Diese Arbeit wurde vom NIH 1R01EY022901, der Career Development Award von Forschung unterstützt, um Blindheit (RPB) Verhütung, CMReeves &amp; MA Reeves-Stiftung, E. Matilda Ziegler-Stiftung für Blinde, Templer Eye Foundation, und eine nicht zweckgebundene Zuwendung an die Abteilung für Augenheilkunde an der University of Utah aus RPB. Balamurali Ambati Wir danken für die technische Unterstützung auf dem Spectralis Multi-Modalität Imaging System und Tao Zhang für Diskussionen und Kommentare zum Manuskript.

1. Vorbereitung der Instrumente <ol><li> Das Verfahren wird in einem Behandlungsraum in einer Tiereinrichtung durchgeführt wird. </li><li> Tragen Gesichtsmasken, Haarhauben, OP-Kittel, sterile Abdeckungen Fuß-und Handschuhe vor dem Beginn des Experiments. </li><li> Hitze Wasser in einem Becher auf ~ 40 ° C auf einer Heizplatte. </li><li> Legen Sie eine sterile blauen Pad auf einem Heizkissen, die später verwendet werden, um die Körpertemperatur der Maus während der Bildgebung halten wird. Schalten Sie das Heizkissen. </li><li> Bereiten Sie die Imaging-System: <ol><li> Entfernen Sie die Staubschutzabdeckung und schalten Sie den Laser. </li><li> Nehmen Sie die 55 °-Objektiv und montieren sie auf die Maschine. </li><li> Öffnen Sie die Imaging-Software aus dem Computer und geben Sie die Daten von der Maus für die Bildgebung unter Blatt eines neuen Patienten (zB Genotyp, Alter, etc.). Unter &quot;Gerätetyp&quot;, wählen Sie Infrarot (IR)-Modus. </li></ol></li></ol> Anmerkung: Es wurde berichtet, dass die Verwendung of eine externe Doppel asphärische Linse können die Bildqualität zu verbessern 17-20 obwohl wir haben kein Problem bei der Beschaffung von qualitativ hochwertigen Bildern von IV-ICGA ohne externe Linsen (siehe repräsentative Ergebnisse, Abbildungen 1-4). 2. Schwanzveneninjektion von ICGA <ol><li> Dilate Maus Augen mit 1% Tropicamide Augenlösung und warten 5 Minuten. </li><li> Wiegen Sie die Maus, um die Menge der Narkose (Ketamin / Xylazin / Acepromazine 65-100/10-20/1-3 mg / kg) benötigt bestimmen. </li><li> Abrufen einer sterilen 1-ml-Spritze mit einer 32 G-Nadel. Spritzen Sie die Maus intraperitoneal mit Anästhetika (13 mg / ml Ketamin, 2,6 mg / ml Xylazin, 0,3 mg / ml Acepromazine in sterilem PBS). Warten Sie, bis die Maus vollständig betäubt wird (~ 5 min). </li><li> Platzieren Sie die Maus Schwanz in 40 ° C warmem Wasser, um Vasodilatation der Vene führen. </li><li> Abrufen einer 1 ml-Spritze mit einer 32 G-Nadel. Ziehen Sie die gewünschte Menge an ICG, typischerweise 50 &amp;mgr; l 1 mg / ml ICG, die steril mit einem 0,2 &amp;mgr; m-Spritzenfilter filtriert, in ein steriles Röhrchen, für eine 25 g-Maus (2 mg / kg) ist. Achten Sie darauf, um die Luft in die Spritze einzuführen. </li><li> Wischen Sie den Schwanz mit einem Alkoholtupfer, um den Bereich injiziert werden sterilisieren. </li><li> Halten den Schwanz mit einer Hand, so daß die laterale Schwanzvene steigend. Mit der Abschrägung der Nadel nach oben, die Nadel zu injizieren ~ 2 mm in die Vene zu einem minimalen Winkel. Achten Sie darauf, um die Vene zu perforieren. Zeichnen Sie wieder auf die Spritze leicht und suchen nach Spuren des Blutflusses in die Nadel-Hub, der anzeigt, dass die Nadel wurde erfolgreich in die Vene eingeführt. </li><li> Langsam spritzen ICG in die Vene. Es sollte minimal sein Widerstand bei der Injektion. Entfernen Sie die Nadel und wenden Sie einen Alkoholtupfer direkt auf die Injektionsstelle für ca. 5-10 Sekunden, um Blutungen zu stoppen. Die Maus ist dann bereit für die Bildgebung. Um die Frühphase (0-4 min nach der Injektion) zu fangen, ist es essential für Bild mit der Maus schnell. </li></ol> Hinweis: Mouse Augen können leicht trocken und kann Katarakt unter Narkose zu entwickeln. Es ist wichtig, das Auge feucht indem sterile PBS während des Verfahrens zu halten. Wischen Sie überschüssiges PBS mit einem sterilen Wattestäbchen, bevor ICGA Aufnahme. Andere Labors haben eine Kontaktlinse eingesetzt, um Austrocknung der Hornhaut 17-20 vermeiden. 