Name: isolation of retinal arterioles for ex vivo cell physiology studies
Uploaded: 2018-07-14T15:00:00.000+00:00
Duration: 12 min 42 s
Description: Watch this Scientific Journal Video about Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies at JoVE.com

Die Protokolle, die in diesem Dokument beschriebenen wurde unterstützt durch Zuschüsse aus folgenden Förderinstitutionen: BBSRC (BB/I026359/1), Kampf um Anblick (1429 und 1822), die Juvenile Diabetes Research Foundation (2-2003-525), Wellcome Trust (074648/Z/04) British Heart Foundation (PG/11/94/29169), HSC R & D Division (STL/4748/13) und MRC (MC_PC_15026).

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Dr. Joanna Kur ist jetzt ein Mitarbeiter von Medtronic (Doppelstädte, Minnesota, USA), ihre gegenwärtige Arbeit nicht in Konflikt mit dem hier vorgestellten.

Die oben beschriebenen Protokolle sollte erfordern Übung jedoch mit minimalen Fehlerbehebung erreicht werden können. An einem durchschnittlichen Tag würden wir erhalten 6-8 nutzbare Arteriolen aus der Isolation und 3-4 erfolgreiche Experimente erreichen. Wenn Probleme auftreten, jedoch gibt es einige Schritte, die zur Verbesserung der Erfolgsraten getroffen werden können. Gelegentlich haben wir gefunden, insbesondere bei Verwendung von jüngeren Ratten (< 8 Wochen), dass die Ausbeute an Arteriolen kann niedrig sein. Um dieses Problem zu umgehen, empfehlen wir, Zentrifugieren netzhautgewebe (10-30 s bei 500 X g) zwischen den einzelnen die Verreibung in Schritten 1.12-1.14 Schritte. Dies oft hilft, um den Ertrag von Schiffen zu verbessern, sondern erhöht auch die Menge der Zelle Ablagerungen innerhalb der Vorbereitung.
Wenn Schiffe für Druck Myographie Studien Metallspirale ist es wichtig, die Arteriolen sorgfältig für jede Seitenäste zu überprüfen, die nah an der Bifurkation wurde gespalten werden kann. Dies ist eine häufige Ursache für Leckage und Druckverlust während der Experimente. Die wichtigste Frage, die mit der [Ca2 +]ich Protokolle entstehen kann ist das Niveau der Farbstoff beladen. Armen Farbstoff Belastung führt zu niedrigen Signal-Rausch-Verhältnis bei Überlastung führt zu Störungen der normalen Ca2 + Homöostase. Um die Wahrscheinlichkeit, dass diese Aspekte zu reduzieren, kann eine kleine aliquoten von retinalen Homogenat entfernt werden, und isolierte Schiffe überprüft in regelmäßigen Abständen während des laden-Protokolls. Wenn Unternehmen Patch-Clamp Experimente, die Erfolgsquote der Gigaseal Bildung ist stark abhängig von wie gut hat der Basallamina verdaut wurde. Wann kann dieses Protokoll zunächst testen oder mit einer neuen Vielzahl von Enzymen die Konzentration/Dauer Enzym Verdauung anpassen erforderlich. Sehr aufmerksam die Trennung der Endothelzellen und glatten Muskelzellen Schichten sorgen für ausreichende Verdauung genug basale entfernen laminal zugreifen. Sorgfältige Überwachung ist auch notwendig, über Verdauung, zu vermeiden, die manifestieren kann, als das Schiff beginnt zu verengen. In diesem Fall sind die glatten Muskelzellen oft zu schwach für Patch-Clamp-Aufnahme. Bei Anwendung von Enzymen, ist es wichtig einzuschließende DNAse I Sicherstellung Entfernung der Stränge der DNA befreit Geschädigte Zellen bei der Isolierung. DNA-Fragmente sind klebrig und dazu führen, dass die Zange zur Einhaltung der arteriola während der Endphase des Reinigungsprozesses (Schritt 4,4), die oft zum Schiff Verlust. Reinigung von Schiffen ist technisch schwierig und erfolgt am besten bei hoher Vergrößerung (20 X) mit sanft geschwungenen Bewegungen der geschlossenen Zange von der feinsten möglich Kopfkreis. Zwischen fegt reinigen Sie die Zange mit Roll-Labor.
Wie früher war eine wesentliche Motivation hinter der Entwicklung der Protokolle in diesem Manuskript beschrieben, besser zu verstehen, warum Blutfluss während retinalen Gefäßerkrankungen gestört wird. Unsere Arbeit konzentriert sich auf Diabetische Auge Krankheit28,33. Arteriolen können von der Netzhaut der experimentellen Nager-Modelle von Diabetes, die mit den in Abschnitt 1 beschriebenen Methoden isoliert werden. Beim auf isolierte retinalen Arteriolen von diabetischen Tieren zu experimentieren, ist es wichtig zu versuchen, eng der hyperglykämischen Bedingungen, durch die Schiffe in Vivozu replizieren. Aus diesem Grund würden wir normalerweise D-Glukose-Spiegel im unsere Isolation und experimentelle Lösungen für 25 mM erhöhen. Verdickung der vaskulären Keller Membranen ist ein bekanntes Phänomen in der Netzhautgefäße bei Diabetes34,35. Wenn Tiere mit längeren Diabetes (> 1 Monaten Krankheitsdauer) verwenden, sind erhöhte Enzymkonzentrationen oder Verdauung Mal oft erforderlich, um die Anwendung der Patch-Clamp Aufnahmemethoden.
Eine wichtige Einschränkung der Verwendung von ex-Vivo isolierte retinale Arteriolen, retinale vaskuläre Physiologie zu studieren und Pathophysiologie ist der Verlust der umgebenden Netzhaut Neuropile. Zwar Entfernung der Netzhaut glialen und neuronalen Zellen einfachen Zugriff auf die Netzhaut vaskulären glatten Muskelzellen für Zelle Physiologie Studien ermöglicht, kann die Reaktion der Gefäße auf vasoaktive Mediatoren dramatisch in Abwesenheit und Anwesenheit der Netzhaut verändern. Gewebe. Die Aktionen der Adenosin-Tri-Phosphat (ATP), geben Sie beispielsweise, ein gutes Beispiel für diesen Punkt. In isolierten Ratte retinalen Arteriolen, Zugabe von ATP löst eine robuste Verengung der Gefäße36, während in der Gegenwart von einer intakten Neuropile, die Gefäße erweitern sich37. Deshalb, wo immer möglich, wir versuchen in der Regel überprüfen die wichtigsten Ergebnisse unserer isolierten arteriola Präparate mit ex Vivo Netzhaut ganz-Halterungen und in Vivo Messungen des Schiffs Durchmesser und Blut fließen16,37 . Hinweis haben vor kurzem neue Methoden für die Untersuchung kleiner Arteriolen und Kapillaren im gesamten PERFUNDIERTEN porcinen Netzhaut ex Vivo38,39entstanden. Solche Präparate werden voraussichtlich erheblich besser verstehen wie die Netzhaut Neuropile retinalen Arteriolen und Kapillare Ton regelt und wie Veränderungen der Netzhaut Hämodynamik modulieren neuronalen Aktivität in der Netzhaut.
Obwohl in diesem Artikel beschriebenen Verfahren auf die Verwendung von isolierten Ratte retinalen Arteriolen für Verständnis Arteriolen Glattmuskel Zellphysiologie ausgerichtet sind, entwickeln wir derzeit Protokolle, um auch das Studium der endothelial Zelle Funktion aktivieren Diese Schiffe. In Vorarbeiten ist es uns gelungen bei der Modifizierung der enzymatischen Verdauung der retinalen Arteriolen Segmente zu lebensfähigen Endothelzellen Röhren hervorbringen, die Ca2 + Imaging und Patchclamp Aufnahme Studien zugänglich sind. Kanülierung der isolierten Arteriolen an beiden Enden ermöglichen Intraluminal Lieferung von Medikamenten, könnte in Zukunft auch aktivieren Endothel-abhängige gefäßerweiternde Antworten in diesen Gefäßen untersucht werden.

