New Method to Quantify Angiogenesis in vivo Using
Multi-photon Imaging
B. J. Herron
1

2
, J. S. Smith
1
, and R. W. Cole
1

2
*
1
Wadsworth Center, New York State Department of Health, PO Box 509, Albany, NY 12201
2
Department of Biomedical Sciences, School of Public Health, State University of New York at Albany,
Empire State Plaza, Albany, NY 12201
* rcole@wadsworth.org
Introduction inflammation. Our goal in this study was to develop a method
Efforts to understand the basic mechanisms of that would minimize the limitations of the conventional in vivo
angiogenesis, that is, the formation of new blood vessels from angiogenesis assay while permitting maximal quantitation. To
existing vasculature, have been limited by the methods that this end we have developed a method that uses multi-photon
are currently used to measure vessel growth. Although in microscopy to quantify subdermal vasculature in mice over a
vivo assays provide the best environment in which to track several-day time span.
angiogenesis, inherent difficulties in obtaining reproducible
data limit the power of this approach. Limitations include:
Methods
environmental variations between experimental animals,
Animals. All animals used in this study were handled
induction of inflammatory responses by surgical methods, and
in accordance with the guidelines of the Wadsworth Center’s
labor-intensive blood vessel quantification procedures. A better
Institutional Animal Care and Use Committee. FVB/N-Tg
assay would measure vessel growth in one animal at multiple
(TIE2-lacZ) or A/J mice were used in all experiments. Mice
time points and would focus on minimization of artifacts
were anesthetized either by exposure to the inhalation agent
induced by experimental manipulation.
isoflurane or through an intraperitoneal injection of 8 mg/mL
Angiogenesis is essential for normal embryonic
ketamine combined with 1 mg/mL xylazine. Once a mouse
development, wound healing, and post-ischemic tissue repair;
was anesthetized, 15 mL of 2X concentration BD Matrigel™
it is also associated with such pathological conditions as cancer,
(BD Biosciences) containing 100 ng/mL rhVEGF165, 300 ng/
diabetes, and psoriasis. If we can develop means to control this
mL bFGF (R&D Systems Inc.), and 25 ng/mL heparin was
process, we acquire important therapeutic tools to combat a
injected into the hind leg, proximal to the hind paw, via an
number of diseases [1]. Thus, angiogenic research is an active
intradermal injection. Control mice were injected with 15 mL of
area of investigation in both preclinical and clinical settings.
only the 2X concentration BD Matrigel™ alone. Additionally, to
Because virtually all adult tissues maintain a well-developed
evaluate whether BD Matrigel™ by itself induces vessel growth,
vasculature, it is difficult to measure angiogenesis in vivo.
we injected another group of control mice with 100 ng/mL
Several strategies have been developed that either follow vessel
rhVEGF165, 300 ng/mL bFGF, and 25 ng/mL heparin in 1X
growth into an avascular tissue of an experimental animal, as
phosphate-buffered saline.
in the corneal implant assay [2], or that follow vessel growth
Once injections had been administered, mice were returned
into an avascular substrate, as in the Matrigel implant assay [3].
to their cages and allowed to recover for approximately 24 hours
Although these approaches have enabled important progress
before imaging. We chose to evaluate the anterior glaborous
in angiogenic research, they are limited by the associated high
skin of the hind leg proximal to the subtalar joint because this
experimental variance and undesirable host responses, such as
region is easily isolated on the microscope stage and does not
require hair removal prior to imaging. Immediately prior to
imaging, anesthetized mice were given a 100ul lateral tail vein
injection of 70-kDa FITC-conjugated dextran (Sigma Aldrich)
in sterile 200mM dextrose in 0.9% NaCl. Vital signs, including
pulse, respiration, and oxygen saturation were monitored with
the MouseOx system (Starr Life Science Corp.) throughout all
procedures.
Imaging. Individual mice were restrained and imaged on a
custom-machined microscope stage as shown in Figure 1. The
temperature of each mouse was maintained by flowing 37ºC
water through tubing embedded on the stage. Mice were imaged
on day 1, 6, and 9 post injection with a Leica SP5 confocal
equipped for spectral and multi-photon imaging (Mai Tai laser,
Figure 1: Demonstration of an anesthetized mouse on the microscope stage, Spectra Physics). The MP laser was tuned to 820 nm to image
undergoing vessel imaging. The sensor for the MouseOx monitoring system, as
the FITC-labeled dextran and 810 nm to image expressed GFP.
well as the rectal temperature probe, are visible.
Non-descan detectors were used to collect all image data. These
24 doi: 10.1017/S1551929509000339
www.microscopy-today.com • 2009 September
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