Name: in vivo vascular permeability detection in mouse submandibular gland
Uploaded: 2022-08-04T14:00:00
Description: Watch this Scientific Journal Video about In Vivo Vascular Permeability Detection in Mouse Submandibular Gland at JoVE.com

Diese Studie wurde von der National Natural Science Foundation of China (Zuschüsse 31972908, 81991500, 81991502, 81771093 und 81974151) und der Beijing Natural Science Foundation (Grant 7202082) unterstützt.

Alle experimentellen Verfahren wurden von der Ethikkommission für Tierforschung des Gesundheitswissenschaftlichen Zentrums der Universität Peking genehmigt und entsprachen dem Leitfaden für die Pflege und Verwendung von Labortieren (NIH-Publikation Nr. 85-23, überarbeitet 1996). Für die vorliegende Studie wurden männliche Wildtyp-Mäuse (WT) in der Altersgruppe von 8-10 Wochen verwendet. Die Versuchstiere wurden sorgfältig behandelt, um ihre Schmerzen und Beschwerden zu minimieren.
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similarities+and+differences+in+the+regulation+of+leukocyte+extravasation+and+vascular+permeability.">Similarities and differences in the regulation of leukocyte extravasation and vascular permeability.</a> Seminars in Immunopathology. 36 (2), 177-192 (2014).</li><li>Schulte, D., 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=Stabilizing+the+VE-cadherin-catenin+complex+blocks+leukocyte+extravasation+and+vascular+permeability.">Stabilizing the VE-cadherin-catenin complex blocks leukocyte extravasation and vascular permeability.</a> The EMBO Journal. 30 (20), 4157-4170 (2011).</li><li>Uhl, B., 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=A+novel+experimental+approach+for+in+vivo+analyses+of+the+salivary+gland+microvasculature.">A novel experimental approach for in vivo analyses of the salivary gland microvasculature.</a> Frontiers in Immunology. 11, 604470 (2020).</li></ol>

Die Aufrechterhaltung und Regulierung der endothelialen Barrierefunktion sind essentiell für die vaskuläre Homöostase. Endothelzellen und ihre interzellulären Verbindungen spielen eine entscheidende Rolle bei der Aufrechterhaltung und Kontrolle der vaskulären Integrität12. Die Scherkraft des Blutflusses, Wachstumsfaktoren und Entzündungsfaktoren können Veränderungen der vaskulären Permeabilität verursachen und somit am Auftreten und der Entwicklung systemischer Erkrankungen wie Bluthoch...