Verstehen, wie der Blutfluss in der Netzhaut gesteuert wird ist ein wichtiges Ziel, da abnormale Blutfluss in der Pathogenese von einer Vielzahl von bedrohlichen Anblick verwickelt hat Netzhauterkrankungen1,2,3, 4. Die retinale Zirkulation, die inneren Netzhaut Neuronen und Gliazellen versorgt, hat eine Ende Arterie Anordnung, mit all dem Blut aus der retinalen Arterien und Arteriolen vorbei durch die Kapillaren der Netzhaut Venolen und schließlich Venen5. Durchblutung der Netzhaut wird durch den Ton der retinalen Arterien und Arteriolen sowie die kontraktilen Aktivität die perizyten befindet sich an den Wänden der retinalen Kapillaren und Post-Kapillaren Venolen6,7, geregelt. 8. die Kontrolle des retinalen Gefäßtonus ist komplex und wird durch eine Reihe von Eingaben aus dem Herz-Kreislauf-System und umliegende netzhautgewebe, Blutgase, zirkulierenden Moleküle und Hormone, einschließlich moduliert und vasoaktive Substanzen freigesetzt aus der Netzhaut vaskulären Endothel und Macroglia9,10,11. Der retinalen Arteriolen sind kleine arterielle Zweige der Netzhaut und bestehen aus einer einzigen Schicht von vaskulären glatten Muskelzellen und ein Innenfutter von längs angeordnet Endothelzellen12,13, 14. diese Schiffe bilden die Haupt-Website der Gefäßwiderstand innerhalb der retinalen Zirkulation und spielen daher eine wichtige Rolle in der lokalen Kontrolle des retinalen Blutflusses. Retinale Arteriolen regulieren Kapillare Durchblutung der Netzhaut durch Erweiterung oder Verengung der luminalen Durchmesser, vermittelt durch Veränderungen der vaskulären glatten Muskelzellen Kontraktilität10,15,16. Das Verständnis der molekularen Mechanismen durch die retinalen Arteriolen regulieren retinaler Perfusion daher Vorbereitungen, erfordert wo die Arteriolen glatten Muskelzellen zugänglich und studierte in Bedingungen so nahe wie möglich physiologische.
Ex-Vivo -Vorbereitungen des isolierten Netzhautgefäße bieten Zugriff auf die vaskulären glatten Muskelzellen Beibehaltung noch ihre Funktionalität und Vernetzung mit dem zugrunde liegenden Endothel. Die meisten Studien bisher mittels isolierter Schiffe konzentrierten sich auf großen Rindern oder Schweinen arteriellen Gefäßen (60-150 µm). Diese können in handelsübliche Draht- oder Myograph Drucksysteme ermöglichen pharmakologische Verhör von vaskulären glatten Muskelzellen Zelle kontraktilen Mechanismen17,18montiert werden. Solche Präparate haben nach unserer Kenntnis der Netzhaut vaskuläre Physiologie unter normalen Bedingungen wesentlich beigetragen. Einige Studien haben retinale Arteriolen isoliert von kleinen Labortieren wie ihre kleineren Durchmesser verwendet (~ 8-45 µm) verhindert den Einsatz in herkömmlichen Myographie Systemen19,20,21,22. Ein wichtiger Vorteil ist jedoch die breite Verfügbarkeit von gentechnisch veränderten, transgenen und Netzhaut Krankheitsmodelle von mit Schiffen von kleinen Labortieren. Auch sind kleine Versuchstiere zugänglicher für in Vivo Interventionsstudien.
Hier beschreiben wir einfache Protokolle zum isolieren und Metallspirale Ratte retinalen Arteriolen für Druck Myographie Experimente. Ca2 + Imaging und Elektrophysiologie Protokolle mittels diese Schiffe sind auch aufgeführt. Diese bieten weitere Einblicke in die Regulierung der vaskulären glatten Muskelzellen Kontraktilität und Durchblutung der Netzhaut.

