Supported by NIH award R01 DK093525 (GFG). We greatly appreciate the scientific review and grammatical editing of the manuscript by Dr. Amber Shaffer (University of Pittsburgh) and thank Michael Burcham for assistance in preparation of figures.

HINWEIS: Dieses Protokoll wurde überprüft und von der University of Pittsburgh Institutional Animal Care und Verwenden Committee genehmigt. 1. Herstellung von modifiziertem Krebs-Lösung und Test-Arzneimittel Aliquots <ol><li> Make 6 L modifizierte Krebs-Lösung, die (in mM) enthält: 117,9 NaCl, 4,7 KCl, 25 NaHCO 3, 1,3 NaH 2 PO 4, 1,2 MgSO 4, 2,5 CaCl 2, 11.1 D-Glucose, 2 Natriumbutyrat 20 Natriumacetat , 0,004 Nifedipin (spontane Muskelkontraktionen blockieren) und 0,003 Indometacin (die Synthese von endogenem Prostaglandin blockieren). Verwenden eiskalt und warmen Krebs Lösungen für Gewebedissektion und Einzelfaser-Aufnahme auf. </li><li> Bereiten keine chemischen Lösungen (zB, IS, Capsaicin, ATP) in Aliquots bei gewünschten Konzentrationen. </li></ol> 2. Dissektion der Kolorektums-Nervengewebe <ol><li> Betäuben und euthanize männliche Mäuse (6 - 8 Wochen alt, 20 - 30 g) inein CO 2 Kammer mit einer Strömungsgeschwindigkeit, die mit 10 verdrängt - 30% des Kammervolumens pro Minute bis Mäusen atmen aufhören vollständig wie durch das Fehlen von Brustbewegung angegeben. </li><li> Unmittelbar nach Euthanasie, exsanguinate durch Aufschneiden der Thoraxkammer, Perforation des rechten Vorhofs und das Eintauchen der Maus Karkasse in einem großen Volumen (~ 500 ml) eiskaltem Krebs (4 ° C) Lösung geblasen mit Carbogen (95% O 2 und 5% CO 2). </li><li> Alle inneren Organe, sondern auch die Dickdarm-und Beckenorgane vorsichtig entfernen. Transekt mit der Maus in die Hälfte über dem Wirbelsegment T12 leicht über dem Zwerchfell und übertragen Sie die Schwanz Hälfte zu einer Dissektion Kammer, eiskalt, sprudelte Krebs-Lösung. </li><li> Unter einem Stereomikroskop, entfernen Sie die Blase und die Fortpflanzungsorgane durch Durchtrennung an ihren Verbindungsstellen an der Harnröhre, und entfernen Sie den absteigenden / Bauchaorta, bis sie in gemeinsamen Beckenarterien gabelt. Befreien Sie den PN oder die LSN von ihren surrounding Gewebe stumpf und folgen Sie den Nerv von außerhalb des Beckenkamm bis zu seiner ventralen Einstieg in die L6 und S1 Wirbelsäule (PN) oder T13 und L1 Wirbelsäule (für LSN). </li><li> Schneiden Sie die Symphyse und rechts und links Pfannen Gelenke, und entfernen Sie das Darmbein. Sorgfältig kostenlos entweder die PN oder LSN aus dem beigefügten Muskels und des Bindegewebes aus der Nähe des colorectal, bis wo der Nerv in die Wirbelsäule. </li><li> Sorgfältig Resektion des Darmbein, um den distalen Kolorektums aussetzen. Präparieren Sie die distale Kolorektums mit dem angebrachten PN oder LSN im Kontinuum. </li><li> Übertragen Sie die Kolorektums mit dem Nerv in das Bad Fach der Gewebekammer angebracht. Überschüssiges Bindegewebe durch weitere Dissektion, und öffnen Sie die Kolorektums Längsrichtung entlang des Anti mesenterialen Grenze. </li><li> Mit Schleimhautseite nach oben, stecken Sie die mesenterialen