Verschiedene Speicheldrüsen produzieren Speichel, der in erster Linie als erste Verteidigungslinie gegen Infektionen fungiert und die Verdauung unterstützt und dadurch eine wesentliche Rolle für die Mund- und allgemeine Gesundheit spielt1. Die Blutversorgung ist entscheidend für die Speicheldrüsensekretion, da sie ständig Wasser, Elektrolyte und Moleküle liefert, die den primären Speichel bilden. Die endotheliale Barrierefunktion, die durch den Tight Junction (TJ)-Komplex reguliert wird, begrenzt streng und empfindlich die Permeation von Kapillaren, die für Wasser, gelöste Stoffe, Proteine und sogar Zellen, die sich von den zirkulierenden Blutgefäßen in das Speicheldrüsengewebe bewegen, sehr durchlässig sind 2,3. Wir haben bereits festgestellt, dass die Öffnung der endothelialen TJs als Reaktion auf einen cholinergen Reiz die Speichelsekretion erleichtert, während die Beeinträchtigung der endothelialen Barrierefunktion mit Hyposekretion und lymphatischer Infiltration in den submandibulären Drüsen (SMGs) beim Sjögren-Syndrom4 verbunden ist. Diese Daten deuten darauf hin, dass dem Beitrag der endothelialen Barrierefunktion in Bezug auf eine Vielzahl von Speicheldrüsenerkrankungen genügend Aufmerksamkeit geschenkt werden muss.
Ein Zwei-Photonen-Laser-Scanning-Mikroskop ist ein leistungsfähiges Werkzeug, um die Dynamik von Zellen in intaktem Gewebe in vivo zu beobachten. Einer der Vorteile dieser Technik besteht darin, dass Nahinfrarotlicht (NIR) eine tiefere Gewebedurchdringung aufweist als sichtbares oder ultraviolettes Licht, wenn die Proben durch NIR angeregt werden, und unter geeigneten Bedingungen keine offensichtlichen Lichtschäden an Geweben verursacht 5,6. Tatsächlich sind die Speicheldrüsen ein sehr homogenes und oberflächliches Gewebe, in dem die oberflächlichen Azinuszellen nur etwa 30 μm von der Drüsenoberfläche entfernt sind 7,8. Es wurde gezeigt, dass die intravitale konfokale Mikroskopie die exokrine Sekretion und das Aktinzytoskelett in lebenden Mausspeicheldrüsen mit subzellulärer Auflösung untersuchen kann8. Die Zwei-Photonen-Laser-Scanning-Mikroskopie hat jedoch nicht nur den Vorteil der konventionellen konfokalen Mikroskopie, sondern kann auch verwendet werden, um tieferes Gewebe und Bild klarer zu erkennen. Hier können fluoreszenzmarkierte Dextrans, die häufig als parazelluläre Permeabilitätstracerate eingesetzt werden und den Vorteil unterschiedlicher Größen haben, verwendet werden, um die Größe der TJ-Pore9 zu testen. In der vorliegenden Studie wird eine intravitale Echtzeit-Zwei-Photonen-Laser-Scanning-Mikroskopie-Technik zur in situ Bewertung der endothelialen Barrierefunktion in Maus-SMGs etabliert. Jeder Arbeitsschritt zur in vivo vaskulären Permeabilitätsdetektion in Maus-SMGs ist im aktuellen Protokoll beschrieben. Hier ist ein Beispiel für die Detektion der endothelialen Barrierefunktion im SMG-Gangligationsmodell der Maus.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-photon microscope (TCS-SP8 DIVE)</td>
			<td>Leica, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 kDa FITC-labeled dextran</td>
			<td>Sigma Aldrich</td>
			<td>46944</td>
			<td></td>
		</tr>
		<tr>
			<td>70 kDa rhodamine B-labeled dextran</td>
			<td>Sigma Aldrich</td>
			<td>R9379</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt tissue separation nickel</td>
			<td>Bejinghuabo Company</td>
			<td>NZW28</td>
			<td></td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Veet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable sterile syringe</td>
			<td>Zhiyu Company</td>
			<td></td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>Becton, Dickinson and Company</td>
			<td>0253316</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Leica Application Suite X software</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microtubes</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline 1x</td>
			<td>Servicebio</td>
			<td>G4207-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue scissors</td>
			<td>Bejinghuabo Company</td>
			<td>M286-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Tribromoethanol</td>
			<td>JITIAN Bio</td>
			<td>JT0781</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo vascular permeability detection in mouse submandibular gland

Speichel spielt eine wichtige Rolle für die Mund- und allgemeine Gesundheit. Die intakte endotheliale Barrierefunktion der Blutgefäße ermöglicht die Speichelsekretion, während die Endothelbarriere-Dysfunktion mit vielen sekretorischen Speicheldrüsenstörungen zusammenhängt. Das vorliegende Protokoll beschreibt eine in vivo parazelluläre Permeabilitätsnachweismethode zur Bewertung der Funktion von endothelialen Tight Junctions (TJs) in submandibulären Drüsen (SMG) der Maus. Zunächst wurden fluoreszenzmarkierte Dextrans mit unterschiedlichen Molekulargewichten (4 kDa, 40 kDa oder 70 kDa) in die eckigen Venen von Mäusen injiziert. Danach wurde das einseitige SMG seziert und in der kundenspezifischen Halterung unter einem Zwei-Photonen-Laser-Scanning-Mikroskop fixiert, und dann wurden Bilder für Blutgefäße, Akini und Kanäle aufgenommen. Mit dieser Methode wurde die dynamische Echtzeit-Leckage der unterschiedlich großen Tracer von Blutgefäßen in die basalen Seiten der Acini und sogar über die Azinusepithelien in die Kanäle überwacht, um die Veränderung der endothelialen Barrierefunktion unter physiologischen oder pathophysiologischen Bedingungen zu bewerten.

