Name: intraluminal drug delivery to the mouse arteriovenous fistula endothelium
Uploaded: 2016-03-04T15:00:00
Description: Watch this Scientific Journal Video about Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium at JoVE.com

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.

Approval by the appropriate Institutional Animal Care and Use Committee is obtained.
1. Anesthesia and Pre-operative Procedures
<ol>
	<li>Anesthetize male C57Bl/6 ice, aged 8 weeks, with vaporized 3% isoflurane and 0.8 L/min oxygen administered into an acrylic induction chamber.</li>
	<li>Confirm adequate anesthesia by lack of reaction to toe pinch. Position the mouse supine on the operation table and position a silicone mask to deliver vaporized 2 - 3% isoflurane by continuous inhalation.</...

<ol>
	<li>Yamamoto, K., Li, X., Shu, C., Miyata, T., Dardik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+Aspects+of+the+Mouse+Aortocaval+Fistula.">Technical Aspects of the Mouse Aortocaval Fistula.</a> J. Vis. Exp. (77), e50449 (2013).</li><li>Yamamoto, K., 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=The+mouse+aortocaval+fistula+recapitulates+human+arteriovenous+fistula+maturation.">The mouse aortocaval fistula recapitulates human arteriovenous fistula maturation.</a> Am. J. Physiol.: Heart Circ. Physiol. 305 (12), H1718-H1725 (2013).</li><li>Escobar-Ch&#225;vez, J. J., L&#242;pez-Cervantes, M., Na&#239;k, A., Kalia, Y. N., Quintanar-Guerrero, D., Ganem-Quintanar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+thermo-reversible+pluronic+F-127+gels+in+pharmaceutical+formulations.">Applications of thermo-reversible pluronic F-127 gels in pharmaceutical formulations.</a> J. Pharm. Pharm. Sci. 9 (3), 339-358 (2006).</li><li>Almeida, H., Amaral, M. H., Lob&#227;o, P., Lobo, J. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluronic&#174;+F-127+and+Pluronic+Lecithin+Organogel+(PLO):+main+features+and+their+applications+in+topical+and+transdermal+administration+of+drugs.">Pluronic&#174; F-127 and Pluronic Lecithin Organogel (PLO): main features and their applications in topical and transdermal administration of drugs.</a> J. Pharm. Pharm. Sci. 15 (4), 592-605 (2012).</li><li>Karper, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+4+is+involved+in+human+and+mouse+vein+graft+remodeling,+and+local+gene+silencing+reduces+vein+graft+disease+in+hypercholesterolemic+APOE*3Leiden+mice.">Toll-like receptor 4 is involved in human and mouse vein graft remodeling, and local gene silencing reduces vein graft disease in hypercholesterolemic APOE*3Leiden mice.</a> Arterioscler., Thromb., Vasc. Biol. 31 (5), 1033-1040 (2011).</li><li>Redmond, E. M., Hamm, K., Cullen, J. P., Hatch, E., Cahill, P. A., Morrow, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+patched-1+prevents+injury-induced+neointimal+hyperplasia.">Inhibition of patched-1 prevents injury-induced neointimal hyperplasia.</a> Arterioscler., Thromb., Vasc. Biol. 33 (8), 1960-1964 (2013).</li><li>Wong, D. J., 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=Ephrin+type-B+receptor+4+activation+reduces+neointimal+hyperplasia+in+human+saphenous+vein+in+vitro.">Ephrin type-B receptor 4 activation reduces neointimal hyperplasia in human saphenous vein in vitro.</a> J. Vasc. Surg. , (2014).</li><li>Matsumoto, T., 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=Hemagglutinating+virus+of+Japan-liposome-mediated+gene+transfer+of+endothelial+cell+nitric+oxide+synthase+inhibits+intimal+hyperplasia+of+canine+vein+grafts+under+conditions+of+poor+runoff.">Hemagglutinating virus of Japan-liposome-mediated gene transfer of endothelial cell nitric oxide synthase inhibits intimal hyperplasia of canine vein grafts under conditions of poor runoff.</a> J. Vasc. Surg. 27 (1), 135-144 (1998).</li><li>Petrofski, J. A., 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=Gene+delivery+to+aortocoronary+saphenous+vein+grafts+in+a+large+animal+model+of+intimal+hyperplasia.">Gene delivery to aortocoronary saphenous vein grafts in a large animal model of intimal hyperplasia.</a> J. Thorac. Cardiovasc. Surg. 127 (1), 27-33 (2004).</li><li>Hata, J. A., 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=Modulation+of+phosphatidylinositol+3-kinase+signaling+reduces+intimal+hyperplasia+in+aortocoronary+saphenous+vein+grafts.">Modulation of phosphatidylinositol 3-kinase signaling reduces intimal hyperplasia in aortocoronary saphenous vein grafts.</a> J. Thorac. Cardiovasc. Surg. 129 (6), 1405-1413 (2005).