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.</li>
	<li>Remove ventral hair from the neck to lower abdomen using a chemical depilatory cream.</li>
	<li>Perform Doppler ultrasound examination prior to AVF surgery to record baseline characteristics of arterial and venous flow and vessel diameter at the areas of interest.1,2</li>
	<li>Attach a 1 ml syringe to a 25 G needle and load the syringe with the desired drug. Bend the needle to a 60 degree angle approximately 4 mm from the needle tip. Grasp the 25 G needle with a curved needle holder.</li>
</ol>
2. Operative Procedures
<ol>
	<li>Prepare the incision site with a topical antiseptic and apply a surgical drape. Use sterile gloves and instruments to maintain aseptic technique throughout the surgery.</li>
	<li>Make a midline abdominal incision with a scalpel extending from the level of the lower liver edge to just above the pubis.</li>
	<li>Insert a retractor and eviscerate all bowels from the abdominal cavity toward the right side. Wrap the bowels in gauze soaked with saline. Dissect the membrane connecting the retro-peritoneum and lower colon to obtain full view of the aorta and IVC.</li>
	<li>Dissect the infra-renal aorta and IVC from surrounding tissues, preparing for proximal and distal clamping.</li>
	<li>Place a single microsurgery clip across both the proximal aorta and the proximal IVC at the level just below the left renal vein. Place a second microsurgery clip across both the distal aorta and the distal IVC.</li>
	<li>Grasp the connective tissue surrounding the aorta and rotate medially so that the dorsal surface of the aorta is slightly exposed for the arterial puncture, as described previously.1</li>
	<li>Quickly expose the puncture site. The puncture site will be at the caudal aspect of the vessels, approximately three quarters of the distance from the left renal vein to the aortic bifurcation. Keeping the aorta in a rotated position with the left hand, dissect the left lateral margin of the aorta so that there is ample exposure to allow puncture with the right hand. Be careful not to dissect between the aorta and the IVC.</li>
	<li>Maintain the aorta in a rotated position and puncture the aorta through into the IVC using a 25 G needle with an attached 1 ml syringe containing drug solution. (Figure 1A)</li>
	<li>Infuse the drug solution (100 - 200 &#181;l) using the left hand. The needle can be seen through the dilated and thin IVC wall when the transparent drug solution displaces the venous blood out of the IVC (Figure 1B,C). Remain still and maintain the needle in position for 15 min.</li>
	<li>Remove the distal microsurgery clip to de-clamp only the distal aorta and the distal IVC.</li>
	<li>Remove the needle and then cover the puncture site of the aorta by pulling up adjacent retro-peritoneal tissue.</li>
	<li>Remove the proximal microsurgery clip to de-clamp the proximal aorta and the proximal IVC. Upon de-clamping, arterial blood is observed flowing into the IVC instead of dark venous blood flow. Keep covering the puncture hole for 1 min.</li>
	<li>After confirmation of hemostasis by observation for 30 sec without compression, return the bowels into their natural position and close the abdomen with a running suture according to your approved animal protocol.&#160;</li>
</ol>
3. Post-operative Procedures
<ol>
	<li>After closure of the abdomen, discontinue anesthesia. Apply post-operative care including analgesia and wound care in accordance with instructions recommended by the Institutional Animal Care and Use Committee.&#160;For analgesia we use buprenorphine at 0.1 mg/kg intrasmuscularly every 12 hr for 24 hr following the surgical procedures.</li>
	<li>On the first day after operation, perform Doppler ultrasound to confirm patency of the AVF. In addition, measure other vessel and flow characteristics serially and compare for changes from pre-operative baseline values.1,2</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/53905/53905fig1.jpg" /> 
Figure 1.&#160;(A) Operative Photo Showing Intraluminal Delivery during AVF Surgery. Clamp the proximal and distal aorta, as well as the IVC by applying microsurgery clips. Puncture the aorta through into the IVC using a 25 G needle with attached syringe containing drug solution. (B) A Higher Power Picture (4X magnification) of the Punctured IVC before Infusion. The needle tip is obscured by dark colored venous blood. Yellow arrowheads denote the wall-to-wall diameter of the IVC. (C) A Higher Power Picture (4X magnification) of the Punctured IVC after Infusion. The needle tip (black arrow) can be seen through the gently distended and thin IVC wall (yellow arrowheads) as the transparent drug solution displaces the venous blood.

<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>

The authors have nothing to disclose.

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.

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

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

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9.3K Views.  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.

Video: 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.

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 ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

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 ...

Murine Model of Central Venous Stenosis using Aortocaval Fistula with an Outflow Stenosis

Central venous stenosis is an important entity contributing to arteriovenous fistula (AVF) failure. A murine AVF model was modified to create a partial ligation of the inferior vena cava (IVC) in the outflow of the fistula, mimicking central venous stenosis. Technical aspects of this model are introduced. The aorta and IVC are exposed, following an abdominal incision. The infra-renal aorta and IVC are dissected for proximal clamping, and the distal aorta is exposed for puncture. The IVC at the midpoint between the left renal vein and the aortic bifurcation is carefully dissected to place an 8-0 suture beneath the IVC. After clamping the aorta and IVC, an AVF is created by puncturing the infra-renal aorta through both walls into the IVC with a 25 G needle, followed by ligating a 22 G intra-venous (IV) catheter and IVC together. The catheter is then removed, creating a reproducible venous stenosis without occlusion. The aorta and IVC are unclamped after confirming primary hemostasis. This novel model of central vein stenosis is easy to perform, reproducible, and will facilitate studies on AVF failure.