<table><tbody><tr><td>Beakers</td><td>Fisherbrand</td><td>15409083</td><td>Or any equilavent product</td></tr><tr><td>Curved Scissors</td><td>Fisher Scientific</td><td>50-109-3542</td><td>Or any equilavent product</td></tr><tr><td>Disposable plastic pipette/ transfer</td><td>Sarstedt</td><td>86.1174</td><td>6mL is best but other sizes are acceptable; remove tip to widen aperture</td></tr><tr><td>Dissecting microscope</td><td>Brunel Microscopes LTD</td><td></td><td>Or any equilavent product</td></tr><tr><td>Forceps</td><td>World Precision Instruments</td><td>Dumont #5 14095</td><td>Any equivalent fine forceps</td></tr><tr><td>Pasteur pipette</td><td>Fisherbrand</td><td>11546963</td><td>Fire polished to reduce friction but not enough to narrow the tip</td></tr><tr><td>Petri dish</td><td>Sigma-Aldrich</td><td>P7741</td><td>Or any equilavent 10cm product</td></tr><tr><td>Pipette teat</td><td>Fisherbrand</td><td>12426180</td><td>Or any equilavent product</td></tr><tr><td>Purified water supply</td><td>Merek</td><td>Milli-Q Integral Water Purification System</td><td>Or any equilavent system</td></tr><tr><td>Round bottomed test tube</td><td>Fisherbrand</td><td>14-958-10 B</td><td>5 mL; any equilavent product</td></tr><tr><td>Seratted forceps</td><td>Fisher Scientific</td><td>17-467-230</td><td>Or any equilavent product</td></tr><tr><td>Single edge blades</td><td>Agar Scientific</td><td>T585</td><td>Or any equilavent product</td></tr><tr><td>Sylgard</td><td>Dow Corning</td><td>184</td><td>Or any pliable surface&amp;nbsp;</td></tr><tr><td>Testtube rack</td><td>Fisherbrand Derlin</td><td>10257963</td><td>Or any equilavent product</td></tr><tr><td>Pressure myography</td><td></td><td></td><td></td></tr><tr><td>3-axis mechanical manipulator</td><td>Scientifica</td><td>LBM-7&amp;nbsp;</td><td>x2 (or any equilavent product)</td></tr><tr><td>3-way taps</td><td>Cole-Parmer</td><td>UY-30600-02</td><td>Or any equilavent product</td></tr><tr><td>Air table</td><td>Technical Manufacturing Corporation&amp;nbsp;</td><td>Clean Bench</td><td>Or any equilavent product</td></tr><tr><td>Analysis software&amp;nbsp;</td><td>Image J</td><td>https://downloads.imagej.net/fiji/</td><td>Free imaging software</td></tr><tr><td>Analysis plugin</td><td>Myotraker&amp;nbsp;</td><td>https://doi.org/10.1371/journal.pone.0091791.s002</td><td>Custom plugin for imageJ Freely available for download</td></tr><tr><td>Cannulation pipette glass</td><td>World Percision instruments</td><td>TW150F-4</td><td>Or any equilavent product</td></tr><tr><td>Computer</td><td>Dell</td><td>Optiplex 7010</td><td>Or any equilavent product</td></tr><tr><td>Digital Thermometer</td><td>RS</td><td>206-3722</td><td>Or any equilavent product</td></tr><tr><td>Fluo-4-AM</td><td>Thermo Fisher Scientific</td><td>F14201</td><td>Make 1 mM stock in DMSO</td></tr><tr><td>Forceps</td><td>World Precision Instruments</td><td>Dumont #7 14097</td><td>Any equivalent fine forceps</td></tr><tr><td>Fura-2-AM</td><td>Thermo Fisher Scientific</td><td>F1221</td><td>Make 1 mM stock in DMSO</td></tr><tr><td>Glass bottomed recording chamber</td><td>Warner Instruments</td><td>64-0759</td><td>Or any equilavent product</td></tr><tr><td>Helper pipette glass</td><td>World Percision instruments</td><td>IB150F-3</td><td>Or any equilavent product</td></tr><tr><td>In-line heater</td><td>Custom made</td><td></td><td>Commerical equilavent SH-27B Solution In-Line Heater from Harvard Apparatus</td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclypse TE 300</td><td>Or any equilavent product</td></tr><tr><td>Manometer</td><td>Riester</td><td>LF1459</td><td>Or any equilavent product</td></tr><tr><td>Microelectrode puller</td><td>Sutter instruments</td><td>P97</td><td>Or