirnschranke Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks performed by the RPE are phagocytosis and processing of outer photoreceptor segments through lysosome-derived organelles. These degradation products, stored and referred to as lipofuscin granules, are composed partially of bisretinoids, which have broad fluorescence absorption and emission spectra that can be detected clinically as fundus autofluorescence with confocal scanning laser ophthalmoscopy (cSLO). Lipofuscin accumulation is associated with increasing age, but is also found in various patterns in both acquired and inherited degenerative diseases of the retina. Thus, studying its pattern of accumulation and correlating such patterns with changes in the overlying sensory retina are essential to understanding the pathophysiology and progression of retinal disease. Here, we describe a technique employed by our lab and others that uses cSLO in order to quantify the level of RPE lipofuscin in both healthy and diseased eyes.

The retinal pigment epithelium (RPE) supports the sensory retina through recycling visual cycle byproducts, which accumulate as lipofuscin. These products are autofluorescent and can be qualitatively imaged in vivo. Here, we describe a method to quantitatively image RPE lipofuscin using confocal scanning laser ophthalmoscopy.

Quantitative Fundus-Autofluoreszenz für die Beurteilung von Netzhauterkrankungen

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

Eine Schritt für Schritt-Protokoll für Subretinale Chirurgie in Kaninchen

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Verbesserte Methode für die Einrichtung einer In-vitro- Blut - Hirn-Schranke Modell auf porcinen Endothelzellen Gehirnzellen basiert

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity is disturbed in gastrointestinal, cardiovascular and metabolic diseases, which may result in easier access of biologically active compounds, such as gut bacterial metabolites, to the bloodstream. Thus, the permeability of the GBB may be a marker of both intestinal and extraintestinal diseases. Furthermore, the increased penetration of bacterial metabolites may affect the functioning of the entire organism.
Commonly used methods for studying the GBB permeability are performed ex vivo. The accuracy of those methods is limited, because the functioning of the GBB depends on intestinal blood flow. On the other hand, commonly used in vivo methods may be biased by liver and kidney performance, as those methods are based on evaluation of urine or/and peripheral blood concentrations of exogenous markers. Here, we present a direct measurement of GBB permeability in rats using an in vivo method based on portal blood sampling, which preserves intestinal blood flow and is virtually not affected by the liver and kidney function. 
Polyurethane catheters are inserted into the portal vein and inferior vena cava just above the hepatic veins confluence. Blood is sampled at baseline and after administration of a selected marker into a desired part of the gastrointestinal tract. Here, we present several applications of the method including (1) evaluation of the colon permeability to TMA, a gut bacterial metabolite, (2) evaluation of liver clearance of TMA, and (3) evaluation of a gut-portal blood-liver-peripheral blood pathway of gut bacteria-derived short-chain fatty acids. Furthermore, the protocol may also be used for tracking intestinal absorption and liver metabolism of drugs or for measurements of portal blood pressure.

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The access of nutrients, microbiota metabolites and medicines to the circulation is controlled by the gut-blood barrier (GBB). We describe a direct method for measuring the GBB permeability in vivo, which, in contrast to commonly used indirect methods, is virtually not affected by liver and kidney functions.

Eine In Vivo -Methode zur Bewertung der Darm-Blut-Schranke und LEBERSTOFFWECHSEL Mikrobiota Produkte

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

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

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

Sauerstoffinduzierte Retinopathie-Modell für ischämische Netzhauterkrankungen bei Nagetieren

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

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to differentiate into all types of retinal cells, even mini-retinal tissues, hold huge promises for patients with these diseases and many opportunities in disease modeling and drug screening. However, the induction process from hPSCs to retinal cells is complicated and time-consuming. Here, we describe an optimized retinal induction protocol to generate retinal tissues with high reproducibility and efficiency, suitable for various human pluripotent stem cells. This protocol is performed without the addition of retinoic acid, which benefits the enrichment of cone photoreceptors. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction. With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate downstream applications, such as disease modeling and cell therapy.

Here we describe an optimized retinal organoid induction system, which is suitable for various human pluripotent stem cell lines to generate retinal tissues with high reproducibility and efficiency.

Retinale Organoid-Induktionssystem zur Ableitung von 3D-Netzhautgewebe aus humanen pluripotenten Stammzellen

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

Retinale Pigmentepitheltransplantation in einem nicht-menschlichen Primatenmodell für degenerative Netzhauterkrankungen

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...