</li><li>Ohta, S., 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=Intraluminal+gene+transfer+of+endothelial+cell-nitric+oxide+synthase+suppresses+intimal+hyperplasia+of+vein+grafts+in+cholesterol-fed+rabbit:+a+limited+biological+effect+as+a+result+of+the+loss+of+medial+smooth+muscle+cells.">Intraluminal gene transfer of endothelial cell-nitric oxide synthase suppresses intimal hyperplasia of vein grafts in cholesterol-fed rabbit: a limited biological effect as a result of the loss of medial smooth muscle cells.</a> Surg. 131 (6), 644-653 (2002).</li><li>Baldwin, Z. K., 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=Modulation+of+vascular+remodeling+induced+by+a+brief+intraluminal+exposure+to+the+recombinant+R7020+strain+of+Herpes+simplex-1.">Modulation of vascular remodeling induced by a brief intraluminal exposure to the recombinant R7020 strain of Herpes simplex-1.</a> J. Vasc. Surg. 41 (1), 115-121 (2005).</li><li>Eslami, M. H., 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=Gene+delivery+to+in+situ+veins:+differential+effects+of+adenovirus+and+adeno-associated+viral+vectors.">Gene delivery to in situ veins: differential effects of adenovirus and adeno-associated viral vectors.</a> J. Vasc. Surg. 31 (6), 1149-1159 (2000).</li><li>Globerman, A. S., 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=Efficient+transgene+expression+from+naked+DNA+delivered+into+an+arterio-venous+fistula+model+for+kidney+dialysis.">Efficient transgene expression from naked DNA delivered into an arterio-venous fistula model for kidney dialysis.</a> J. Gene Med. 13 (11), 611-621 (2011).</li><li>Yang, 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=Adventitial+transduction+of+lentivirus-shRNA-VEGF-A+in+arteriovenous+fistula+reduces+venous+stenosis+formation.">Adventitial transduction of lentivirus-shRNA-VEGF-A in arteriovenous fistula reduces venous stenosis formation.</a> Kidney Int. 85 (2), 289-306 (2014).</li><li>Brahmbhatt, A., 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=The+role+of+Iex-1+in+the+pathogenesis+of+venous+neointimal+hyperplasia+associated+with+hemodialysis+arteriovenous+fistula.">The role of Iex-1 in the pathogenesis of venous neointimal hyperplasia associated with hemodialysis arteriovenous fistula.</a> PLoS ONE. 9 (7), e102542 (2014).</li><li>Karram, T., 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=Induction+of+cardiac+hypertrophy+by+a+controlled+reproducible+sutureless+aortocaval+shunt+in+the+mouse.">Induction of cardiac hypertrophy by a controlled reproducible sutureless aortocaval shunt in the mouse.</a> J. Investig. Surg. 18 (6), 325-334 (2005).</li><li>Perry, G. J., 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=Genetic+variation+in+angiotensin-converting+enzyme+does+not+prevent+development+of+cardiac+hypertrophy+or+upregulation+of+angiotensin+II+in+response+to+aortocaval+fistula.">Genetic variation in angiotensin-converting enzyme does not prevent development of cardiac hypertrophy or upregulation of angiotensin II in response to aortocaval fistula.</a> Circ. 103 (7), 1012-1016 (2001).</li><li>Guzman, R. J., Krystkowiak, A., Zarins, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+and+sustained+medial+cell+activation+after+aortocaval+fistula+creation+in+mice.">Early and sustained medial cell activation after aortocaval fistula creation in mice.</a> J. Surg. Res. 108 (1), 112-121 (2002).</li><li>Alexander, J. H., 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=Efficacy+and+safety+of+edifoligide,+an+E2F+transcription+factor+decoy,+for+prevention+of+vein+graft+failure+following+coronary+artery+bypass+graft+surgery:+PREVENT+IV:+a+randomized+controlled+trial.">Efficacy and safety of edifoligide, an E2F transcription factor decoy, for prevention of vein graft failure following coronary artery bypass graft surgery: PREVENT IV: a randomized controlled trial.</a> JAMA. 294 (19), 2446-2454 (2005).</li><li>Khaleel, M. S., 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=High-pressure+distention+of+the+saphenous+vein+during+preparation+results+in+increased+markers+of+inflammation:+a+potential+mechanism+for+graft+failure.">High-pressure distention of the saphenous vein during preparation results in increased markers of inflammation: a potential mechanism for graft failure.</a> Ann. thorac. surg. 93 (2), 552-558 (2012).</li></ol>

This modification of the murine AVF model incorporates intraluminal drug delivery to the venous endothelium at the time of AVF creation. An AVF was created by puncturing the infra-renal aorta with a 25 G needle and extending&#160;the puncture through the opposite aortic wall into the IVC, followed by injection of drug solution through the same needle. The solution is maintained intra-caval, i.e., in the venous limb of AVF, until de-clamping. What distinguishes this model from other murine AVF models17-19</s...