Dem Protokoll folgend, wurde das einseitige SMG an einem speziell angefertigten Halter befestigt und die Drüse so weit wie möglich vom Mauskörper entfernt gehalten, um zu verhindern, dass die Atmung Bewegungsartefakte verursacht. Der schnelle Fluss der roten Blutkörperchen (schwarze Punkte) in Blutgefäßen wurde unter dem Mikroskop beobachtet. Nachdem man das Gewebefeld unter einer Augenlinse gefunden hat, muss man umschalten, um die Mikroskopsoftware zu manipulieren. In der Kontrollgruppe befanden sich beide Tracer...

Watch this Scientific Journal Video about In Vivo Vascular Permeability Detection in Mouse Submandibular Gland at JoVE.com

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

Im vorliegenden Protokoll wurde die endotheliale Barrierefunktion der submandibulären Drüse (SMG) durch Injektion verschiedener molekulargewichteter Fluoreszenztracer in die Winkelvenen von Versuchstiermodellen in vivo unter einem Zwei-Photonen-Laser-Scanning-Mikroskop bewertet.

Im lebenden Organismus Nachweis der vaskulären Permeabilität in der submandibulären Drüse der Maus

Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the ...

Das vorliegende Protokoll beschreibt ein in vivo parazelluläres Permeabilitätsnachweismaß zur Bewertung der Funktion enger endothelialer Verbindungen in submandibulären Drüsen der Maus. Die Zwei-Photonen-Laser-Scanning-Mikroskopie hat jedoch nicht nur den Vorteil der konventionellen konfokalen Mikroskopie, sondern kann auch verwendet werden, um tieferes Gewebe zu erkennen und klarer abzubilden. Dieses Experiment bietet ein gutes methodisches Maß zur Beurteilung der vaskulären Permeabilität verschiedener Gewebe, insbesondere der Oberflächengewebe und Organe von Tieren.Beginnen Sie mit der Auswahl geeigneter Tracer wie Fluorescein, Isothiocyanat, markiertem Dextran und Rhodamin B, markiertem Dextran mit ausgeprägten Anregungsemissionsspektren, um die Interferenz zwischen Fluoreszenzsignalen für den Permeabilitätstest zu minimieren. Verdünnen Sie die Spuren zu steriler phosphatgepufferter Kochsalzlösung in 100 Milligramm pro Milliliter und lagern Sie die vor Licht geschützten Aliquots bei minus 20 Grad Celsius. Nach der Betäubung der 8 bis 10 Wochen alten männlichen Wildtyp-Maus halten Sie vorsichtig Kopf und Hals der Maus, damit eine Seite des Augapfels leicht hervorsteht.Führen Sie eine Insulinspritze mit 100 Mikrolitern fluoreszierender Tracerlösungsmischung entlang des Augenwinkels im rechten Winkel zum Auge ein. Nach der Injektion legen Sie die Maus schnell in Rückenlage mit Klebeband und geklebtem Kopf auf den Karton. Für die submandibuläre Drüse oder SMG-Isolierung schneiden Sie unter einem stereoskopischen Mikroskop die Epidermis des Halses mit einer allgemeinen Gewebeschere ab, um beide Seiten der SMGs freizulegen.Als nächstes verwenden Sie eine stumpfe Gewebetrennungsnadel, um die Kapsel sanft von der Drüsenoberfläche zu trennen, ohne das Drüsengewebe, die Blutgefäße und die Drüsenstruktur zu beschädigen. Schließen Sie die kundenspezifische Halterung an das Unterdruckgerät an und prüfen Sie im Vorfeld, ob die Unterdruckwirkung richtig erreicht wird. Legen Sie das freiliegende SMG vorsichtig auf den maßgeschneiderten Halter und saugen Sie das Gewebe durch Unterdruckabsaugen so weit wie möglich vom Mauskörper ab, um Bewegungsartefakte zu reduzieren, die durch Atmung und andere Bedingungen verursacht werden.Beginnen Sie mit der Bildgebung, kurz nachdem die Stelle der Drüse ausgewählt wurde. Erfassen Sie sequentielle Bilder entlang der Z-Achse, die zwei Mikrometer von der Oberfläche der Drüse entfernt sind, bis zu 70 bis 140 Mikrometer in das Gewebe, um eine dreidimensionale Konstruktion zu erzeugen. Als nächstes erfassen Sie eine Zeitrafferaufnahme der Gefäße, um die vaskuläre Permeabilität im SMG zu messen.Klicken Sie im Explorer-Dialog auf "Neuen Ordner hinzufügen" und ändern Sie den Dateinamen. Kehren Sie dann zur Spalte "Erfassung" zurück. In XY ist das Format 512 x 512 und die Geschwindigkeit beträgt 400 Hertz.Schalten Sie Bidirektional X" ein, stellen Sie den Zoomfaktor auf 0,75 und 1,5 für den Zoom ein. Als nächstes wählen Sie XYT unter "Aufnahmemodus", um Zeitrafferaufnahmen aufzunehmen. Stellen Sie die Dauer je nach experimentellem Bedarf auf 20 Sekunden, 30 Minuten oder länger ein.Nachdem Sie die Bedingungen eingestellt haben, klicken Sie auf "Live", um die vaskuläre Bildgebung von zwei Kanälen und überlappenden Kanälen im Fotoformungsrahmen zu beobachten und die Interessengebiete auszuwählen. Klicken Sie auf "Start", um die Bildgebung zu starten. In-vivo-Gefäßpermeabilitätstest und dreidimensionale Bilder von Blutgefäßen in Mäusen.Submandibuläre Drüsen sind hier dargestellt. In der Kontrollgruppe befanden sich beide Tracer in den Blutgefäßen der SMG. Aufgrund seines geringen Molekulargewichts konnte FD4 aus den Blutgefäßen zu den basalen Seiten von Acini und Kanälen austreten und so die Form der Acini und Gänge klar darstellen.Das RD70 wurde in großen Blutgefäßen und Mikrogefäßen verteilt. In der Duct-Ligationsgruppe wurden sowohl FD4 als auch RD70 auf die basalen Seiten von Acini extravasiert, was darauf hindeutet, dass die Duct-Ligatur die endotheliale Barrierefunktion stören und die Permeabilität für große Moleküle erhöhen könnte. Darüber hinaus bestätigten die semiquantitativen FD4- und RD70-Fluoreszenzintensitätsergebnisse diese Beobachtungen.Die dreidimensionalen Bilder zeigten eine viel verdecktere Fluoreszenz von FD4 und RD70 um Blutgefäße in der Ligationsgruppe im Vergleich zur Kontrollgruppe. Eine sorgfältige SMG-Isolierung und geeignete Platzierung sowie das Mikroskop sind wesentliche Voraussetzungen für eine erfolgreiche Detektion. Nach den Verfahren kann die Blutflussrate berechnet werden, indem die Bewegung der roten Blutkörperchen geheiratet und die Infiltration der fluoreszenzmarkierten Blutzellen verfolgt werden kann.

