S och Drottning Viktorias Frimurarstiftelse: Diese Arbeit wurde von der Karolinska Institutet KID-Finanzierung, Swedish Radiation Protection Authority, Karolinska Institutet Eye Research Foundation, Gun och Bertil Stohnes Stiftelse, St. Eriks Eye Hospital Research Foundation, Ögonfonden, Konung Gustav V unterstützt.

Ein. Exposition gegenüber ultravioletter Strahlung <ol><li> 15 min vor der Belichtung anästhesieren einen weiblichen Sprague-Dawley-Ratten mit einem Gemisch von 90 mg / kg Ketalar (Ketamin) und 10 mg / kg Rompun (Xylazin) durch intraperitoneale Injektion. </li><li> Platzieren des Tieres in einem Ratten Restrainer und ziehen die Bänder bis Immobilisierung der Ratte ohne einen Stamm Squeeze 13. </li><li> Instill Mydriacyl (Tropicamid), 10 mg / ml, in beiden Augen des Ratten zu induzieren Mydriasis. </li><li> Platzieren des Tieres, so daß ein Auge gegen einen schmalen Strahl von UVR bei 300 nm 10 nm 14 mit voller Breite bei halbem Maximum positioniert und schirmen das kontralaterale Auge mit schwarzer Folie. </li><li> Setzen Sie den rat einseitig zu sub-Schwellendosis 1 kJ / m 2 UVR-B bei 300 nm für 15 min 15. </li></ol> 2. Präparation <ol><li> Nach vorgegebenen Nachbelichtung Intervall (1, 5, 24 und 120 Stunden), opfern die 6 Wochen alten Ratte (Gewicht &lt;200 g) von carbon Kohlendioxid Ersticken und Dislokation der Halswirbelsäule. </li><li> Entkernen Augen. Danach legte die Augenhornhaut unten, hinteren Seite nach oben. Dann Stempels eine minimale Öffnung durch Anlegen einer 27 Gauge Kanüle und schieben tangential an der Sklera um eine Beschädigung der Linse, die sehr nahe an der Sklera ist. Dann nutzen Sie ein Paar der Augenchirurgie Schere Umfang geschnitten direkt hinter dem Limbus, bis der hintere Teil der Sklera abgehoben werden kann. Dann heben Sie das Objektiv mit einem stumpfen gebogenen Pinzette. </li><li> Entfernen Sie Reste des Ziliarkörpers von der Linsenäquator unter einem Mikroskop, mit dem Objektiv in einem ausgewogenen Salzlösung nicht länger als 5-10 min. </li></ol> 3. Quantitative RT-PCR <ol><li> Platzieren einer Linse in einer Mischung von 350 ul Lysepuffer RA1 (NucleoSpin RNA II Gesamt-RNA Isolation Kit) und 3,5 ul β-Mercaptoethanol in einem 2 ml Eppendorf-Röhrchen. </li><li> Halten Sie die Linse in diesem Mix während 30 min bei Raumtemperatur. </li><li> Homogenisieren Sie das Objektiv miteine Nadel, bis nur harte Kern der Linse wird von der Linse (Cortex und Linsenkapsel im Lysepuffer) verlassen. Entfernen Sie den Kern aus dem Mix. </li><li> Lagern Sie die Proben sofort bei -70 ° C. </li><li> Tauen Sie die Proben und folgen Sie das Protokoll &quot;Total RNA Aufreinigung aus kultivierten Zellen und Gewebe&quot; (NucleoSpin RNA II Gesamt-RNA Isolation Kit). </li><li> Shop-RNA-Proben bei -70 ° C. </li><li> Um eine ausreichende Entfernung von DNA aus jeder Probe zu überprüfen, führen eine PCR unter den folgenden Bedingungen (Schritt 1: 95 ° C für 2 min; Schritt 2 während 40 Zyklen: 95 ° C für 20 sec, 55 ° C für 20 sec, 72 ° C für 20 Sek.; Schritt 3: 72 ° C für 7 min) mit p53 DNA-spezifischen Primern, 5&#39;-Vorwärts ACCCTCTGACCTTTTTCCCA-3 &#39;und 5&#39;-Reverse TGCTGGGATCTTAGGCACTC-3&#39; und Taq DNA-Polymerase (dNTPack), nach der Herstellung Protokoll. Die erwartete PCR-Produkt ist 243 Basenpaare. </li><li> Ausführen eines 1,5% Agarose-Gel-Elektrophorese DNA-spezifische PCR-Produkt von 243 Basenpaaren