An aortocaval fistula was created by puncturing the murine infra-renal aorta through both walls into the inferior vena cava and was followed by creation of a stenosis in its outflow via partial ligation of the inferior vena cava. This reproducible model can be used to study central venous stenosis.

Central venous stenosis is an important entity contributing to arteriovenous fistula (AVF) failure. A murine AVF model was modified to create a partial ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

A Murine Model of Hemodialysis Access-Related Hand Dysfunction

Chronic kidney disease is a major public health problem, and the prevalence of end-stage renal disease (ESRD) requiring chronic renal replacement therapies such as hemodialysis continues to increase. Autogenous arteriovenous fistula (AVF) placement remains a primary vascular access option for ESRD patients. Unfortunately, approximately half of the hemodialysis patients experience dialysis access-related hand dysfunction (ARHD), ranging from subtle paresthesia to digital gangrene. Notably, the underlying biologic drivers responsible for ARHD are poorly understood, and no adequate animal model exists to elucidate the mechanisms and/or develop novel therapeutics for the prevention/treatment of ARHD. Herein, we describe a new mouse model in which an AVF is created between the left common iliac artery and vein, thereby facilitating the assessment of limb pathophysiology. The microsurgery includes vessel isolation, longitudinal venotomy, creation of arteriovenous anastomosis, and venous reconstruction. Sham surgeries include all the critical steps except for AVF creation. Iliac AVF placement results in clinically relevant alterations in central hemodynamics, peripheral ischemia, and impairments in hindlimb neuromotor performance. This novel preclinical AVF model provides a useful platform that recapitulates common neuromotor perturbations reported by hemodialysis patients, allowing researchers to investigate the mechanisms of ARHD pathophysiology and test potential therapeutics.

This protocol details the surgical steps of murine common iliac arteriovenous fistula creation. We developed this model to study hemodialysis access-related limb pathophysiology.

Chronic kidney disease is a major public health problem, and the prevalence of end-stage renal disease (ESRD) requiring chronic renal replacement ...

Drug Treatment by Central Venous Catheter in a Mouse Model of Angiotensin II Induced Abdominal Aortic Aneurysm and Monitoring by 3D Ultrasound

Since pharmaceutical treatment options are lacking in the clinical management of abdominal aortic aneurysm (AAA), animal models, in particular mouse models, are applied to advance the understanding of the disease pathogenesis and to identify potential therapeutic targets. Testing novel drug candidates to block AAA growth in these models generally requires repeated drug administration during the time course of the experiment. Here, we describe a compiled protocol for AAA induction, insertion of an intravenous catheter to facilitate prolonged therapy, and serial AAA monitoring by 3D ultrasound. Aneurysms are induced in apolipoprotein E (ApoE) deficient mice by angiotensin II release over 28 days from osmotic mini-pumps implanted subcutaneously into the mouse back. Subsequently, the surgical procedure for external jugular vein catheterization is conducted to allow for daily intravenous drug treatment or repeated blood sampling via a subcutaneous vascular access button. Despite the two dorsal implants, the monitoring of AAA development is readily facilitated by sequential semi-automated 3D ultrasound analysis, which yields comprehensive information on the expansion of aortic diameter and volume and on aneurysm morphology, as illustrated by experimental examples.

This protocol describes the consecutive implantation of an osmotic pump to induce abdominal aortic aneurysm by angiotensin II release in apolipoprotein E (ApoE) deficient mice and of a vascular access port with a jugular vein catheter for repeated drug treatment. Monitoring of aneurysm development by 3D ultrasound is effectively conducted despite dorsal implants.

Since pharmaceutical treatment options are lacking in the clinical management of abdominal aortic aneurysm (AAA), animal models, in particular mouse ...

A Modified Technique for Arteriovenous Fistula Construction in Rabbits

Juxta-anastomotic stenosis is a challenging problem that often causes non-maturation and decreases the patency of an arteriovenous fistula (AVF). Injury to the veins and arteries during the operation and hemodynamic changes can lead to intimal hyperplasia, leading to juxta-anastomotic stenosis. To reduce injury to the veins and arteries during the operation, this study proposes a new modified no-touch technique (MNTT) for AVF construction that can decrease the rate of juxta-anastomotic stenosis and improve the AVF patency. To unravel the hemodynamic changes and mechanisms of the MNTT, this study presented an AVF procedure using this technique. Although this procedure is technically challenging, 94.4% procedural success was achieved after adequate training. Ultimately, 13 out of 34 rabbits had a functional AVF 4 weeks after the surgery, leading to a 38.2% AVF patency rate. However, at 4 weeks, the survival rate was 86.1%. Ultrasonography showed active blood flow through AVF anastomosis. Furthermore, the spiral laminar flow was observed in the vein and artery near the anastomosis, suggesting that this technique may improve the hemodynamics of the AVF. On histological observation, significant venous intimal hyperplasia was observed at the AVF anastomosis, whereas no significant intimal hyperplasia was observed at the proximal external jugular vein (EJV) of the anastomosis. This technique will improve the understanding of the mechanisms underlying the use of MNTT for AVF construction and provide technical support for the further optimization of the surgical approach in AVF construction.

The present protocol proposes the creation of an arteriovenous fistula in rabbits using a modified no-touch technique. The technique involves the side-to-side anastomosis of the common carotid artery and external jugular vein without the dissection of the perivenous tissues or cutting off the artery.

Juxta-anastomotic stenosis is a challenging problem that often causes non-maturation and decreases the patency of an arteriovenous fistula (AVF). Injury ...