Rand des Kolorektums neben dem Aufnahmeraum in die Silikonbasis der chamber und befestigen Sie den antimesenteriale Länge des Kolorektums an einen Rake von Haken mit einem Kraftstellglied verbunden (in Abbildung 1 veranschaulicht und in 2A fotografiert). </li><li> Erweitere PN oder LSN in die Aufnahmefach, das zu dem Bad Raum durch eine Mausloch und dem Gate verbunden ist. Legen Sie vorsichtig den Nervenstamm auf einen kleinen Glasspiegel im Aufnahmeraum, der eine hydrophile Oberfläche für den Mut, zu halten bietet. Superfuse das Baderaum mit warmem (30 bis 32 ° C), mit Sauerstoff angereicherte Krebs-Lösung und füllt die Aufnahmefach mit Mineralöl. </li></ol> 3. Einzelfaser-Aufnahme und Lokalisierung der rezeptiven Feld <ol><li> Schieben Sie vorsichtig die Epineurium (Nervenscheide) der PN oder LSN unter dem Stereomikroskop bei hoher Vergrößerung (50 - 60X). Mit einer feinen Pinzette, necken den Nervenstamm in 5-8 Nervenbündel von ~ 100 &amp; mgr; m Dicke. </li><li> Legen Sie die Platin-Iridium-ReferenzElektrode in Kontakt mit dem Krebs-Lösung in das Gewebe Kammer. Sequentielles Platzieren der einzelnen Nervenbündel auf die Aufzeichnungselektrode aus dem gleichen Material hergestellt. </li><li> Mit einem weichen Pinsel, um APs von den Dick Afferenzen durch sanft streichelte oben und unten der kolorektalen Oberfläche hervorrufen. Suchen Sie den Nervenbündels (n), die den Doppelpunkt durch nachweisbare AP (Aktionspotential) Aufnahmen innervieren. HINWEIS: Die PN und LSN auch die Harnblase und andere Beckenorgane innervieren. </li><li> Verwenden Sie ein Paar 30 G Nadelspitzen weiter zu spalten die Nervenbündels in feine Faszikel Filamente ~ 10 &amp; mgr; m Dicke und legen Sie einen individuellen Filament auf die Aufzeichnungselektrode. </li><li> Legen Sie die rund Spitze konzentrischen Elektrode senkrecht zur Schleimhautoberfläche zu afferenten Enden am Überschwellenreizstärke (10 mA Stärke, 0,5 ms Dauer @ 0,3 Hz), die eine mm Radius von aktuellen Ausbreitung ~ 2 produziert elektrisch erregt. Bewegen Sie den Elektroden systematisch (~ 1,5 mm Schritten) Entlang der Länge und Breite des abgeflachten colorectum empfänglich Digungen lokalisieren. </li><li> Wenn ein afferenten Ende ist aufgeregt, stellen Sie die Elektrodenposition, um den Ort der Aktivierung (rezeptiven Feld, HF), die minimale Reizstärke (Reizschwelle) erfordert genau zu bestimmen. Entsorgen Enden mit einer Reizschwelle&gt; 3 mA 19. </li><li> Berechnen der Leitungsgeschwindigkeit (CV) von 1) der Abstand zwischen der Stimulationselektrode an dem Empfangsfeld (RF) und der Aufzeichnungsstelle, und 2) die Leitungsverzögerung (zB, 2B) zwischen dem Reizartefakt und dem Einsetzen des Aktionspotentials . CV (m / sec) = Abstand (mm) / Leitungsverzögerung (msec). </li></ol> 4. Funktions Einstufung mechanosensitiven Colorectal Afferenzen <ol><li> Nach dem Auffinden eines RF durch elektrische Stimulation, gelten die folgenden drei mechanische Reize an den HF: <ol><li> Führen Sie die Sondierung Reiz, indem Sie die Spitze eines calibrated von Frey-like Nylonmonofilament (0,4 und 1 g Kraft), die senkrecht auf die