This work was supported in part by the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Program Merit Review Award I01-BX002336, the National Institute of Health grant R56-HL095498, as well as with the resources and the use of facilities at the VA Connecticut Healthcare System, West Haven, CT.

Die Genehmigung durch die entsprechende Institutional Animal Care und Use Committee erhalten. 1. Anästhesie und präoperative Verfahren <ol><li> Anesthetize männlichen C57Bl / 6 Eis im Alter von 8 Wochen mit verdampfte 3% Isofluran und 0,8 l / min Sauerstoff in eine Acrylinduktionskammer verabreicht. </li><li> Bestätigen ausreichende Anästhesie durch den Mangel an Reaktion auf Zehe Prise. 3% Isofluran durch kontinuierliche Inhalation - Bewegen Sie die Maus in Rückenlage auf dem Operationstisch und positionieren, um eine Silikonmaske zu liefern 2 verdampft. </li><li> Entfernen ventralen Haare vom Hals zum Unterbauch eine chemische Enthaarungscreme verwenden. </li><li> Führen Sie Doppler - Ultraschall - Untersuchung vor der AVF Chirurgie Baseline - Charakteristika der arteriellen und venösen Rückstrom und Gefäßdurchmesser in den Bereichen von Interesse zu erfassen. 1,2 </li><li> Bringen Sie eine 1-ml-Spritze mit einer 25 G-Nadel und laden Sie die Spritze mit dem gewünschten Medikament. Biegung der Nadel zu einem Winkel von 60 Grad ungefähr 4 mm von der Notwendigkeitle Spitze. Fassen Sie die 25 G-Nadel mit einem gebogenen Nadelhalter. </li></ol> 2. Operative Verfahren <ol><li> Bereiten Sie die Inzisionsstelle mit einem Antiseptikum und ein Operationstuch anwenden. Verwenden Sie sterile Handschuhe und Instrumente zur aseptischen Bedingungen in der gesamten Operation erhalten. </li><li> Machen Sie eine Mittellinie Bauchschnitt mit einem Skalpell aus dem Niveau der unteren Leberrand erstreckt, knapp über dem Schambein. </li><li> Legen Sie eine Aufroller und ausweiden alle Eingeweide aus der Bauchhöhle zur rechten Seite. Wickeln Sie die Eingeweide in Gaze, getränkt mit Kochsalzlösung. Präparieren Sie die Membran, die die retro-Peritoneum verbindet und unteren Kolon vollen Blick auf die Aorta und IVC zu erhalten. </li><li> Präparieren Sie die infra-renalen Aorta und IVC aus den umliegenden Geweben, die Vorbereitung für proximale und distale Klemm. </li><li> Legen Sie eine einzelne Mikroclip über beide proximalen Aorta und dem proximalen IVC auf der Ebene direkt unterhalb der linken Nierenvene. Legen Sie eine zweite Mikroclip über beide distal Aorta und das distale IVC. </li><li> Fassen des Bindegewebes die Aorta umgibt und medial zu drehen , so dass die Rückenfläche der Aorta leicht für den arteriellen Einstich ausgesetzt ist, wie zuvor beschrieben. 1 </li><li> Schnell die Einstichstelle aus. Die Punktionsstelle wird von der linken Nierenvene zur Aortenbifurkation am kaudalen Aspekt der Gefäße, etwa drei Viertel des Abstandes sein. Halten Sie die Aorta in einer gedrehten Position mit der linken Hand, sezieren die linke seitliche Rand der Aorta, so dass es genügend Belichtung ist Einstich mit der rechten Hand zu ermöglichen. Achten Sie darauf, nicht zwischen der Aorta und der IVC zu sezieren. </li><li> Pflegen Sie die Aorta in einer gedrehten Position und durchstechen die Aorta durch in die IVC unter Verwendung einer 25 G-Nadel mit einem daran befestigten 1 ml Spritze mit Wirkstofflösung. (1A) </li><li> Infuse die Wirkstofflösung (100-200 ul) mit der linken Hand. Die Nadel kann durch die erweiterte und dünn zu sehenIVC Wand , wenn die transparente Arzneimittellösung aus dem IVC (1B, C) ​​das venöse Blut verdrängt. Bleiben Sie ruhig und die Nadel in Position für 15 Minuten aufrechtzuerhalten. </li><li> Entfernen Sie die distale Mikrochirurgie Clip zu de-Klemme nur die distale Aorta und das distale IVC. </li><li> Entfernen Sie die Nadel und dann bedecken Sie die Einstichstelle der Aorta durch benachbarte retroperitonealen Gewebe nach oben ziehen. </li><li> Entfernen Sie den proximalen Mikrochirurgie Clip zu de-klemmen die proximale Aorta und den proximalen IVC. Bei der De-Klemm wird arterielle Blut in den IVC beobachtet fließt, anstatt dunkel venösen Blutfluss. Halten Sie das Einstichloch für 1 min abdecken. </li><li> Nach der Bestätigung der Hämostase durch Beobachtung für 30 Sekunden ohne Kompression, kehren die Därme in ihre natürliche Position und schließen Sie den Bauch mit einem fortlaufenden Naht nach Ihren zugelassenen Tier Protokoll. </li></ol> 3. Post-operative Verfahren <ol><li> Nach dem Schließen des Bauches, beenden anesthesia. Bewerben postoperative Pflege einschließlich Analgesie und Wundversorgung in Übereinstimmung mit den Anweisungen von der Institutional Animal Care und Use Committee empfohlen. Analgesieprüfung verwenden wir Buprenorphin bei 0,1 mg / kg intrasmuscularly alle 12 h für 24 Stunden, die chirurgische Verfahren folgende. </li><li> Am ersten Tag nach der Operation durchführen Doppler-Ultraschall-Durchgängigkeit des AVF zu bestätigen. Darüber hinaus messen die anderen Behälter und Fließeigenschaften seriell und für Änderungen von der präoperativen Ausgangswerten zu vergleichen. 1,2 </li></ol><img alt="Abbildung 1" src="/files/ftp_upload/53905/53905fig1.jpg" /> Zeige intraluminale Lieferung bei AVF Chirurgie Abbildung 1. (A) Operative Foto. Klemmen Sie den proximalen und distalen Aorta sowie die IVC von Mikrochirurgie Clips anwenden. Die Punktion der Aorta durch in die IVC unter Verwendung einer 25 G - Nadel mit angebrachtem Spritze mit Wirkstofflösung. (B) Ein Higihre Leistung Bild (4-fache Vergrößerung) des punktierten IVC vor der Infusion. Die Nadelspitze wird durch dunkel gefärbte venösem Blut verdeckt. Gelbe Pfeile bezeichnen die Wand-zu-Wand - Durchmesser des IVC. (C) eine höhere Leistung Bild (4 - fache Vergrößerung) des punktierten IVC nach der Infusion. Die Nadelspitze (schwarzer Pfeil) durch die sanft aufgetrieben und dünne IVC Wand (gelbe Pfeile) als die transparente Arzneimittellösung verdrängt das venöse Blut zu sehen.