nachzuweisen. Zur Vorbereitung1,5% Agarosegel in TBE-Puffer nehmen 3,75 g Agarose und lösen es in 250 ml TBE-Puffer. Hinzufügen Ethidiumbromid (0,5 mg / ml) 500 ul in 500 ml 1,5% Agarose-Gel-Lösung. Das Gemisch wird in einer Mikrowelle und rühren, um die Agarose zu lösen. </li></ol> Keine der Proben muss keine DNA-spezifischen PCR-Produkte auf 1,5% Agarose-Gelelektrophorese offenbaren. <ol start="9"><li> Messung der Konzentration von RNA-Proben (1 ul) und das Verhältnis der Probe Absorption bei 260 nm (RNA) und 280 nm (Protein) in einer NanoDrop ND-1000 Spektrophotometer. Wenn das Verhältnis zu Protein von RNA-RNA-Probe Absorption ist 2,0 oder mehr wird die RNA-Probe ist rein. Wenn das Verhältnis der Extinktion RNA-Probe geringer ist als 2,0, Redo RNA Reinigungsschritt 3.5. </li><li> Nehmen ein Volumen von RNA-Probe, die 1 ug cDNA zu synthetisieren und entsprechend dem Protokoll 1. Strang cDNA-Synthese Kit (1 st Strang cDNA-Synthese-Kit für RT-PCR). </li><li> Bewahren cDNA-Proben bei -20 ° C(Für einen Zeitraum von 1 und mehr Jahren, bei -70 ° C). </li><li> Führen quantitative real-time PCR auf iCycler MyiQ Single Color Real Time PCR Detection System. Last Triplikate auf 96-Well-Platte mit 1 ul cDNA Probe TaqMan Gene Expression Master Mix, TaqMan Gene Expression Assay für Caspase 3, nach Anleitung zu fertigen. Last Triplikate auf einem anderen 96-Well-Platte mit 1 ul cDNA Probe TaqMan Gene Expression Master Mix, TaqMan Gene Expression Assay für 18s, nach Anleitung (TaqMan Gene Expression Assay Protocol) herstellen. Verdünnen in Serie einen Bruchteil der cDNA aus drei zufällig ausgewählten unbelichteten Linsen. Führen Reihenverdünnungen zusammen mit der cDNA aus Proben in einem 96-Well-Platte. </li><li> Verwenden Sie die Standardkurve Methode zur Quantifizierung der Ergebnisse. Die Zahl der Zyklen bei Schwellenwert Fluoreszenz wird als die Messung in MyiQ Software verwendet. Eine Standardkurve Ausdruck Anzahl der Zyklen an der Schwelle als Funktion der relativen Konzentration des Kalibrator ist established für die seriellen Verdünnungen in jeder Platte. cDNA aus drei nicht behandelten Linse Proben wurde für die Kalibrierung verwendet. Betreffen die Schwelle Anzahl von Zyklen für jede Probe mit der Standardkurve, um die relative Konzentration der Probe gemessen cDNA erhalten. Schließlich erhalten Expressionsniveau der Zielgene durch über die relative Konzentration der Ziel-cDNA, um die interne Kontrolle 18s cDNA. </li></ol> 4. Immunhistochemischen Färbung <ol><li> Fixierung <ol><li> Sezieren das Auge in PBS (Phosphate Buffered Saline, pH 7.4). </li><li> Setz das Auge in einem Rohr mit 4% Paraformaldehyd (PFA) in eiskaltem 20 min gefüllt. Bereiten PFA 4% am Tag zuvor. Lagerung bei 4 ° C bis zur Verwendung. PFA ist für etwa eine Woche frisch. </li><li> Entfernen PFA mit einer Mikropipette. Dann, mit eiskaltem PBS für 20 min waschen. </li><li> Setzen Sie das Auge in Saccharose 30% bei +4 ° C über Nacht. </li><li> Setzen Sie das Auge in einer Tasse mit Optimal Cutting Temperature (OCT)-Medium gefüllt (Tissue-Tek). Setzen Sie das Auge in eine geeignete Position zum Schneiden. </li><li> Frieren in OCT-Medium über Trockeneis. </li><li> Lagerung bei -70 ° C bis zur Verwendung. </li></ol></li><li> Schnitte <ol><li> Positionieren Sie den Blick für Kryo-Schnitte in angemessener Ebene. </li><li> Geschnitten drei 5 um dicke mittleres Sagittalschnitten von einem