RF auf der abgeflachten Kolorektums. </li><li> Führen Sie den Reiz von streichelte sanft streichelte den kolorektalen Schleimhaut mit einer feinen Nylonfaden Strang (10 mg Kraft), eine kleine Oberflächenscherspannung an der RF zu erzeugen. </li><li> Führen Sie die Umfangsdehnung mit Hilfe eines computergesteuerten Kraftantrieb, die eine rampenförmige Strecke Kraft liefert (0-170 mN bei 5 mN / sec) in Umfangsrichtung entlang der Anti mesenterialen Rand des Kolorektums über den Rechen des in Schritt 2.8 beschrieben Haken . </li></ol></li><li> Bewerte Afferenzen als Serosa (reagieren nur auf stumpf Sondieren), Schleimhaut (reagieren auf Schleimhaut streicheln und stumpf Sondieren), muskulös (zum Umfangsdehnung reagieren und stumpf Sondieren) Muskel / Schleimhaut (zum Umfangsdehnung, Schleimhaut Streicheln reagieren und stumpf Sondieren), oder MIA (nicht in Reaktion auf jede der drei mechanischen Reizen). </li><li> Für mesenterialen Afferenzen (nur indie LSN Innervation), die schwierig sind, um selektiv durch elektrische Stimulation zu aktivieren, suchen ihre rezeptiven Enden durch mechanische Streicheln / Sondierung des Mesenteriums. </li><li> Für Stretch-sensitive Afferenzen (Muskel-und Muskel-Schleimhaut), bestimmen die Ansprechschwelle, die als die Kraft, die die erste AP während der geneigte Strecke erinnert definiert ist. </li><li> Für serösen Afferenzen, notieren ihre Antworten auf aufsteigend Ebenen der punktförmige Sondieren des rezeptiven Feldes durch die computergesteuerte Kraftstellglied angetrieben. </li></ol> 5. Chemische Anwendung / Modulation der Empfang Endings <ol><li> Zeichnen Sie eine Baseline-Reaktion auf einen mechanischen Reiz (dh Reaktion auf geneigten Strecke, punktförmige Sondieren oder Schleimhaut Streicheln). </li><li> Bestreichen Sie die Unterkante eines Schlauchstück (Messing oder Edelstahl, 10 mm Höhe und 4 x 4 mm 2 quadratisch oder 4-5 mm Durchmesser) mit Vaseline und legen Sie es über dem rezeptiven Feld auf der Kolorektums. </li><li> Entfernendie Krebs-Lösung im Inneren des Schlauchs, und setzen die aufnahme Endung für 3 - 5 min zu 170 ul der Lösung, die die Chemikalie (n) zu prüfen. </li><li> Beobachten Sie die Reaktion des afferenten bei der chemischen Anwendung (einige Afferenzen sind chemosensitiven). </li><li> Entfernen Sie die chemische Lösung und die Schläuche, die Wirkung der chemischen beenden. In 4 - 6 min, testen die afferenten Reaktion auf denselben mechanischen Stimulus, wie in der Grundreaktion. </li><li> Wenden Sie die mechanischen Reiz wieder nach ausreichenden Zeitraum Auswaschen (&gt; 15 min). </li></ol> 6. Aufnahme und Diskriminierende AP Spikes <ol><li> Digitalisieren der elektrischen Signale von Axonen bei 20 kHz aufgenommen und speichern Sie die Daten auf einen Computer. Überwachen Sie die Signal online durch eine Audio-Überwachung. </li><li> Analysieren Sie die AP Spitzen off-line und diskriminieren einzelne Einheiten basierend auf der Hauptkomponentenanalyse einzelner Dorn 25 Wellenformen. HINWEIS: Ein Datensatz sollte nicht mehr th enthaltenein zwei leicht unterscheidbar aktiven Einheiten. </li></ol>