3. ICG-Angiographie <ol><li> Start, um Bilder aufzunehmen 30-40 sec nach ICG-Injektion, die die Erfassung der frühen Phase der Aderhautfüllung ermöglicht, bis Netzhaut-und Aderhautkreisläufe sind bei maximaler Helligkeit (0-4 min). Die Netzhautgefäßsystem wird am besten bei Fokus ~ 35-45 Dioptrien und Aderhautgefäßsystem wird bei 10-15 Dioptrien sichtbar visualisiert. Hinweis: Während der ersten Untersuchung eines Tiermodells, wird empfohlen, um Bilder aus allen Winkeln (nasal, temporal, dorsalen und ventralen) zu erfassen, um alle möglichen abno identifizierenrmalities in das Gefäßsystem. Während der frühen Phase sind die beiden mittleren und großen choroidalen Arterien und Venen gut visualisiert. Im Tiermodell in diesem Protokoll verwendet wird, kann choroidalen Läsionen (zB polypoidal Streckungen) beginnen, 1 min in der frühen Phase angezeigt. </li><li> Stellen Sie die Bildschärfe über das Gefäßsystem. Steuerung für Helligkeit und Fokus mit dem Steuermodul und Fokusknopf auf. Diese Werte sind digital einstellbar und leicht konstant gehalten. Den Abstand von der Maus Auge des Kameraobjektivs konstant mit der hier beschriebenen Technik die Bildqualität zu gewährleisten, ist reproduzierbar. Hinweis: Da das Gerät nur Bild ein Teil des hinteren Augen, versuchen wir, den Fokus, Helligkeit und den Abstand zwischen Linse und Auge der Maus konstant zu halten, da wir das gesamte Bild hinteren Augen aus verschiedenen Blickwinkeln. Der Schlüssel dazu ist, um die kreisförmige Lumineszenz durch die ICG durch das Auge mit dem Feld auszurichten vie emittiertenw der Kamera. Dies wird durch das von links nach rechts durchgeführt, oben und unten, und in-und aus Einstellungen der Kameraposition, bis das gesamte Bild hat keine dunklen Stellen. Wenn die Lumineszenz und das Sichtfeld der Kamera aufgereiht sind, wird der Abstand vom Auge auf die Linse reproduzierbar sein für den nächsten Satz von Bildern als auch in einem optimalen Abstand zur Bildqualität. </li><li> Sobald das Gefäßsystem im Fokus ist, erfassen die Bildrahmen durch Drücken der runden schwarzen Knopf auf der Erfassungsmodul. Die runde schwarze Taste kann auch verwendet werden, zu reduzieren oder zu verbessern das Signal des ICG für beste Bildqualität werden. </li><li> Bestimmen Sie den optimalen Blickwinkel und Fokussierung Tiefe Bild choroidalen Läsionen. Es ist wichtig, die Position des Auges, die Fokussierungstiefe und andere Geräteeinstellungen für den gesamten Zeitverlauf ICGA fixiert zu halten. Die Bilder werden durch Drücken erwerben Taste auf dem Touch-Screen-Panel des Erfassungsmodul gespeichert. </li><li> Erwerben Sie Bilder in der mittleren Phase bei 15.06 min achternäh Injektion. Hinweis: Sowohl die Aderhaut und Netzhautgefäße werden weniger deutlich. Aderhautgefäßsystem erscheint als diffuse Fluoreszenz. Aderhautläsionen aufweisen Hyper entstehen im Gegensatz zu den Fading umgebenden normalen Hintergrundfluoreszenz. </li><li> Erwerben Sie Bilder in der Spätphase bei 17-25 Minuten nach der Injektion. Hinweis: Hyperfluoreszenz verblasst. Beide Aderhaut-und Netzhautgefäße sind nicht mehr sichtbar. Der Sehnervenkopf wird schwarz. Hyperfluoreszente choroidalen Läsionen maximale Kontrast mit dem Verblassen Hintergrund. </li><li> Nach Abschluss der Übernahme von Bildern, gelten eine klare Schmiermittel Augen-Gel auf Maus Augen und lassen Sie die Maus auf einem Heizkissen für die Verwertung. </li><li> Zurück Mäuse in ihre Käfige und Haltebereich. Export Bilder als Tiff oder JPEG-Dateien zur weiteren Analyse. </li></ol> Anmerkung: Der Zeitpunkt für jede Phase ist nicht absolut. Wir fanden, dass der Zeitpunkt der each Phase kann je nach der Menge des eingespritzten ICG ändern. Mehr ICG neigt dazu, jede Phase zu verlängern. Der beste Weg, um eine Phase zu definieren, ist nach den wichtigsten Funktionen der einzelnen oben aufgeführten Phase.

