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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

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

Published: June 29th, 2013

DOI:

10.3791/50364

1The Texas Heart Institute at St. Luke's Episcopal Hospital, 2Department of Vascular Surgery, The Ninth People's Hospital, School of Medicine, Shanghai Jiao Tong University
* These authors contributed equally

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 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.

Peripheral arterial disease (PAD) is an atherosclerotic disease that causes insufficient blood supply in the legs4. It affects 8 to 12 million Americans, and current medical treatments offer only limited relief5,6. Novel therapeutic agents that improve blood circulation in legs would not only restrain disease progression but also enhance quality of life. The incidence of PAD is higher in people over the age of 50 years, so local pharmacologic therapy is a more desirable treatment modality because the reduced kidney and liver function often seen in older patients can decrease drug metabolism and increase side effects with systemic administration.<....

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1. Priming the Osmotic Pump

  1. Use sterile techniques (e.g. gloves, laminar flow hood) when preparing the pumps. Remove the pump and flow moderator from the packaging. Weigh the empty pump and record the weight (mg).
  2. Fill the pump slowly, to avoid creating air bubbles, by using a small 1cc syringe and blunt-tipped 27G filling tube. When the solution reaches the top of the pump, stop filling.
  3. Wipe off the excess solution and weigh the filled pump. For most aqueous solutions, the weigh.......

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The animation demonstrates the osmotic pump insertion and hindlimb ischemia surgery detailed in the protocol. Figure 1 shows laser Doppler images of perfusion of the hindlimb, confirming ischemia. After vascular casting with Microfil, a 3D micro-CT image of the vascular network shows that Microfil can fill the vessels effectively (Figure 2A) but that discontinuities can occur (Figure 2B) due to various procedural factors (e.g., air bubbles, lack of pressure, vis.......

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Here we present a method for osmotic drug/substance delivery in a mouse model of hindlimb ischemia. In addition, we describe a casting technique in which we have used Microfil to produce a 3D reconstruction for analysis of the vascular network.

The level or severity of ischemia varies according to where the arterial ligation/excision is made. We created a double ligation in the common femoral artery proximal to the origin of the profunda femoris and epigastrica arteries; this approac.......

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The authors would like to thank Keith Michel of the MD Anderson Small Animal Imaging Facility for his technical assistance with micro-CT imaging, Edward T.H. Yeh, MD, for surgical assistance, and Rebecca Bartow, PhD, for editorial assistance. This work was supported in part by the American Heart Association.

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Name Company Catalog Number Comments
Reagent/Material
Surgical tools Fine Science Tools Type: Tool
Puritan sterile cotton swabs Fisher Scientific 22-029-499 Type: Tool
Betadine (povidone-iodine) Fisher Scientific 19-065534 Type: Reagent
70% Alcohol pads Fisher Scientific NC9926371 Type: Reagent
Phosphate buffered saline Lonza 17-516F Type: Reagent
6-0 prolene suture Cardinal Health 8709 Type: Tool
8-0 prolene suture Cardinal Health 2775 Type: Tool
Depilatory cream Nair Type: Tool
Osmotic pump ALZET 1002 Type: Tool, 14 day release
Vinyl catheter ALZET 7760 Type: Tool
Heparinized saline (0.9%) Baxter 2B0944 Type: Reagent
Neutral buffered formalin Richard-Allan Scientific 5705 Type: Reagent
Microfil (silicone rubber contrast agent) Flowtech MV-112 Type Reagent, Microfil White
Cal-Ex II (formic acid solution) Fisher Scientific CS511-1D Type: Reagent
Buprenex CIII 7571 Type: Analgesic
Bupivicaine Hospira, Inc. 381 Type: Analgesic
Equipment
Dissecting microscope Carl Zeiss Microimaging Zeiss Stemi 2000-C Type:Equipment
Laser Doppler perfusion imager Perimed Inc. Periscan PIM3 Type:Equipment
Micro-CT imaging system GE Healthcare Explore Locus SP Type:Equipment

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