<ol>
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The authors have nothing to disclose.

Die hier beschriebene in vitro-Nerven Kolorektums Vorbereitung hat sich als leistungsfähiges Konzept für neuronale Kodierung Funktionen einzelner kolorektalen Afferenzen, die schön ergänzt andere nicht-funktionale Ansätze (zB Zell-, Molekular- und histologische Untersuchungen) auf viszeralen sensorischen Neuronen (studieren siehe Bewertung 27 für Details). Neuronalen Mechanismen Beitrag zur Nozizeption und langfristig colorectal Überempfindlichkeit festgestellt worden sind und pharmak...

Schmerzüberempfindlichkeit sind die vorherrschenden Beschwerden von Patienten mit funktionellen gastrointestinalen Störungen einschließlich Reizdarmsyndrom (IBS), die in Abwesenheit des sichtbaren pathobiologischen Ursache oder Gewebeschäden vorliegen. Zum Beispiel weisen IBS-Patienten Überempfindlichkeit, einschließlich erhöhter Antworten auf rektale Ballon Spannungsgefühl und erhöhte Empfindlichkeit während des normalen Darmfunktion sowie Überempfindlichkeit der somatischen Weisung (dh Druckschmerz im Bauchbereich) 1. Da Targeting kolorektalen Afferenzen hat sich als wirksam bei der Linderung von Schmerzen und Überempfindlichkeit in IBS-Patienten (zB intrarektale Einträufeln von Lokalanästhetika 2,3; orale Aufnahme des Guanylatzyklase-C-Agonisten linaclotide 4-6) sein, ein besseres Verständnis der afferenten Innervation Kolorektum ist wichtig. Visceral Afferenzen, darunter Dickdarm- AfferenzenIn der Lage sind zu reagieren, um chemische / Nähr- und thermischen Behandlungsformen (zum Beispiel 7-9). Allerdings viszerale Afferenzen Reaktion auf mechanische Reize (dh mechanosensitive Afferenzen) waren die am besten untersuchte, weil mechanische Reize (zB Lumen Blähungen, Dehnung) sind diejenigen, die in der Regel führen zu bewussten Empfindungen, einschließlich Beschwerden und Schmerzen 10-16. Darüber hinaus werden die Eingeweide auch mechanisch unempfindlich Afferenzen (MIAs) innerviert, allgemein als stille oder Schlaf Nozizeptoren 17. Unter normalen physiologischen Bedingungen, müssen MIAs nicht auf mechanische Reize reagieren und haben eine sehr hohe Ansprechschwellen 18, sondern kann aktiv werden und erwerben Mechanosensitivität in pathophysiologischen Bedingungen und dazu beitragen, Überempfindlichkeit. Unter Verwendung der in vitro Herstellung und hier beschriebene Protokoll, entwickelt und verwendet ein elektrisches Stimulus-Strategie in See wir<html> rch für aufnahmeEnden und ermöglicht unvoreingenommene Identifizierung der beiden mechanosensitive und MIA Enden in der Kolorektums 19. Das kolorektale Innervation aus Lenden splanchnischen (LSN) und Beckennerv (PN) Wege abgeleitet und enthält kolorektalen Afferenzen, die in fünf mechanosensitive Klassen (seröse, Schleimhäute, muskulös, Muskel-Schleimhaut, mesenterialen) und eine MIA-Klasse 20 eingestuft werden können. Mit dieser In-vitro-Vorbereitung, fanden wir, dass kolorektalen MIAs erworben Mechanosensitivität (Sensibilisierung) nach kurzzeitiger ihres rezeptiven Felder zu einer entzündlichen Suppe (IS), die 71% der MIAs in der PN-Bahn und 23% der MIAs im LSN Weg sensibilisiert 19. Wir haben auch dokumentiert Langzeitsensibilisierung (bis 28 Tage) von MIA in Zusammenhang mit langanhaltenden Verhaltens viszerale Hypersensitivität (dh in Mäusen intrakolische Behandlungen mit Zymosan 21 oder 2,4,6-Trinitrobenzolsulfonsäure (TNBS) 22) . jove_content &quot;&gt; Unter mechanosensitive Afferenzen, muskulös und Muskel-Schleimhaut-Afferenzen sind die einzigen Klassen, die tonisch kodieren Umfangsdehnung des colorectal (dh sind Stretch-und Kleinschreibung) und dienstbar die Kodierung der schädlichen kolorektale Dehnung 23,24. Mit Hilfe eines computergesteuerten Kraftaktuator wir mit einem Standard, homogene und reproduzierbare gefahren Dehnung in der Umfangsrichtung des abgeflachten kolorektales Gewebe und weiter kategorisiert dehnungsempfindlichen Afferenzen als Niedrigschwellenwert und hohem Schwellenwert 23. Darüber hinaus den Zeitverlauf der Sensibilisierung von Dehnungs sensitive Afferenzen nach intrakolische Zymosan 21 oder TNBS 22 Behandlung entspricht der Beginn, Ausdauer, und / oder Wiederherstellung von Verhaltens viszerale Hypersensitivität, was auf eine Rolle von Dehnungsempfindlichen Dick Afferenzen in viszeralen Schmerzen und Überempfindlichkeit.