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YF ist Erfinder von zwei Patentanmeldungen, die für die AMD-Mausmodell in dieser Arbeit verwendet werden. SK, ZB, ADJ und haben nichts zu offenbaren.

In dieser Studie haben wir gezeigt, die Verwendung von ICGA für Bild choroidalen Läsionen in HTRA1 transgenen Mäusen. Die Eigenschaften der frühen, mittleren und späten Phasen der ICGA in unserem Mausmodell entsprechen den Zeitverlauf auch in Studien am Menschen ein. Dies ist wichtig, um bessere Vergleiche zwischen der menschlichen Pathologie und Tier Phänotypen, die von unschätzbarem Wert für die Erforschung der pathophysiologischen Mechanismen und Behandlungsstrategien von Bedingungen, die der Aderh...

Indocyaningrün-Angiographie (ICGA) ist ein diagnostischer Test zur Imageprobleme, die Blutgefäße in den Augen stehen. Das Absorptionsspektrum der ICG Bereiche 790-805 nm, während das Emissionsspektrum im Bereich von 770 bis 880 nm mit der Peakemission bei 835 nm 1. Dies unterscheidet sich von den anderen bekannten Farbstoff Fluorescein-Natrium, dessen Spektrum liegt im sichtbaren Bereich. Das Infrarotspektrum ermöglicht ICG durch retinale Pigmentepithel (RPE), serosanguineous Flüssigkeit und Fettausscheidungen, die alle leicht zu blockieren Visualisierung durch Natrium-Fluorescein-basierte Fluoreszenzangiographie (FA) zu durchdringen. ICG ist zu 98% an Proteine ​​gebunden im Gefäßsystem, was zu weniger Extravasation, so dass verbesserte Bildgebung von Aderhautgefäße und choroidalen Läsionen 1,2. ICGA ist fast die einzige Wahl, um choroidalen Gefäßsystem, die hinter RPE visualisieren. Abbildung 1 zeigt den Vergleich der ICGA und FA in bildgebenden Gefäßsystem in der Maus die Augen. FA kann be zur Abbildung der Netzhautgefäßsystem gut, aber nicht der choroidalen Gefäßsystem. Im Gegensatz dazu können die Bild ICGA sowohl retinalen und choroidalen Gefäßsystem verwendet werden. ICGA ist mit hochauflösenden Digital-Imaging-Systeme oder Scanning-Laser-Ophthalmoskop (SLO) zusammen mit infrarotempfindlichen Videokameras, die wir in dieser Studie verwenden durchgeführt. In der Klinik hat ICGA bei der Diagnose eine Reihe von chorioretinale Störungen mit Beteiligung des Aderhautgefäßsystem einschließlich polypoidale choroidalen Vaskulopathie (PCV), Netzhaut angiomatöse Proliferation (RAP), angioiden Streifen, vitelliformen Makuladystrophie, Zentral serosa, choroidalen Hämangiom, Blutungen der Netzhaut empfohlen arteriolar Makroaneurysmen, choroidale Tumoren und bestimmte Formen von Uveitis posterior 1,3. Die Kombination von ICGA mit FA und Optische Kohärenztomographie (OCT) bieten leistungsstarke Tools für die Ärzte in der Diagnose und Behandlung der exsudativen altersbedingten Makula-Degeneration (AMD) 10.04. ICGA ist besonders nützlich für die Diagnose von Bedingungen, die die Aderhaut. In der Tat ist ICGA als der Goldstandard für die Diagnose von PCV, eine Variante der exsudativen AMD 11-13. PCV wird von einem Netzwerk von verzweigenden