any equilavent product</td></tr><tr><td>Microforge +&amp;nbsp; Olympus CX 31 microscope</td><td>Glassworks</td><td>Fine Point F-550</td><td>Or any equilavent product</td></tr><tr><td>Micromanipulator</td><td>Sutter instruments</td><td>MP-285</td><td>Or any equilavent product</td></tr><tr><td>Multi-channel delivery manifold&amp;nbsp;</td><td>Automate Scientific</td><td>Perfusion Pencil</td><td>Or any equilavent product</td></tr><tr><td>Pipette filler&amp;nbsp;</td><td>BD Plastipak</td><td>1mL</td><td>Syringe heated and pulled to internal diameter of pipette</td></tr><tr><td>Pipette holder</td><td>Molecular Devices</td><td>1-HL-U</td><td>Or any equilavent product</td></tr><tr><td>Suction pump</td><td>Interpet</td><td>AP1</td><td>Converted aeration pump by reversing bellows</td></tr><tr><td>Syringe</td><td>BD Plastipak</td><td>20mL Luer-lok</td><td>Or any equilavent product</td></tr><tr><td>Tungsten wire</td><td>Advent</td><td>W558818</td><td>75&amp;mu;m diameter</td></tr><tr><td>Tygon Tubing</td><td>VWR</td><td>miscellaneous sizes</td><td>Or any equilavent product</td></tr><tr><td>USB camera</td><td>&amp;nbsp;Logitech&amp;nbsp;</td><td>HD Pro Webcam C920</td><td>Or any equilavent product</td></tr><tr><td>Video capture software</td><td>Hypercam</td><td>version 2.28.01</td><td>Or any equilavent product</td></tr><tr><td>Water bath</td><td>Grant</td><td>Sub Aqua 12 Plus</td><td>Or any equilavent product</td></tr><tr><td>x20 lens</td><td>Nikon</td><td>20x/0.40 WD 3.8, &amp;infin;/0.17</td><td>Or any equilavent product</td></tr><tr><td>x4 lens</td><td>Nikon</td><td>4x/0.10, WD 30, &amp;infin;/-</td><td>Or any equilavent product</td></tr><tr><td>Y-connectors</td><td>World Percision Instruments</td><td>14012</td><td>Or any equilavent product</td></tr><tr><td>Patch clamping</td><td></td><td></td><td></td></tr><tr><td>3-way taps</td><td>Cole-Parmer</td><td>UY-30600-02</td><td>Or any equilavent product</td></tr><tr><td>3-axis mechanical manipulator</td><td>Scientifica</td><td>LBM-7&amp;nbsp;</td><td>Or any equilavent product</td></tr><tr><td>Air table</td><td>Technical Manufacturing Corporation&amp;nbsp;</td><td>Clean Bench</td><td>Or any equilavent product</td></tr><tr><td>Amphotericin B</td><td>Sigma-Aldrich</td><td>A2411</td><td>3 mg disolved daily in 50 &amp;mu;L of DMSO (sonicate to dissolve)</td></tr><tr><td>Amplifier</td><td>Molecular Devices</td><td>Axopatch 200a</td><td>Or any equilavent product</td></tr><tr><td>Analogue digital converter</td><td>Axon</td><td>Digidata 1440A</td><td>Or any equilavent product</td></tr><tr><td>Collagenase Type 1A</td><td>Sigma-Aldrich</td><td>C9891&amp;nbsp;</td><td>0.1mg/mL in LCH</td></tr><tr><td>Computer</td><td>Dell</td><td>Optiplex 7010</td><td>Or any equilavent product</td></tr><tr><td>Digital Thermometer</td><td>RS</td><td>206-3722</td><td>Or any equilavent product</td></tr><tr><td>DNAse I</td><td>Millipore</td><td>260913</td><td>Working stock 1 MU mL-1 (10 MU diluted in 10 mL LCH)&amp;nbsp;</td></tr><tr><td>Faraday cage</td><td>Custom made</td><td></td><td>Any equilavent commerical product</td></tr><tr><td>Fine forceps</td><td>Dumont</td><td>No. 7</td><td>Any superfine forcep</td></tr><tr><td>Glass bottomed recording chamber</td><td>Warner Instruments</td><td>64-0759</td><td>Or any equilavent product</td></tr><tr><td>Headstage</td><td>Molecular Devices</td><td>CV203BU</td><td>Or any equilavent product</td></tr><tr><td>In-line heater</td><td>Custom made</td><td></td><td>Commerical equilavent SH-27B Solution In-Line Heater from Harvard Apparatus</td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclypse TE 300</td><td>Or any equilavent product</td></tr><tr><td>Microelectrode puller</td><td>Sutter