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			<td height="21" style="height:21px;width:375px;">Name of Material/ Equipment</td>
			<td style="width:291px;">Company</td>
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			<td style="width:372px;">Comments/Description</td>
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			<td height="21" style="height:21px;">Leica MZ16 stereo microscope&#160;</td>
			<td>Leica Microsystems Inc.</td>
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			<td></td>
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			<td height="21" style="height:21px;">Leica IC D camera</td>
			<td>Leica Microsystems Inc.</td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Amplifier</td>
			<td>World Precision instruments, Inc.</td>
			<td>SYS-DAM80&#160;</td>
			<td>Low-noise differential amplifier</td>
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			<td height="21" style="height:21px;width:375px;">Two-compartment tissue chamber</td>
			<td style="width:291px;">Custom made</td>
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			<td></td>
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			<td height="21" style="height:21px;width:375px;">Power1401</td>
			<td>Cambridge Electronic Design Limited</td>
			<td style="width:137px;">Power1401</td>
			<td>Data acquisition, analog signal input/out</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Spike2 v5.02</td>
			<td>Cambridge Electronic Design Limited</td>
			<td style="width:137px;"></td>
			<td>Software package that works with the Power1401</td>
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			<td height="21" style="height:21px;width:375px;">Audio monitor</td>
			<td>Natus</td>
			<td style="width:137px;">Am 8</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Square pulse stimulator</td>
			<td>Natus</td>
			<td style="width:137px;">S48</td>
			<td>To deliver electrical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Photoelectric isolation unit</td>
			<td>Natus</td>
			<td style="width:137px;">PSIU6</td>
			<td>Stimulus isolation to reduce noise</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Concentric bipolar microelectrode</td>
			<td style="width:291px;">FHC Inc.</td>
			<td style="width:137px;">CBFFG75</td>
			<td>To deliver electrical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dual-mode lever system</td>
			<td>Aurora Scientific Inc.</td>
			<td style="width:137px;">Series 300C&#160;</td>
			<td>To deliver mechanical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td>forceps with fine tips</td>
		</tr>
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in vitro functional characterization of mouse colorectal afferent endings

Dieses Video zeigt im Detail ein in vitro-Einzelfaser elektrophysiologische Ableitung Protokoll mit einer Maus Kolorektums-Nerven Vorbereitung. Der Ansatz ermöglicht unvoreingenommene Identifizierung und funktionelle Charakterisierung einzelner kolorektalen Afferenzen. Extrazelluläre Ableitungen von propagierten Aktionspotentiale (APs), die von einem oder wenigen afferenten stammen (dh Einzelfaser) rezeptiven Felder (RFS) in Kolorektum aus gehänselt Nervenfaser Faszikel gemacht. Die Kolorektums ist entweder mit dem Becken (PN) oder Lenden splanchnischen (LSN) Nerven befestigt und in Längsrichtung geöffnet entfernt. Das Gewebe wird in einer Aufnahmekammer gebracht, fixiert flach und mit Sauerstoff angereichertem Krebs-Lösung perfundiert. Brenn elektrischen Stimulation verwendet, um die kolorektale afferenten Endungen, die durch drei verschiedene mechanische Stimuli getestet lokalisieren (blunt Sondieren, Schleimhaut Streichen und Umfangsdehnung) funktionell zu kategorisieren Afferenzen in fünf mechanosensitive Klassen. Endings Reaktion auf keine dieser mechanische Reize werden als mechanisch unempfindlich Afferenzen (MIAs) kategorisiert. Sowohl mechanosensitive und MIAs kann Sensibilisierung (dh, verbesserte Reaktion, reduziert Schwelle und / oder Erwerb von Mechanosensitivität) bewertet werden, indem die lokale Exposition des RFs Chemikalien (zB entzündliche Suppe (IS), Capsaicin, Adenosintriphosphat (ATP)). Wir beschreiben die Geräte und Kolorektums-Nerven Aufnahme Vorbereitung, Ernte Kolorektums mit angeschlossenem PN oder LSN, die Identifizierung von RFs im Kolorektums, Einzelfaser-Aufnahme von Nervenfaszikel und lokalisierte Anwendung von Chemikalien auf die RF. Außerdem werden Probleme der Herstellung und Anwendung von standardisierten mechanischen Stimulation ebenfalls diskutiert.

Der Aufbau ist in Abbildung 1 dargestellt ist. Es umfasst eine maßgeschneiderte Gewebekammer, die die colorectum in einem Silikon-Bad ausgekleideten Kammer und der beigefügten Nerv in einem zusammenhängenden Mineralöl gefüllten Abteil untergebracht ist. Die Zwei-Kammer-Kammer wurde aus einem massiven Block aus Acrylkunststoff mit einer CNC-Maschine bearbeitet; der Boden der beiden Kompartimenten wurde anschließend mit festen Silikon ausgekleidet, um einfach Stift nach unten des kolorektalen Gewebe...

Watch this Scientific Journal Video about In vitro Functional Characterization of Mouse Colorectal Afferent Endings at JoVE.com

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

In-vitro-Funktionelle Charakterisierung von Maus Colorectal Afferente Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

in-vitro-functional-characterization-mouse-colorectal-afferent

Research

JoVE Journal

Medicine

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

9.5K Aufrufe.  University of Pittsburgh. This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation. 