in-vivo-vascular-permeability-detection-in-mouse-submandibular-gland

Research

JoVE Journal

Biology

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Einzel-Zell-Saug-Recordings aus Maus Zapfen-Photorezeptoren

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Forschung

Biologie

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

In-vivo-Imaging und therapeutische Behandlungen in einem orthotopen Mausmodell des Eierstockkrebs

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Isolierung von Maus Speicheldrüsen Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

Evaluation der Brustdrüse Entwicklung und Funktion in Mausmodellen

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

Multi-Parameter-Messung der Durchlässigkeit Transition Pore Opening in isolierten Maus-Mitochondrien

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

Intraduktale Injection für Lokalisierte Drug Delivery auf die Maus Milchdrüse

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Messung des osmotischen Wasserdurchlässigkeit Koeffizient (P<sub&gt; F</sub&gt;) Von sphärischen Zellen: Isolierte Pflanzenprotoplasten als Beispiel

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Messung der Steifigkeit von<em&gt; Ex Vivo</em&gt; Maus Aorten Mit Rasterkraftmikroskopie

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions. However, due to the complexity of the process of SUMOylation in vivo, it is more challenging to detect and analyze protein SUMOylation in cells, especially when under endogenous conditions. A recent study by the authors of this paper revealed that endogenous retinoblastoma (Rb) protein, a tumor suppressor that is vital to the negative regulation of the cell cycle progression, is specifically SUMOylated at the early G1 phase. This paper describes a protocol for the detection and analysis of Rb SUMOylation under both endogenous and exogenous conditions in human cells. This protocol is appropriate for the phenotypical and functional investigation of the SUMO-modification of Rb, as well as many other SUMO-targeted proteins, in human cells.

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.

In Vivo Detektion und Analyse von Rb Protein Sumoylierung in menschlichen Zellen

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.