Gefäßen mit endständigen polypoidal Streckungen in der choroidalen Gefäß 11-13 gekennzeichnet. PCV wird häufig mit rezidivierenden serosanguineous Abteilungen der RPE und Netzhaut mit Leck und Blutungen aus den Komponenten polypoidal 11,14,15 verbunden. Vor kurzem berichteten wir die Erzeugung des ersten PCV Tiermodell durch menschliche HTRA1, ein multifunktionales Serinprotease transgen exprimiert, in Maus retinalen Pigmentepithel (RPE) 16. Wir haben gezeigt, dass eine erhöhte HTRA1 induzierten charakteristischen Merkmale der PCV, z. B. polypoidal Läsionen. Hier zeigten wir den Einsatz von Zeitverlauf ICGA durch Schwanzveneninjektion in die Forschung mit unserem AMD HTRA1 Mausmodell. Unsere Daten legen nahe, dassIV-ICGA überlegen ist IP (oder subkutan (SC))-ICGA, die derzeit im Bereich 17,18 für die Charakterisierung von Läsionen in der Aderhaut verwendet werden. Erklärung zum Tierforschung Tierversuche wurden nach den Protokollen, die von Institutional Animal Care und Verwenden Committee (IACUC) genehmigt durchgeführt und wurden in Übereinstimmung mit der ARVO Erklärung für die Nutzung von Tieren in Ophthalmic und Vision Research durchgeführt.

<table border="0" cellpadding="0" cellspacing="0" width="667">
	<tbody>
		<tr height="50">
			<td height="50" style="height:50px;width:216px;">Spectralis Multi-Modality Imaging System</td>
			<td style="width:152px;">Heidelberg Engineering, Germany</td>
			<td style="width:132px;">SPECTRALIS HRA+OCT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tropicamide ophthalmic solution (1%)</td>
			<td style="width:152px;">Bausch &#38; Lomb</td>
			<td style="width:132px;">NDC 24208-585-64</td>
			<td>for dilation of pupils</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GenTeal Gel</td>
			<td style="width:152px;">Genteal</td>
			<td style="width:132px;">NDC 58768-791-15</td>
			<td>&#160;clear lubricant eye gel&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine</td>
			<td style="width:152px;">Vedco Inc</td>
			<td style="width:132px;">NDC 50989-996-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylazine</td>
			<td style="width:152px;">Lloyd Laboratories</td>
			<td style="width:132px;">NADA 139-236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acepromazine</td>
			<td style="width:152px;">Vedco Inc</td>
			<td style="width:132px;">NDC 50989-160-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">32-G Needle</td>
			<td>Steriject</td>
			<td style="width:132px;">PRE-32013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1-ml syringe</td>
			<td style="width:152px;">BD</td>
			<td style="width:132px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Indocyanine Green</td>
			<td style="width:152px;">Pfaltz &#38; Bauer</td>
			<td style="width:132px;">I01250</td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting abnormalities in choroidal vasculature in a mouse model of age-related macular degeneration by time-course indocyanine green angiography