Serie: In vitro Functional Characterization of Mouse Colorectal Afferent Endings

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

Die Verwendung von primären humanen Fibroblasten zur Überwachung Mitochondriale Phänotypen auf dem Gebiet der Parkinson-Krankheit

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Forschung

Medizin

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

In-vitro-Kultur von Organoide Primäre Maus Kolontumoren

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Isolierung und funktionelle Charakterisierung der Menschen ventrikuläre Kardiomyozyten von Fresh Chirurgische Proben

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

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. ...

Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable noninvasive method is essential to assess real-time cardiac morphological and functional changes in animal models of heart disease. Transthoracic echocardiography represents an important tool for noninvasive assessment of cardiac structure and function2. Here we used a high-resolution ultrasound imaging system to monitor myocardial remodeling and heart failure progression over time in a mouse model of TAC. B-mode, M-mode, and Doppler imaging were used to precisely assess cardiac hypertrophy, ventricular dilatation, and functional deterioration in mice following TAC. Color and pulse wave (PW) Doppler imaging was used to noninvasively measure pressure gradient across the aortic constriction created by TAC and to assess transmitral blood flow in mice. Thus transthoracic echocardiographic imaging provides comprehensive noninvasive measurements of cardiac dimensions and function in mouse models of heart disease.

The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

Beurteilung der Herz Morphologische und funktionelle Veränderungen in Maus-Modell der Quer Aortakonstriktion durch Echokardiographie

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

in Längsrichtung<em&gt; In Vivo</em&gt; Imaging des Cerebrovasculature: Bedeutung für ZNS-Erkrankungen

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

Vorbereitung und<em&gt; In Vitro</em&gt; Charakterisierung von Magnetized miR-modifizierten Endothelzellen

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

Entwicklung und Charakterisierung von dendritischen Zellen murinen Tolerogene

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

Functional Characterization of Endogenously Expressed Human RYR1 Variants

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients&#39; cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.

Funktionelle Charakterisierung endogen exprimierter humaner RYR1-Varianten

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia ...

Occlusion of the Great and Small Saphenous Vein Using Copolymeric Glue Based on N-Butyl Cyanoacrylate and Methacryloxy Sulfolane

We present the preliminary results of a longitudinal observational study aimed at evaluating the effectiveness and safety at different short- and long-term follow-ups of vascular occlusion of the great saphenous vein (GSV) and small saphenous vein (SSV) affected by severe pathological reflux, using an innovative modified cyanoacrylate surgical glue composed of N-butyl cyanoacrylate and methacryloxy sulfolane (NBCA+MS). Ninety patients, prospectively recruited for 1 year, underwent the study with EcoColor-Doppler (ECD) to evaluate the maximum diameters of the GSV and SSV in the orthostatic position and the reflux time (RT). An RT greater than 0.5 s was considered pathologic. Clinical, etiology, anatomy, and pathophysiology (CEAP) assessment was used for the complete evaluation of each patient in the study. All the patients were treated by NBCA+MS glue to obtain vein occlusion and observed before treatment (baseline; T0), within 6 h after treatment (T1), 1 month after treatment (T2), 3 months after treatment (T3), 6 months after treatment (T4), and 1 year after treatment (T5). Chi-square (&#967;) analysis was performed to evaluate the effectiveness and safety of the treatment. All the patients participated in the entire duration of the study. Complete occlusion was maintained in 100% of patients at T1, 98.9% at T2 and T3, and 97.8% at T4 and T5 (p &lt; 0.001). None of the patients suffered from post-surgical thrombosis. No blue hyperpigmentation, or paresthesia was observed during the entire observation period. Immediately after treatment, 7.7% of patients needed painkillers; 1 week after treatment, 100% of patients returned to normal life. Vascular occlusion of the great or small saphenous vein using NBCA+MS glue is a safe procedure with persistent benefits after a 1 year follow-up. This procedure can be performed with local anesthesia, allowing a quick return to normal life. Thanks to its low invasiveness, the treatment is not painful.

Here, we present a protocol to treat the great saphenous vein (GSV) and the small saphenous vein (SSV) affected by severe pathological reflux, using an original sclerosing and embolizing cyanoacrylate-based glue composed of N-butyl cyanoacrylate and methacryloxy sulfolane (NBCA+MS).