instruments</td><td>P97</td><td>Or any equilavent product</td></tr><tr><td>Microforge +&amp;nbsp; Olympus CX 31 microscope</td><td>Glassworks</td><td>Fine Point F-550</td><td>Or any equilavent product</td></tr><tr><td>Micromanipulator</td><td>Sutter instruments</td><td>MP-285</td><td>Or any equilavent product</td></tr><tr><td>Multi-channel delivery manifold&amp;nbsp;</td><td>Automate Scientific</td><td>Perfusion Pencil</td><td>Or any equilavent product</td></tr><tr><td>Patching software</td><td>Molecular Devices</td><td>Pclamp v10.2</td><td>Or any equilavent product</td></tr><tr><td>Pipette filler&amp;nbsp;</td><td>BD Plastipak</td><td>1mL</td><td>Syringe heated and pulled to internal diameter of pipette</td></tr><tr><td>Pipette holder</td><td>Molecular Devices</td><td>1-HL-U</td><td>Or any equilavent product</td></tr><tr><td>Protease Type XIV</td><td>Sigma-Aldrich</td><td>P5147&amp;nbsp;</td><td>0.01mg/mL in LCH</td></tr><tr><td>Single channel pipette glass</td><td>World Percision instruments</td><td>IB150F-3</td><td>Or any equilavent product</td></tr><tr><td>Suction pump</td><td>Interpet</td><td>AP1</td><td>Converted aeration pump by reversing bellows</td></tr><tr><td>Syringe</td><td>BD Plastipak</td><td>20mL Luer-lok</td><td>Or any equilavent product</td></tr><tr><td>Tungsten wire</td><td>Advent</td><td>W557418</td><td>50&amp;mu;m diameter</td></tr><tr><td>Tygon Tubing</td><td>VWR</td><td>miscellaneous sizes</td><td>Any equilavent product to fit</td></tr><tr><td>USB camera</td><td>&amp;nbsp;Logitech&amp;nbsp;</td><td>HD Pro Webcam C920</td><td>Or any equilavent product</td></tr><tr><td>Water bath</td><td>Grant</td><td>Sub Aqua 12 Plus</td><td>Or any equilavent product</td></tr><tr><td>Whole cell pipette glass</td><td>Warner Instruments</td><td>GC150TF-7.5</td><td>Or any equilavent product</td></tr><tr><td>x20 lens</td><td>Nikon</td><td>20x/0.40 WD 3.8, &amp;infin;/0.17</td><td>Or any equilavent product</td></tr><tr><td>x4 lens</td><td>Nikon</td><td>4x/0.10, WD 30, &amp;infin;/-</td><td>Or any equilavent product</td></tr><tr><td>x40 lens</td><td>Nikon</td><td>40x/0.55, WD 2.1, &amp;infin;/1.2</td><td>Or any equilavent product</td></tr><tr><td>Y-connectors</td><td>World Percision Instruments</td><td>14012</td><td>Or any equilavent product</td></tr><tr><td>Ca&lt;sup&gt;2+&lt;/sup&gt; imaging</td><td></td><td></td><td></td></tr><tr><td>3-way taps</td><td>Cole-Parmer</td><td>UY-30600-02</td><td>Or any equilavent product</td></tr><tr><td>3-axis mechanical manipulator</td><td>Scientifica</td><td>LBM-7&amp;nbsp;</td><td>Or any equilavent product</td></tr><tr><td>Acquisition software&amp;nbsp;</td><td>Cairn Research Ltd.</td><td>Acquisition Engine V1.1.5</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>Air table</td><td>Technical Manufacturing Corporation&amp;nbsp;</td><td>Clean Bench</td><td>Or any equilavent product</td></tr><tr><td>Analysis software for confocal imaging</td><td>Image J</td><td>https://downloads.imagej.net/fiji/</td><td>Free imaging software</td></tr><tr><td>Computer</td><td>Dell</td><td>Optiplex 7010</td><td>Or any equilavent product</td></tr><tr><td>Confocal microscope</td><td>Leica Geosystems</td><td>SP5&amp;nbsp;</td><td>Or any equilavent product; confocal</td></tr><tr><td>Digital Thermometer</td><td>RS</td><td>206-3722</td><td>Or any equilavent product</td></tr><tr><td>Fine forceps</td><td>Dumont</td><td>No. 7</td><td>Any superfine forcep</td></tr><tr><td>Glass bottomed recording chamber</td><td>Warner Instruments</td><td>64-0759</td><td>Or any equilavent product</td></tr><tr><td>In-line heater</td><td>Custom made</td><td></td><td>Commerical equilavent SH-27B Solution In-Line Heater from Harvard Apparatus</td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclipse TE2000</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>Monochromator&amp;nbsp;</td><td>Cairn Research Ltd.