The murine aortovenous fistula (AVF) puncture model between the aorta and the inferior vena cava (IVC) is now an established technique.1 In this model, both walls of the infra-renal aorta are punctured with a 25 G needle, exiting into the adjoining infra-renal vena cava; the anterior aortic entrance hole is repaired with simple compression, and does not require suture repair. Serial follow-up examination by high-resolution Doppler ultrasound and histological analysis shows the AVF to have a maturation phase and then a failing phase, recapitulating the known pathophysiology of human AVF.2
To explore mechanisms that modulate AVF maturation, improved methods for delivery of therapeutic agents to the maturing AVF endothelium are needed. Delivery of therapeutic agents to vessels can be either via endovascular delivery to the lumen, or via external delivery to the adventitia. One example of external delivery is the commonly used adventitial application of Pluronic gel. This copolymer is thermo-reversible and transformed from liquid to solid gel when warmed to body temperature. Prior studies have shown sustained drug delivery is achieved when drug mixed in pluronic gel is applied topically in vivo.3,4 Adventitial application of viral vectors or siRNA with Pluronic gel has been reported to be effective as a perivascular delivery system.5,6 We have also reported that treatment of explanted human saphenous veins with adventitial stimulation by peptides resulted in phosphorylation of endothelial receptor proteins.7
On the other hand, investigators have also used intraluminal delivery of both viral and nonviral vectors in canine8-10 and rabbit11,12 models of vein grafts. In these reports, gene transfer was performed ex vivo after vein harvest. Eslami et al. reported endovascular viral gene delivery to carotid veins in situ without creating a bypass.13 Gloverman et al. reported intraluminal and adventitial delivery of naked DNA in rat femoral artery-superficial epigastric vein fistulae.14 The Mayo group reported adventitial drug delivery in mouse carotid artery-jugular vein fistulae.15,16 However, these previously reported models required a sutured anastomosis to create the AVF. In this report, intraluminal drug delivery with simultaneous AVF creation in mice is described, using a suture-less model of AVF creation. By using this modified murine AVF model a simple method for intraluminal drug delivery to the venous limb of the fistula can be performed.

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	<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

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

In a series of 33 mice, survival on the first post-operative day was 97.0%; AVF patency, as determined by ultrasound,&#160;was 84.9%.
Gene transduction efficiency of this endovascular delivery route with the traditional external route was compared. For intraluminal delivery (ILD), immediately after the puncture, 200 &#181;l of Adenovirus-GFP (Ad-GFP) vector solution (1 &#215; 109 PFU/ml) was infused into the IVC via t...

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.