Indocyaningrün-Angiographie (oder ICGA) ist eine Technik, die von Augenärzten durchgeführt, um Anomalien der Aderhaut und Netzhautgefäß verschiedener Augenkrankheiten, wie altersbedingte Makula-Degeneration (AMD) zu diagnostizieren. ICGA ist besonders nützlich, um das hintere Bild choroidalen Gefäßsystem des Auges aufgrund ihrer Eignung zur Durchdringung durch die pigmentierte Schicht mit Infrarotspektrum. ICGA Zeitverlauf kann in frühe, mittlere und späte Phasen unterteilt werden. Die drei Phasen liefern wertvolle Informationen über die Pathologie von Augenproblemen. Obwohl der Zeitverlauf ICGA durch intravenöse (IV)-Injektion ist weit verbreitet in der Klinik für die Diagnose und Behandlung von Aderhautproblemen eingesetzt, ICGA durch intraperitoneale Injektion (IP) wird häufig in Tierversuchen eingesetzt. Hier zeigten wir die Technik, um hochauflösende ICGA Zeitverlauf Bilder in Mäusen durch Schwanzveneninjektion und konfokale Scanning-Laser-Ophthalmoskopie erhalten. Wir nutzten diese Technik, um das Bild choroidalen legen in einem Mausmodell der altersbedingten Makuladegeneration. Obwohl es viel einfacher, ICG zur Maus Vaskulatur von IP einzuführen, zeigen unsere Daten, dass es schwierig ist, reproduzierbare ICGA Zeitverlaufsbildern von IP-ICGA erhalten. Im Gegensatz dazu ICGA über Schwanzveneninjektion bietet hochwertige ICGA Zeitverlauf Bilder vergleichbar mit Studien am Menschen. Darüber hinaus haben wir gezeigt, dass ICGA geführt auf Albinomäuse gibt klarere Bilder von choroidalen Gefäßen als pigmentierter Mäuse durchgeführt. Wir schlagen vor, dass die Zeit-Gänge-IV-ICGA sollte eine gängige Praxis in AMD Forschung auf der Grundlage von Tiermodellen zu werden.

Wir führten ICGA Zeitverlauf in HTRA1 transgenen Mäusen und Kontroll WT Wurfgeschwistern, die beide auf der CD1 Hintergrund. Der Albino CD1 Hintergrund wurde ausgewählt, um Indocyaningrün-Angiographie (ICGA)-Bildgebung (siehe Diskussion) zu erleichtern. Einige Aneurysma wie Streckungen begann in der frühen Phase in der HTRA1 Maus erscheinen (Abbildung 2, eine rote Pfeil zeigt die Erweiterung an der Spitze eines Schiffes und ein roter Kreis zeigt eine Läsion polypoidal Cluster-Typ). Aderhautgefäß...

Watch this Scientific Journal Video about Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography at JoVE.com

Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

Indocyaningrün-Angiographie (ICGA oder) durch Schwanzveneninjektion durchgeführt, bietet hochwertige ICGA Zeitverlauf Bilder, Anomalien in der Maus Aderhaut zu charakterisieren.

Erkennen von Fehlbildungen in Aderhautgefäßsystem in einem Maus-Modell der Altersbedingte Makuladegeneration durch Zeitverlauf Indocyaningrün-Angiographie

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal ...

detecting-abnormalities-choroidal-vasculature-mouse-model-age-related

Research

JoVE Journal

Medicine

13.5K Aufrufe.  University of Utah Health Sciences Center.  Indocyaningrün-Angiographie (ICGA oder) durch Schwanzveneninjektion durchgeführt, bietet hochwertige ICGA Zeitverlauf Bilder, Anomalien in der Maus Aderhaut zu charakterisieren. 