</td><td>Optoscan</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>Multi-channel delivery manifold&amp;nbsp;</td><td>Automate Scientific</td><td>Perfusion Pencil</td><td>Or any equilavent product</td></tr><tr><td>Pipette filler&amp;nbsp;</td><td>BD Plastipak</td><td>1mL</td><td>Syringe heated and pulled to internal diameter of pipette</td></tr><tr><td>Software for confocal microscope</td><td>Leica Geosystems</td><td>LAS-AF version 3.3.</td><td>Or any equilavent product; confocal</td></tr><tr><td>Suction pump</td><td>Interpet</td><td>AP1</td><td>Converted aeration pump by reversing bellows</td></tr><tr><td>Syringe</td><td>BD Plastipak</td><td>20mL Luer-lok</td><td>Or any equilavent product</td></tr><tr><td>Tungsten wire</td><td>Advent</td><td>W557418</td><td>50&amp;mu;m diameter</td></tr><tr><td>Tygon Tubing</td><td>VWR</td><td>miscellaneous sizes</td><td>Any equilavent product to fit</td></tr><tr><td>Water bath</td><td>Grant</td><td>Sub Aqua 12 Plus</td><td>Or any equilavent product</td></tr><tr><td>x100 lens</td><td>Nikon</td><td>x100 N.A. 1.3 oil</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>x20 lens</td><td>Nikon</td><td>20x/0.40 WD 3.8, &amp;infin;/0.17</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>x20 lens</td><td>Leica Geosystems</td><td>HCX PL FLUOTAR, 20x/0.50, &amp;infin;/0.17/D</td><td>Or any equilavent product; confocal</td></tr><tr><td>x4 lens</td><td>Nikon</td><td>4x/0.10, WD 30, &amp;infin;/-</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>x63 lens</td><td>Leica Geosystems</td><td>HCX PL APO, 63x/1.40 - 0.60 OIL CS &amp;infin;/0.17/E</td><td>Or any equilavent product; confocal</td></tr><tr><td>Y-connectors</td><td>World Percision Instruments</td><td>14012</td><td>Or any equilavent product</td></tr><tr><td>&lt;strong&gt;Pipettes&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;Specifications and settings for fabrication of pipettes for experimentation&lt;/strong&gt;</td></tr><tr><td>Helper pipette</td><td>World Percision instruments</td><td>IB150F-3</td><td>Inner diameter: 0.86 mm&lt;br/&gt; Ramp: 261&lt;br/&gt; Pressure: 300&lt;br/&gt; Heat: 280&lt;br/&gt; Pull : 0&lt;br/&gt; Velocity: 56&lt;br/&gt; Time: 250&lt;br/&gt; No of cycles: 4&lt;br/&gt; Tip diameter: 0.5-2 &amp;mu;m&lt;br/&gt; Polishing: yes&lt;br/&gt; Final Resistance: &gt;10 M&amp;Omega;</td></tr><tr><td>Cannulation pipette</td><td>World Percision instruments</td><td>TW150F-4</td><td>Inner diameter: 1.17 mm&lt;br/&gt; Ramp: 290&lt;br/&gt; Pressure: 300&lt;br/&gt; Heat: 280&lt;br/&gt; Pull : 0&lt;br/&gt; Velocity: 58&lt;br/&gt; Time: 150&lt;br/&gt; No of cycles: 4&lt;br/&gt; Tip diameter: 3-10 &amp;mu;m&lt;br/&gt; Polishing: no&lt;br/&gt; Final Resistance: &amp;lt;1 M&amp;Omega;</td></tr><tr><td>On-cell patching</td><td>World Percision instruments</td><td>IB150F-3</td><td>Inner diameter: 0.86 mm&lt;br/&gt; Ramp: 261&lt;br/&gt; Pressure: 300&lt;br/&gt; Heat: 280&lt;br/&gt; Pull : 0&lt;br/&gt; Velocity: 56&lt;br/&gt; Time: 250&lt;br/&gt; No of cycles: 4&lt;br/&gt; Tip diameter: 0.5-2 &amp;mu;m&lt;br/&gt; Polishing: yes&lt;br/&gt; Final Resistance: &gt;5 M&amp;Omega;</td></tr><tr><td>Whole-cell patching</td><td>Warner Instruments</td><td>GC150TF-7.5</td><td>Inner diameter: 1.17 mm&lt;br/&gt; Ramp: 296&lt;br/&gt; Pressure: 200&lt;br/&gt; Heat: 287&lt;br/&gt; Pull : 0&lt;br/&gt; Velocity: 50&lt;br/&gt; Time: 250&lt;br/&gt; No of cycles: 4&lt;br/&gt; Tip diameter: 2-3 &amp;mu;m&lt;br/&gt; Polishing: helpful but not necessary&lt;br/&gt; Final Resistance: 1-2 M&amp;Omega;</td></tr></tbody></table>