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

The overall goal of this procedure is to modify the murine arteriovenous fistula, or AVF model, into a simple and intergrated method for drug delivery to the fistula endothelium. This method can help answer key questions about the role of the endothelium in venous remodeling. After exposure to the arterial enivroment and in the development of neointimal hyperplasia.The main advantage of this technique is that it uses a single needle to simultaneously create an AVF while delivering the drug of interest to the endothelium. Before beginning the procedure, autoclave all surgical instruments and vascular clamps. Equip a one-milliliter syringe with a 25 gauge needle and load the syringe with the drug of interest.Then bend the needle to a 60-degree angle approximately four millimeters from the needle tip and grasp the needle with the curved needle holder. Next, clean the incision site of an eight-week old anesthetized C57 black 6 mouse with a topical antiseptic and place a surgical drape over the animal. Using a scalpel make a mid-line abdominal incision extending from the lower liver edge to just above the pubis and insert a retractor.Then, transfer the bowels from the abdominal cavity to the right side and wrap them in saline soaked gauze Dissect the membrane connecting the retroperitoneum and lower colon to obtain a full view of the aorta and IVC. To prepare the tissue for the proximal and distal clamp, dissect the infrarenal aorta and IVC from the surronding tissues and place a single microsurgery clip across both the proximal aorta and IVC just below the left renal vein. Place a second microsurgery clip across both the distal aorta and IVC.Then grasp the connective tissue surrounding the aorta and rotate it medially so that the dorsal surface of the aorta is slightly exposed for the arterial puncture. Keeping the aorta in a rotated position with the left hand, dissect the left lateral margin of the aorta for three quarters of the distance from the left renal vein to the aortic bifurcation so there is ample room to make the puncture with the right hand. Maintaining the aorta in the rotated position, puncture the vessel into the IVC with a curved 25 gauge needle and infuse 100 to 200 microliters of the drug of interest.The needle will be visible through the dilated thin wall of the IVC as the transparent drug solution displaces the venous blood out of the IVC. Carefully maintain the needle in position for 15 minutes without moving, then remove the distal microsurgery clip from the distal aorta and IVC only. When the distal vessel have been declamped, remove the needle and cover the puncture site of the aorta with the adjacent retroperitoneal tissue.Remove the proximal microsurgery clip from the proximal aorta and IVC. The arterial blood will be observed flowing into the IVC instead of the usual dark venous blood flow. Take care to provide careful compression when covering the puncture site to avoid occlusion of the AVF and thrombosis.Finally, after observing 30 seconds of blood flow without compression return the bowels to the abdominal cavity and close the abdomen with a running suture. After closure of the abdomen, apply post-operative care including analgesia, thermal support, and monitoring every 15 minutes until sternal recumbency. In these images, the gene transduction efficiency of this endovascular delivery route with the traditional external route was compared.As evident, en face observation demonstrated a stronger expression of GFP in the IVC wall in mice treated by intraluminal compared to adventitial delivery of the virus with a characteristic augmentation observed around the fistula at 24 hours post adminstration. Cross sectional histological analysis with anti-GFP antibody showed more robust and site-specific GFP expression along the endothelium of the IVC in mice treated with ILD compared with AD.To confirm these results, infrarenal IVCs treated with adenovirus GFP by either route of delivery were harvested for western blood analysis. Indeed, GFP protein expression was sufficient for detection only after intraluminal delivery, suggesting a superior viral drug delivery to the IVC wall via the intraluminal route at 24 hours post transfection.Once mastered, this technique can be completed in 25 minutes, including the 15 minute incubation time if it's performed properly.

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

Research

JoVE Journal

Medicine

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.

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

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.

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.

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 ...

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.

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

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.

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.

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.

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.

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 ...

Intratracheal Administration of Dry Powder Formulation in Mice

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper introduces a noninvasive method of intratracheal delivery of dry powder formulation in mice. A dry powder loading device that consists of a 200 &#181;L gel loading pipette tip connected to an 1 mL syringe via a three-way stopcock is presented. A small amount of dry powder (1-2 mg) is loaded into the pipette tip and dispersed by 0.6 mL of air in the syringe. Because pipette tips are disposable and inexpensive, different dry powder formulations can be loaded into different tips in advance. Various formulations can be evaluated in the same animal experiment without device cleaning and dose refilling, thereby saving time and eliminating the risk of cross-contamination from residual powder. The extent of powder dispersion can be inspected by the amount of powder remaining in the pipette tip. A protocol of intubation in mouse with a custom-made light source and a guiding cannula is included. Proper intubation is one of the key factors that influences the intratracheal delivery of dry powder formulation to the deep lung region of the mouse.

Dry powder formulations for inhalation have great potential in treating respiratory diseases. Before entering human studies, it is necessary to evaluate the efficacy of the dry powder formulation in preclinical studies. A simple and noninvasive method of the administration of dry powder in mice through the intratracheal route is presented.

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper ...

Direct Drug Delivery to Kidney via the Renal Artery

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research field. Accumulation of drugs in certain organs may give rise to adverse and unwanted effects, depending on the nature of injectate. To minimize spillover and/or accumulation in other tissues, the herein described method directs the formulation into the renal artery bloodstream by inserting a catheter in the infra renal aorta, just below where it branches into the renal artery, resulting in the kidney as first reached organ and distributing of formulation throughout the kidney.
This manuscript provides a detailed description of the method, as well as its challenges and difficulties. It guides the experimenter to become skillful with this type of microsurgery that requires accuracy under sterile conditions. Speed is crucial for minimizing the ischemia and practicing the procedure will increase the chance of successful injections without adverse effects. By modulating the time between injection and reperfusion as well as the injected volume, the risk of spillover to other organs is mitigated. 
Note that this technique is suitable for single dosing strategies.

This manuscript describes a method for targeted delivery to a single kidney via a catheter placed in the infrarenal abdominal aorta in the mouse.

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research ...