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The authors have nothing to disclose.

Diese Modifikation des murinen AVF Modell enthält intraluminal Arzneimittelabgabe an das venöse Endothel zum Zeitpunkt der AVF-Erstellung. Ein AVF wurde durch Punktierung der infra-renalen Aorta mit einer 25 G-Nadel und zur Verlängerung der Einstich durch die gegenüberliegende Aortenwand in die IVC, gefolgt von Injektion von Arzneimittellösung durch die gleiche Nadel geschaffen. Die Lösung wird beibehalten inner caval, dh., Im venösen Schenkel des AVF, bis de-Klemm. Was unterscheidet dieses Modell von an...

Die murine aortovenous Fistel (AVF) Einstich Modell zwischen der Aorta und der unteren Hohlvene (IVC) ist jetzt eine etablierte Technik. 1 In diesem Modell beide Wände der infra-renale Aorta sind mit einer 25 G Nadel durchstochen, in das austretende angrenzenden Infranieren Hohlvene; die vordere Aorten-Eingangsloch ist mit einfachen Kompression repariert, und erfordert keine Naht Reparatur. Serien Follow-up - Prüfung von hochauflösenden Doppler - Ultraschall und histologische Analyse zeigt die AVF eine Reifephase haben und dann eine versagende Phase, die bekannte Pathophysiologie menschlicher AVF rekapituliert. 2 Mechanismen erforschen, die AVF Reifung modulieren, verbesserte Verfahren zur Abgabe von therapeutischen Mitteln an die Reifungs AVF Endothels benötigt. Lieferung von therapeutischen Wirkstoffen auf Schiffe können entweder über endovaskuläre Lieferung an das Lumen oder über externe Lieferung an die Adventitia sein. Ein Beispiel für eine externe Lieferung ist der comhäufigsten verwendeten adventitiellen Anwendung von Pluronic-Gel. Dieses Copolymer wird thermo-reversibel und vom flüssigen in den festen Gel umgewandelt wenn sie auf Körpertemperatur erwärmt. Frühere Studien haben gezeigt , aufrechterhalten Arzneimittelabgabe erreicht wird , wenn die Droge in Pluronic - Gel gemischt topisch in vivo angewendet wird. 3,4 adventitiellen Anwendung von viralen Vektoren oder siRNA mit Pluronic - Gel wurde als perivaskuläre Abgabesystem wirksam zu sein berichtet. 5,6 Wir haben auch berichtet , dass die Behandlung von explantierten humanen Vena saphena mit adventitiellen Stimulation durch Peptide , die in der Phosphorylierung der endothelialen Rezeptorproteine ​​führte. 7 Auf der anderen Seite haben Forscher auch intraluminale Lieferung von beiden viralen und nicht - viralen Vektoren in canine 8-10 und Kaninchen 11,12 Modelle von Venentransplantaten verwendet. In diesen Berichten wurde Gentransfer durchgeführt ex vivo nach der Ernte Vene. Eslami et al. Berichtet endovaskuläre viralen Gens lieferny Venen in situ zu Karotis ohne einen Bypass zu schaffen. 13 Gloverman et al. berichtete intraluminale und adventitiellen Lieferung von nackter DNA in Ratte Femoralarterie-V. epigastrica superficialis Fisteln. 14 Die Mayo - Gruppe berichtet adventitiellen Arzneimittelabgabe in Arterie-Halsvene Fisteln Maus Carotis. 15,16 Allerdings sind diese bereits berichtet Modelle eine genähte Anastomose , die zum Erstellen die AVF. In diesem Bericht wird intraluminale Medikamentenabgabe bei gleichzeitiger AVF Schöpfung bei Mäusen beschrieben, eine Naht lose Modell der AVF Schöpfung verwenden. Durch die Verwendung dieses modifizierte murine AVF Modell eine einfache Methode zur intraluminale Medikamentenabgabe an den venösen Schenkel der Fistel durchgeführt werden.