zentralen Abschnitt jeder Linse. Entsorgen Sie mindestens 6 Abschnitte zwischen aufeinanderfolgenden Abschnitten zu vermeiden Färbung die gleiche Kerne in verschiedenen Abschnitten. </li><li> Sobald ein Abschnitt geschnitten worden ist, sanft legen eine Folie oben drauf. Der Abschnitt sollte dann auf der Folie haften. </li><li> Lassen Sie die Folien an der Luft trocknen vor dem Gebrauch. Dias können bei -20 ° C gelagert werden, bis sie benötigt. </li></ol></li><li> Fluoreszenz Immunhistochemie <ol><li> Lassen Proben erreichen Raumtemperatur (5-10 min). </li><li> Zeichnen Ränder um die Proben mit einem PAP-pen (Invitrogen). </li><li> Lassen Sie die Grenze trocken für eine Weile (10 min). </li><li> Beschriften Sie die Objektträger. </li><li> Rehydrieren in 1 × PBS für 15 min. </li><li> Permeabilisieren in Standard-Baustein-Lösung für 30 min. </li><li> Fügen primären Antikörper (polyklonaler Kaninchen, gespalten Caspase-3-Antikörper Asp175 9661; Zelluläre Signalübertragung Technology, Inc) verdünnt 1:3000 in Standard-Baustein-Lösung. </li><li> Speicher in einer befeuchteten Kammer bei 4 ° C über Nacht. </li><li> Waschen in PBS durch Pipettieren (3 × 5 min) oder durch Eintauchen in eine große Badewanne (&gt; 15 min, 1-2 Änderungen). </li><li> Fügen sekundären Antikörper (anti-Kaninchen sekundären Antikörper mit einer spezifischen Absorption / Emissionsspektrum) verdünnt 1:300 in der Standard-Baustein-Lösung. </li><li> Waschen in PBS durch Pipettieren (3 × 5 min) oder durch Eintauchen in eine große Badewanne (&gt; 15 min, 1-2 Änderungen). </li><li> Wischen Sie PAP-pen Abstriche. </li><li> Speicherung in einer befeuchteten Kammer bei Raumtemperatur während 3 Stunden. Vermeiden Sie Licht. </li><li> Fügen Sie einen Tropfen Vectashield und legen Sie eine Deckglas auf dem Objektträger. Vermeiden Sie Luftblasen. </li><li> Lassen Sie die Vectashield aushärten (~ 20 min). </li><li> Halten Abschnitten in den dunklen bis zur Analyse. </li><li> Alle Steuer-Abschnitte werden in Abwesenheit von primären Antikörper verarbeitet. </li><li> Achten Sie auf die Ergebnisse innerhalb weniger Stunden unter einem Fluoreszenzmikroskop. </li><li> Graf Epithelzellen &#39;Kerne von einer Seite Kernbogen zur anderen Seite Kernbogen jeder Linse. Anwenden der Standard Blaufilter alle Linsenepithelzellen Kerne in Blau und einen Standard-Filter, der die Emission des Sekundärantikörpers paßt, um die Caspase-3-positiven Kernen im grünen sehen. </li><li> Aufzeichnung der Anzahl aller Linsenepithelzellen Kerne und die Anzahl der positiven Kernen. Zählen Sie die Zellen dreimal für jeden Abschnitt. </li></ol></li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+epidemiologic+evidence+linking+ultraviolet+radiation+and+cataracts.">A review of the epidemiologic evidence linking ultraviolet radiation and cataracts.</a> Dev. Ophthalmol. 35, 21-31 (2002).</li><li>Li, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lens+epithelial+cell+apoptosis+appears+to+be+a+common+cellular+basis+for+non-congenital+cataract+development+in+humans+and+animals.">Lens epithelial cell apoptosis appears to be a common cellular basis for non-congenital cataract development in humans and animals.</a> Journal of Cell Biology. 130, 169-181 (1995).</li><li>Michael, R., Vrensen, G., van Marle, J., Gan, L., S&#246;derberg, P. 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Keine Interessenskonflikte erklärt.