isolation of retinal arterioles for ex vivo cell physiology studies

Die Netzhaut ist ein hoch metabolisch aktives Gewebe, die erfordert eine erhebliche Blut-Versorgungsmaterial. Die retinale Zirkulation unterstützt die inneren Netzhaut, während die aderhautgefäße Photorezeptoren liefern. Veränderungen der Netzhaut Perfusion zur zahlreiche Anblick lebensbedrohlichen Erkrankungen, einschließlich diabetischen Retinopathie, Glaukom und retinal Branch Vene Gefäßverschlüsse. Verständnis der molekularen Mechanismen der Kontrolle der Durchblutung der Netzhaut und wie diese geändert werden, während augenfällige Krankheit könnte die Identifizierung neuer Ziele für die Behandlung dieser Bedingungen. Retinale Arteriolen sind die wichtigsten Widerstand Gefäße der Netzhaut, und folglich eine zentrale Rolle bei der Regulierung der Netzhaut Hämodynamik durch Änderungen in der luminalen Durchmesser. In den letzten Jahren entwickelten wir Methoden zur Isolierung von Arteriolen von der Ratte Netzhaut, die für eine Vielzahl von Anwendungen einschließlich Zelle Physiologie Studien geeignet sind. Dieses Präparat hat bereits begonnen, neue Einblicke in wie Blutfluss wird in der Netzhaut gesteuert und hat uns erlaubt, einige der wichtigsten Änderungen zu identifizieren, die während der okulären Erkrankungen auftreten. In diesem Artikel Wir beschreiben Methoden für die Isolierung von Ratte retinalen Arteriolen und Protokolle für den Einsatz in Patch-Clamp Elektrophysiologie, Kalzium Imaging und Druck Myographie Studien. Diese Schiffe sind auch für den Einsatz in der PCR, western Blot und Immunohistochemistry-basierten Studien zugänglich.

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Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

Dieses Manuskript beschreibt eine einfache Protokoll für die Isolierung von Arteriolen von der Netzhaut der Ratte, die in elektrophysiologische, Kalzium Imaging und Druck Myographie Studien verwendet werden können.

Isolierung der retinalen Arteriolen für Ex Vivo Zelle Physiologie Studien

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...

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Research

JoVE Journal

Biology

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

10.2K Aufrufe.  Queen's University of Belfast. Dieses Manuskript beschreibt eine einfache Protokoll für die Isolierung von Arteriolen von der Netzhaut der Ratte, die in elektrophysiologische, Kalzium Imaging und Druck Myographie Studien verwendet werden können.