<table border="0" cellpadding="0" cellspacing="0" width="948">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:343px;">Pluronic F-127</td>
			<td style="width:259px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2443-250G</td>
			<td style="width:231px;">Used as 30% solution in d-water</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:343px;">GFP antibody</td>
			<td style="width:259px;">NOVUS BIOLOGICALS INC</td>
			<td style="width:119px;">NB100-1770</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:343px;">Ad-CMV-GFP</td>
			<td style="width:259px;">VECTOR BIOLABS</td>
			<td style="width:119px;">1060</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:343px;">0.9% Sodium Chloride Irrigation, USP</td>
			<td style="width:259px;">Baxter</td>
			<td style="width:119px;">2F7122</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:343px;">BD PrecisionGlide Needle 25G x 5/8</td>
			<td style="width:259px;">BD</td>
			<td style="width:119px;">305122</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:343px;">BD 1ml Syringe Tuberculin Slip Tip</td>
			<td style="width:259px;">BD</td>
			<td style="width:119px;">309659</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:343px;">Scarpel</td>
			<td style="width:259px;">Surgical Design Inc</td>
			<td style="width:119px;">22079707</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:343px;">6-0 ETHILON P-1 11mm 3/8c Reverse Cutting</td>
			<td style="width:259px;">ETHICON INC</td>
			<td style="width:119px;">697G</td>
			<td></td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:343px;">Vevo 770 ultrasound machine&#160;</td>
			<td style="width:259px;">Visualsonics&#160;</td>
			<td style="width:119px;">20-60 Mhz scan head; RMV-704</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:343px;">Vascular clamp&#160;</td>
			<td style="width:259px;">Roboz Surgical Instrument Co.</td>
			<td style="width:119px;">RS-5424</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:343px;">Clamp applying forceps&#160;</td>
			<td style="width:259px;">Roboz Surgical Instrument Co.</td>
			<td style="width:119px;">RS-5410</td>
			<td></td>
		</tr>
	</tbody>
</table>

intraluminal drug delivery to the mouse arteriovenous fistula endothelium

Abgabe therapeutischer Mittel arteriovenöse Fistel (AVF) zur Verbesserung der Reifung entweder via intraluminalen oder externe Wege verabreicht werden kann. Die einfache murine AVF Modell wurde mit intraluminale Verabreichung von Arzneimittellösung in die venöse Endothel zugleich als Fistel Schaffung kombiniert. Technische Aspekte dieses Modells diskutiert. Unter Vollnarkose wird ein Bauchschnitt gemacht und die Aorta und untere Hohlvene (IVC) ausgesetzt sind. Die Infrarot-Nieren-Aorta und IVC sind zum Klemmen seziert. Nach proximal und distal Klemmung wird der Einstichstelle freigelegt und eine 25 G-Nadel verwendet wird, beide Wände der Aorta und in die IVC zu durchstechen. Unmittelbar nach dem Einstich, ein Reporter-Gen-exprimierenden viralen Vektor wurde in der IVC über die gleiche Nadel infundiert, um 15 min inkubiert. Die intraluminale Verabreichungsmethode ermöglicht robustere virales Gen Lieferung an den venösen Endothel im Vergleich zur Verabreichung durch die externe Route. Diese novel Verfahren zur Lieferung wird Studien erleichtern, die die Rolle des Endothels in AVF Reifung erforschen und zum Zeitpunkt der chirurgischen Operation intraluminal Arzneimittelabgabe ermöglichen.