<html> Dieses Papier beschreibt Methoden, die Untersuchung der molekularen Ereignisse, die während UVR-B induzierte Katarakt ermöglichen. In Anbetracht, dass die meisten Informationen für in vivo UVR induzierten Katarakt aus Experimenten an Albino-Sprague-Dawley-Ratten 7, 16, 17, 18, ​​19 abgeleitet wurde, beschlossen wir, die Albino-Sprague-Dawley-Ratte in der aktuellen Studie zu verwenden. Alter der Ratten betrug sechs Wochen alte. Das Geschlecht wurde gewählt, w...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> Ketalar </td><td> Pfizer </td><td> 150086 </td><td> 50 mg / ml </td></tr><tr><td> Rompun </td><td> Bayer </td><td> 022545 </td><td> 20 mg / ml </td></tr><tr><td> Oculentum Simplex </td><td> Apoteket, Schweden </td><td> 336164 </td><td> 5 g </td></tr><tr><td> Mydriacyl </td><td> Alcon </td><td> 00352 </td><td> 10 mg / ml </td></tr><tr><td> Balanced Salzlösung </td><td> Alcon </td><td> 0007950055 </td><td></td></tr><tr><td> 3,5 ul β-Mercaptoethanol </td><td></td><td></td><td></td></tr><tr><td> NucleoSpin RNA II Gesamt-RNA Isolation Kit </td><td> Macherey-Nagel GmbH &amp; Co, Düren, Deutschland </td><td> 740955,50 </td><td></td></tr><tr><td> p53 DNA spezifischen Primern </td><td> biomers.net GmbH </td><td> Maßgeschneiderte </td><td></td></tr><tr><td> Taq-DNA-Polymerase, dNTPack </td><td> Roche </td><td> 04 728 866 001 </td><td></td></tr><tr><td> Agarose </td><td> Sigma </td><td> A5093 </td><td></td></tr><tr><td> Ethidiumbromidlösung 0,5 mg / ml </td><td> Sigma </td><td> E1385 </td><td></td></tr><tr><td> Nano-Drop ND-1000 Spektralphotometer </td><td> NanoDrop Produkte </td><td></td><td></td></tr><tr><td> 1. cDNA-Synthese-Kit für RT-PCR (AMV) </td><td> Roche Diagnostics GmbH </td><td> 11 483 188 001 </td><td></td></tr><tr><td> iCycler MyiQ Single Color Real Time PCR Detection System </td><td> Bio-Rad Laboratories </td><td></td><td></td></tr> &lt;tr&gt; <td> 96-Well-Platte </td><td> Sarstedt </td><td> 72.1979.202 </td><td></td></tr><tr><td> TaqMan Gene Expression Master Mix </td><td> Applied Biosystems </td><td> 4369016 </td><td></td></tr><tr><td> TaqMan Gene Expression Assay für Caspase 3 </td><td> Applied Biosystems </td><td> Rn00563902_m1 </td><td></td></tr><tr><td> TaqMan Gene Expression Assay für 18s </td><td> Applied Biosystems </td><td> Hs99999901_s1 </td><td></td></tr><tr><td> MyiQ Software </td><td> Bio-Rad Laboratories </td><td></td><td></td></tr><tr><td> Gespaltenen Caspase-3 (Asp175) </td><td> Cell Signaling Technology </td><td> 9661 </td><td></td></tr><tr><td> Anti-Kaninchen-IgG </td><td> Abcam </td><td> Ab6798 </td><td></td></tr><tr><td> Saccharose </td><td> Sigma Aldrich </td><td> 84097 </td><td></td></tr&gt; <tr><td> Vectashield </td><td> Vector Laboratories </td><td></td><td></td></tr><tr><td> Universal-Mikroskop Axioplan 2 Imaging </td><td> Carl Zeiss </td><td></td><td></td></tr><tr><td> Paraformaldehyd </td><td> Sigma Aldrich </td><td> 441244 </td><td></td></tr><tr><td> TBE-Puffer 10x </td><td> Promega </td><td> V4251 </td><td></td></tr></tbody></table>

