Angiogenesis Model for Ultrasound Contrast Research: Exploratory Study

Rationale and Objectives. To optimize an angiogenesis model for imaging research that is stable and can be imaged several times over the angiogenic time course. Materials and Methods. Mice and rats received two injections of 0. 4 mL of extract of basement membrane matrix (Matrigel; Becton Dickinson Labware, Bedford, MA) in the subcutaneous spaces on either side of the spine. One of the two Matrigel plugs in each animal had either 0.1 μg/mL of basic fibroblast growth factor (bFGF) (11 mice), 1.0 μg/mL of bFGF (12 mice, 5 rats), or 1.0 μg/mL of bFGF and 60 U/mL of heparin (11 mice). Three to 12 days after implantation, animals were imaged before and after the administration of up to four injections of 0.1 mL AF0150. Phase inversion imaging was used on a Siemens Elegra (Siemens ultrasound, Issaquah, WA) equipped with a 13 MHz VFX transducer. Three observers subjectively assessed the pattern of enhancement using a four-point scale. The Matrigel plugs were then removed and two observers graded the angiogenic response on a four-point scale. Ten Matrigel plugs, five with 1.0 μg/mL bFGF and five without, were evaluated histologically following immunohistochemical staining with anti-CD31. Results. The angiogenic response was greater in Matrigel plugs with 1.0 than with 0.1 μg/mL of bFGF. Heparin did not increase the angiogenic response. Vessels were predominantly at the periphery of the plugs with variable central penetration. Plugs appeared anechoic and homogeneous on ultrasound. Contrast enhancement within the plug occurred in 44% of mice with an angiogenic response at or after day 6 and the enhancement increased with the angiogenic response. In the others, peripheral enhancement could not be distinguished from the enhancement of surrounding tissues that were also hyperemic. The thicker rat skin interfered with plug assessment. Conclusion. A stable angiogenesis model without the complexity of tumors is described. This model offers the opportunity to image the development and/or inhibition of angiogenesis. Neovasculature in Matrigel was detectable using ultrasound contrast. Quantitative studies correlating the degree of enhancement to microvascular density will be determined in subsequent studies.