In einer Serie von 33 Mäusen, Überleben am ersten postoperativen Tag betrug 97,0%; AVF Durchgängigkeit, wie durch Ultraschall bestimmt wurde, betrug 84,9%. Gene Transduktionswirksamkeit dieser endovaskulären Lieferweg mit der traditionellen externen Route verglichen. Zur intraluminalen Liefer (ILD), unmittelbar nach der Punktion 200 ul Adenovirus-GFP (Ad-GFP) Vektor - Lösung (1 × 10 9 PFU / ml) wurde über die...

Watch this Scientific Journal Video about Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium at JoVE.com

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

Intraluminale Drug Delivery auf die Maus arteriovenöse Fistel Endothel

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

intraluminal-drug-delivery-to-mouse-arteriovenous-fistula

Research

JoVE Journal

Medicine

9.3K Aufrufe.  Yale University. After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

Serie: Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

Osmotic Drug Delivery zu ischämischen Hinterbeine und Perfusion von Blutgefäßen mit Microfil für Mikro-Computertomographie

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Forschung

Medizin

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

Technische Aspekte der Maus aortokavaler Fistel

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

Heterotope Mucosal-Transplantationsverfahren für Direkt Drug Delivery zum Gehirn bei Mäusen

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

Slow-Release-Drug Delivery durch Elvax 40W, um die Ratte Retina: Implikationen für die Behandlung von chronischen Erkrankungen

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

A Novel Mausmodell der arteriovenöse Fistel Ausfall: Der chirurgische Eingriff im Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the ...

Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, pancreatitis, and pancreatic cancer. All three of these diseases mark active areas of study, not only to develop immediate therapy, but also to better understand their pathophysiology. There are few tools to further these areas of study. Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. The technique requires the intricate dissection of the second portion of the duodenum and ampulla, followed by the occlusion of the bile duct and the cannulation of the pancreatic duct. Although the technique is technically challenging at first, the applications are myriad. Ambiguity in the specifics of the procedure between groups highlighted the need for a standard protocol. This work describes the expression of a green fluorescent protein (GFP) within the pancreas after the pancreatic duct infusion of a viral vector expressing GFP versus a sham surgery. The infusion and therefore expression is specific to the pancreas, without expression present in any other tissue type.

Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. A pancreatic duct infusion technique for drug and viral delivery to pancreatic cells is presented here.

Pankreasgang-Infusion: Eine effektive und selektive Methode der Medikamenten- und Virusabgabe

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Optimierte LC-MS/MS-Methode für die Hochdurchsatz-Analyse der klinischen Proben von Ivacaftor, seinen Hauptmetaboliten und Lumacaftor in biologischen Flüssigkeiten von Mukoviszidose-Patienten

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Verbesserte Methode für die Einrichtung einer In-vitro- Blut - Hirn-Schranke Modell auf porcinen Endothelzellen Gehirnzellen basiert

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could interfere with the chemotherapeutic efficacy of the primary drugs. This is especially true for the drug cisplatin, whose anticancer actions are attenuated by antioxidants which provide adequate protection against hearing loss. Other current or potential otoprotective agents could pose a similar problem, if administered systemically. The application of various biologicals or protective agents directly to the cochlea would allow for high levels of these agents locally with limited systemic side effects. In this report, we demonstrate a trans-tympanic method of delivery of various drugs or biological reagents to the cochlea, which should enhance basic science research on the cochlea and provide a simple way of directing the use of otoprotective agents in the clinics. This report details a method of trans-tympanic drug delivery and provides examples of how this technique has been used successfully in experimental animals to treat cisplatin ototoxicity.

We present a technique for localized administration of drugs through the trans-tympanic route into the cochlea. Drug delivery through this route would not interfere with the anti-cancer efficacy of the chemotherapeutic drugs such as cisplatin.

Trans-Trommelfells Drug-Delivery für die Behandlung von Ototoxizität

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

In Vivo Gentransfer in die Kaninchen gemeinsame Halsschlagader Endothel

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...