characterization of molecular mechanisms of in vivo uvr induced cataract

Grauer Star ist die häufigste Ursache für Erblindung in der Welt ein. Die Weltgesundheitsorganisation definiert Katarakt als einer Trübung der Linse des Auges, die die Übertragung von Licht behindert. Grauer Star ist eine multifaktorielle Erkrankung mit Diabetes, Rauchen, UV-Strahlung (UVR), Alkohol, ionisierende Strahlung, Steroide und Hypertonie. Es gibt starke experimentelle 2-4 und epidemiologische Evidenz 5,6, dass UVR Katarakt verursacht. Wir entwickelten ein Tiermodell für UVR B induzierten Katarakt sowohl betäubt 7 und nicht-narkotisierten Tieren 8. Das einzige Heilmittel für die Katarakt-Chirurgie ist aber diese Behandlung ist nicht für alle zugänglich. Es wurde geschätzt, dass eine Verzögerung des Einsetzens von Katarakt für 10 Jahre könnte die Notwendigkeit für die Kataraktchirurgie um 50% 9 zu reduzieren. Um das Auftreten von Katarakt verzögern, ist es notwendig, die Mechanismen der Entstehung von grauem Star zu verstehen und wirksame Prävention Strategiegien. Unter den Verfahren zur Kataraktentwicklung spielt Apoptose eine wichtige Rolle bei Einleitung von Katarakt bei Menschen und Tieren 10. Wir konzentrieren uns wurde kürzlich Apoptose in der Linse als Mechanismus zum Kataraktentwicklung 8,11,12. Es wird erwartet, dass ein besseres Verständnis der Wirkung von UV-Strahlung auf der Apoptoseweg werden Möglichkeiten für die Entdeckung neuer Arzneimittel zu verhindern Katarakt liefern. In diesem Artikel beschreiben wir, wie Katarakt kann experimentell durch in vivo Exposition gegenüber UVR-B induziert. Weitere RT-PCR und Immunhistochemie dargestellt als Werkzeuge zur molekularen Mechanismen von UVR-B induzierte Katarakt studieren.

Die verschiedenen Quellen der Variation in den Messungen wurden mit einer Varianzanalyse geschätzt und es wurde festgestellt, dass unter Berücksichtigung drei Messungen pro Tier die Varianz für Messungen in der Größenordnung von 15% der für Tiere war. Somit, wenn man bedenkt gesamte Linse Analyse ist es nicht möglich, die Genauigkeit zu erhöhen. Orthogonal Tests aufgeklärt einen statistisch signifikanten Unterschied für die Caspase-3-Nachricht zwischen 120 hr Latenzzeit im Vergleich zu kürzeren Latenzzeiten A...

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Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

Grauer Star ist die häufigste Ursache für Erblindung in der Welt. Ultraviolette Strahlung der Sonne (UV) ist der Hauptrisikofaktor für die Katarakt-Entwicklung. Ein Tiermodell von weit UVR-B induzierte Katarakt entwickelt. In diesem Artikel beschreiben wir Methoden für die Untersuchung der Entstehung von grauem Star: Exposition gegenüber UV-Strahlung, quantitative RT-PCR und Immunhistochemie.