Serie: Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

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

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

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

Nicht-invasive Beurteilung der Herzfehlbildungen in experimentellen autoimmunen Myokarditis von Magnetic Resonance Imaging Microscopy in der Maus

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

Forschung

Medizin

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

Ein Mausmodell für Laser-induzierten CNV

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

Das Monoiodoacetate Modell von Osteoarthritis Schmerzen in der Maus

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

Erkennung von Anastasis In Vivo durch CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

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

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

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

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

Regenerative Therapie durch Suprachoroidal Zelle Autotransplantation in trockene altersbedingte Makuladegeneration: vorläufige In Vivo Bericht

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

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Messung der isometrischen Kontraktilität der Maus mesenterialen Arterie mit Draht Myographie

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Ein neonatales BALB/c-Mausmodell der nekrotisierenden Enterokolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...

Robotic Left Hepatectomy using Indocyanine Green Fluorescence Imaging for an Intrahepatic Complex Biliary Cyst

Biliary cysts (BC) are rare congenital dilatations of intra- and extrahepatic parts of the biliary tract and bear a significant risk of carcinogenesis. Surgery is the cornerstone treatment for patients with BC. While total BC excision and Roux-Y hepaticojejunostomy is the treatment method of the choice in patients with extrahepatic BC (i.e., Todani I-IV), patients with intrahepatic BC (i.e., Todani V) benefit the most from a surgical liver resection. In recent years, minimally invasive liver surgery (MILS) including robotic MILS has gained more acceptance as a feasible, safe, and effective procedure for the treatment of both benign and malignant indications. Robotic major MILS is still considered technically demanding and a detailed description of the technical approach during robotic major MILS has only been limitedly discussed in the literature. The current article describes the main steps for a robotic left hepatectomy in a patient with a large BC Todani Type V. The patient is in French position with 5 trocars placed (4 robotic, 1 laparoscopic assistant). After mobilizing the left hemiliver, the left and right hepatic artery are dissected carefully followed by a cholecystectomy. Intraoperative ultrasound is performed to confirm localization and margins of the BC. The Left hepatic artery and left portal vein are isolated, clipped, and divided. Indocyanine green (ICG) fluorescence imaging is used regularly during the entire procedure to visualize and confirm biliary tract anatomy and the BC. Parenchymal transection is performed with robotic cautery hook for the superficial part and robotic cautery spatula, bipolar cautery, and vessel sealer for the deeper parenchyma. The postoperative course was uncomplicated. A robotic left hepatectomy is technically demanding, yet a feasible and safe procedure. ICG-fluorescence imaging aids in delineating the BC and bile duct anatomy. Further, comparative studies are needed to confirm clinical benefits of robotic MILS for benign and malignant indications.

Robotic liver surgery has gained more acceptance as a feasible, safe, and effective procedure for the treatment of both benign and malignant indications. However, robotic left hepatectomy is still technically demanding. We describe our surgical technique of a robotic left hepatectomy using indocyanine green fluorescence imaging for a large biliary cyst.

Roboter-Linkshepatektomie mit Indocyanin-Grünfluoreszenz-Bildgebung für eine intrahepatische komplexe Gallenzyste

Biliary cysts (BC) are rare congenital dilatations of intra- and extrahepatic parts of the biliary tract and bear a significant risk of carcinogenesis. ...