Serie: Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

Isolierung von primären murinen Retinal-Ganglion-Zellen (RGCs) durch Durchflusszytometrie

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Forschung

Biotechnik

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies revealing novel paradigms in vascular biology. Applying these methods to models of vascular development requires the optimization of cell isolation techniques from embryonic and postnatal tissues. Cell yield, viability, and purity all need to be maximal to obtain accurate and reproducible results from next-generation sequencing approaches. The neonatal mouse retinal vascularization model is used by researchers to study mechanisms of vascular development. Researchers have used this model to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Applying next-generation sequencing techniques to study the retinal vascular development model requires optimization of a method for the isolation of retinal endothelial cells that maximizes cell yield, viability, and purity. This protocol describes a method for murine retinal tissue isolation, digestion, and purification using fluorescence-activated cell sorting (FACS). The results indicate that the FACS-purified CD31+/CD45- endothelial cell population is highly enriched for endothelial cell gene expression and exhibits no change in viability for 60 min post-FACS. Included are representative results of next-generation sequencing approaches on endothelial cells isolated using this method, including bulk RNA sequencing and single-cell RNA sequencing, demonstrating that this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. This method of retinal endothelial cell isolation will allow for advanced sequencing techniques to reveal novel mechanisms of vascular development.

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.

Isolierung von retinen retinalen Endothelzellen für die Sequenzierung der nächsten Generation

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies ...

Entwicklungsbiologie

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

Reinigung von Maus-Hirngefäße

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Neurowissenschaften

Isolation and Cannulation of Cerebral Parenchymal Arterioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves &#8212; innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

Isolierung und Kanülierung von Cerebral Parenchymale Arteriolen

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

Vorbereitende Schritte für die Messung der Reaktivität in Maus retinalen Arteriolen Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

Medizin

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina&#160;with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2&#8211;9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day.
The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.

The protocol describes organotypic explants of mouse neuroretina, cultivated together with its retinal pigment epithelium (RPE), in R16 defined medium, free of serum and antibiotics. This method is relatively simple to perform, less expensive, and time-consuming when compared to in vivo experiments, and can be adapted to numerous experimental applications.

Langfristige, serumfreie Kultivierung organotypischer Maus-Retina-Explanten mit intaktem retinalen Pigmentepithel

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Quantifizierung vaskulärer Parameter in der gesamten Netzhaut von Mäusen mit nicht-proliferativen und proliferativen Retinopathien

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

Netzhautexplantat der adulten Mausnetzhaut als Ex-vivo-Modell zur Untersuchung neurovaskulärer Netzhauterkrankungen

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular pericytes are central to ophthalmological research. It is vital to choose an appropriate method of visualizing the microvascular pericytes. We describe retinal microvascular pericyte immunohistochemical staining in cryo-sections, whole-mounts, and hypotonic isolated vasculature using antibodies for platelet-derived growth factor receptor &#946; (PDGFR&#946;) and nerve/glial antigen 2 (NG2). This allows us to highlight advantages and shortcomings of each of the three tissue preparations for the visualization of the retinal microvascular pericytes. Cryo-sections provide transsectional visualization of all retinal layers but contain only a few occasional transverse cuts of the microvasculature. Whole-mount provides an overview of the entire retinal vasculature, but visualization of the microvasculature can be troublesome. Hypotonic isolation provides a method to visualize the entire retinal vasculature by the removal of neuronal cells, but this makes the tissue very fragile.

We demonstrate three different tissue preparation techniques for immunohistochemical visualization of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network.

Netzhaut Cryo-Abschnitte, ganze-Reittiere und hypotone isoliert Gefäßsystem Vorbereitungen für immunhistochemische Visualisierung von Mikrovaskulären Perizyten

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular ...

Biologie

Neuartiger Netzhautschutz-Assay für die Pathogenese-Studie zur diabetischen Retinopathie

An Assay to Detect Protection of the Retinal Vasculature from Diabetes-Related Death in Mice

Diabetic retinopathy (DR) is a complex and progressive ocular disease characterized by two distinct phases in its pathogenesis. The first phase involves the loss of protection from diabetes-induced damage to the retina, while the second phase centers on the accumulation of this damage. Traditional assays primarily focus on evaluating capillary degeneration, which is indicative of the severity of damage, essentially addressing the second phase of DR. However, they only indirectly provide insights into whether the protective mechanisms of the retinal vasculature have been compromised. To address this limitation, a novel approach was developed to directly assess the retina's protective mechanisms - specifically, its resilience against diabetes-induced insults like oxidative stress and cytokines. This protection assay, although initially designed for diabetic retinopathy, holds the potential for broader applications in both physiological and pathological contexts. In summary, understanding the pathogenesis of diabetic retinopathy involves recognizing the dual phases of protection loss and damage accumulation, with this innovative protection assay offering a valuable tool for research and potentially extending to other medical conditions.

Autor im Rampenlicht: Verständnis der Resilienz von retinalen Gefäßen und des Fortschreitens der Krankheit

A protection assay was developed to monitor the retinal vasculature's resilience to death from diabetes/diabetic retinopathy-related insults such as oxidative stress and cytokines.

Ein Assay zum Nachweis des Schutzes des Netzhautgefäßsystems vor diabetesbedingtem Tod bei Mäusen

Diabetic retinopathy (DR) is a complex and progressive ocular disease characterized by two distinct phases in its pathogenesis. The first phase involves ...