Charakterisierung der molekularen Mechanismen der<em&gt; In vivo</em&gt; UVR Induced Cataract

Cataract is the leading cause of blindness in the world 1. The World Health Organization defines cataract as a clouding of the lens of the eye which ...

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Research

JoVE Journal

Medicine

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

9.8K Aufrufe.  Karolinska Institutet. Grauer Star ist die häufigste Ursache für Erblindung in der Welt. Ultraviolette Strahlung der Sonne (UV) ist der Hauptrisikofaktor für die Katarakt-Entwicklung. Ein Tiermodell von weit UVR-B induzierte Katarakt entwickelt. In diesem Artikel beschreiben wir Methoden für die Untersuchung der Entstehung von grauem Star: Exposition gegenüber UV-Strahlung, quantitative RT-PCR und Immunhistochemie.

Serie: Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

In vivo Near Infrared Fluorescence (NIRF) intravaskuläre Molecular Imaging von entzündlichen Plaque, einen multimodalen Ansatz, um Imaging der Atherosklerose

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

Forschung

Medizin

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

Ein In vivo Nager-Modell der Kontraktion-induzierte Verletzung und nicht-invasive Überwachung von Recovery

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In-vivo-19 F-MRT für die Zellverfolgung

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

Molecular Profiling von der invasiven Tumor-Microenvironment in einem 3-Dimensional Model of Colorectal Cancer Cells und<em&gt; Ex-vivo-</em&gt; Fibroblasten

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapie-induzierte vaskuläre Toxizität - Echtzeit-<em&gt; In-vivo-</em&gt; Imaging von Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Aufbau und Charakterisierung von UTI und CAUTI in einem Mausmodell

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

Depletion von Maus-Zellen aus menschlichem Tumorxenografte deutlich verbessert Downstream-Analyse von Zielzellen

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

In Vitro und In Vivo Nachweis von Mitophagy in menschlichen Zellen, C. Elegansund Mäuse

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac performance in the absence of hypertension, valvular, congenital, or ischemic heart diseases, and which may be related to infection, autoimmune or metabolic abnormalities, or family inheritance. It can progress into congestive heart failure with a poor prognosis. Doxorubicin (Dox) is widely employed as a chemotherapeutic drug, but its use is limited because it causes DCM-like changes of the myocardium. Its myocardial toxicity is attributed to oxidative stress, chronic inflammation, and cardiomyocyte apoptosis. A model of DCM exploiting these Dox-induced DCM symptoms has not been established.

Described is a protocol to establish a Doxorubicin-induced dilated cardiomyopathy (DCM) model in mice via long-term intraperitoneal injection of Doxorubicin.

Ein Doxorubicin-induziertes Murinmodell der dilatativen Kardiomyopathie in vivo

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac ...

Generation and Characterization of Right Ventricular Myocardial Infarction Induced by Permanent Ligation of the Right Coronary Artery in Mice

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In contrast to the extensively used mouse myocardial infarction (MI) model generated by left coronary artery ligation, the RVI mouse model is rarely employed due to the difficulty associated with model generation. Research on the mechanisms and treatment of RVI-induced RV remodeling and dysfunction requires animal models to mimic the pathophysiology of RVI in patients. This study introduces a feasible procedure for RVI model generation in C57BL/6J mice. Further, this model was characterized based on the following: infarct size evaluation at 24 h after MI, assessment of cardiac remodeling and function with echocardiography, RV hemodynamics assessment, and histology of the infarct zone at 4 weeks after RVI. In addition, a coronary vasculature cast was performed to observe the coronary arterial arrangement in RV. This mouse model of RVI would facilitate the research on mechanisms of right heart failure and seek new therapeutic targets of RV remodeling.

There are several differences between the right and left ventricles. However, the pathophysiology of right ventricular infarction (RVI) has not been clarified. In the present protocol, a reproducible method for RVI mouse model generation is introduced, which may provide a means to explain the mechanism of RVI.

Erzeugung und Charakterisierung eines rechtsventrikulären Myokardinfarkts induziert durch permanente Ligation der rechten Koronararterie bei Mäusen

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In ...