Fluorescent Laparoscopic Central Hepatectomy for Liver Cancer Using Indocyanine Green Negative Staining

Laparoscopic hepatectomy is an important treatment method for liver cancer. In the past, the resection boundary was usually determined by intraoperative ultrasound, important vascular structures, and surgeon experience. With the development of anatomical hepatectomy, visual surgery technology has gradually been applied to this type of surgery, particularly indocyanine green (ICG)-guided anatomical hepatectomy. As ICG can be specifically ingested by hepatocytes and used for fluorescence tracing, negative staining techniques have been applied according to different tumor positions. Under ICG fluorescent guidance, the surface boundary and deep resection plane can be more accurately displayed during liver resection. Thus, the tumor-bearing liver segment can be anatomically removed, which helps to avoid damage to important vessels and reduce ischemia or congestion of the remaining liver tissue. Finally, the incidence of postoperative biliary fistula and liver dysfunction is reduced; therefore, a better prognosis is obtained after the resection of liver cancer. Centrally located liver cancer is usually defined as a tumor located at segments 4, 5, or 8 that requires resection of the middle section of the liver. These are among the most difficult hepatectomies to perform because of the large surgical wounds and multiple vessel transections. Based on the specific tumor location, we formulated the required resection ranges by designing personalized fluorescent staining strategies. By completing anatomical resection based on the portal territory, this work aims to achieve the best therapeutic effect.

The present protocol describes fluorescent negative staining in laparoscopic central hepatectomy. This technique can make hepatectomy more accurate and precise.

Fluoreszierende laparoskopische zentrale Hepatektomie bei Leberkrebs mit Indocyaningrün-Negativfärbung

Laparoscopic hepatectomy is an important treatment method for liver cancer. In the past, the resection boundary was usually determined by intraoperative ...

Erforschung der quantitativen Autofluoreszenz bei altersbedingter Makuladegeneration

A Workflow to Quantitatively Determine Age-Related Macular Degeneration Lesion-Specific Variations in Fundus Autofluorescence

Fundus autofluorescence (FAF) imaging allows the noninvasive mapping of intrinsic fluorophores of the ocular fundus, particularly the retinal pigment epithelium (RPE), now quantifiable with the advent of confocal scanning laser ophthalmoscopy-based quantitative autofluorescence (QAF). QAF has been shown to be generally decreased at the posterior pole in age-related macular degeneration (AMD). The relationship between QAF and various AMD lesions (drusen, subretinal drusenoid deposits) is still unclear.
This paper describes a workflow to determine lesion-specific QAF in AMD. A multimodal in vivo imaging approach is used, including but not limited to spectral domain optical coherence tomography (SD-OCT) macular volume scanning and QAF. Using customized FIJI plug-ins, the corresponding QAF image is aligned with the near-infrared image from the SD-OCT scan (characteristic landmarks; i.e., vessel bifurcations). The foveola and the edge of the optic nerve head are marked in the OCT images (and transferred to the registered QAF image) for accurate positioning of the analysis grids.
AMD-specific lesions can then be marked on individual OCT BScans or the QAF image itself. Normative QAF maps are created to account for the varying mean and standard deviation of QAF values throughout the fundus (QAF images from a representative AMD group were averaged to build normative standard retinal QAF AMD maps). The plug-ins record the X and Y coordinates, z-score (a numerical measurement that describes the QAF value in relation to the mean of AF maps in terms of standard deviation from the mean), mean intensity value, standard deviation, and number of pixels marked. The tools also determine z-scores from the border zone of marked lesions.&#160;This workflow and the analysis tools will improve the understanding of the pathophysiology and clinical AF image interpretation in AMD.

Autor im Rampenlicht: Verständnis der Pathophysiologie der altersbedingten Makuladegeneration mit dem QAF-Workflow 

This research describes a workflow to determine and compare autofluorescence levels from individual regions of interest (e.g., drusen and subretinal drusenoid deposits in age-related macular degeneration [AMD]) while accounting for varying autofluorescence levels throughout the fundus.

Ein Workflow zur quantitativen Bestimmung altersbedingter Makuladegenerationsläsions-spezifischer Variationen in der Fundus-Autofluoreszenz

Fundus autofluorescence (FAF) imaging allows the noninvasive mapping of intrinsic fluorophores of the ocular fundus, particularly the retinal pigment ...