A P P L I C AT I O N N O T E

Pre-clinical Imaging
Authors
Houari Korideck
Jeffrey D. Peterson, Ph.D.
PerkinElmer, Inc.
Waltham, MA USA

Noninvasive, In Vivo
Quantitation of Asthma
Severity using
Fluorescence Molecular
Tomography

Introduction

Asthma is an inflammatory disease
process characterized by reversible airway
obstruction and airway hyperresponsiveness.
This disease process is driven by activated
T lymphocytes and eosinophils that arerecruited to the lung upon inhalation oftriggering allergens. These cells releaseinflammatory mediators, activate mastcells and epithelial cells and stimulate
mucus secretion, ultimately leading toairway obstruction. The incidence andseverity of asthma is increasing worldwide, elevating the need for clinically
relevant in vivo animal models that can be used to improve the understanding ofasthma biology and the development of effective therapeutics. Here we illustrate
the use of PerkinElmer’s FMT® 2500LX Quantitative Pre-clinical Imaging System
in combination with ProSense® 680 Fluorescent Pre-clinical Imaging Agent, anear-infrared, protease-activatable agent, for the noninvasive in vivo imagingand quantitation of pulmonary inflammation.

Noninvasive quantitative tomography of mouseasthmaAsthma can be induced in female BALB/c mice by provoking
specific immunity to ovalbumin in the lung.1 To do this,we immunized 6- to 8-week-old BALB/c mice on day 0 andday 14 by intraperitoneal injections of 50 μg of ovalbumin
combined with 2 g of aluminum hydroxide as an adjuvant
to enhance the allergic response. Between day 21 and day
24, mice received daily intranasal challenges with 100 μg ofovalbumin solubilized in PBS (pH 7.4) to focus an ovalbuminspecific allergic response within the airways. The allergic
response to ovalbumin induces cytokines and immune factors
typical of those in human asthma (for example, interleukin(IL)-4, histamine and IgE), a large influx of eosinophils andchanges in airway hyperreactivity. Measurement of theseparameters requires surgical procedures (for airway hyperreactivity), assessment of sacrificed mice (for bronchoalveolar
lavage (BAL) eosinophil count) and extensive sample handlingand preparation (for serum and BAL fluid microplate assays).2Figure 1. Noninvasive tomographic imaging of disease-associated proteaseactivity in ovalbumin-induced mouse asthma. The asthmatic mouse (left)
shows broad distribution of protease-activated ProSense agent fluorescencein the lung, with little fluorescence seen in the lung region of a control mouse(right). The box marks the region of interest established for analysis.

To quantitate the inflammation associated with asthma
progression, we injected the mice with 5 nmol of ProSense
680 agent at day 24, 4 hours after the final intranasal
administration of ovalbumin; that is, 24 hours before
imaging. On the day of imaging, we anesthetized the miceand positioned them in the imaging chamber of the FMT
2500LX system. This system provides both tomographic (fordeep-tissue fluorescence) and two-dimensional fluorescencereflectance imaging (for skin fluorescence and assessmentof excised organs and tissues). Tomographic imaging ofasthmatic mice yielded clear fluorescence signal in lungs ofasthmatic mice, but not in control mice that received PBS
instead of ovalbumin intranasal challenge (Figure 1). Analysisof the tomographic imaging data sets measured a highly
significant increase in activated ProSense agent fluorescencein the lung (>70 pmol/lung) as compared to <5 pmol in thelungs of control mice (P = 0.002; Figure 2a). These resultscorrelate well with the eosinophil count from BAL samples(Figure 2b).
Asthmatic lungs removed from euthanized mice showedwidespread fluorescence (Figure 3a), with minimal fluorescentbackground evident in control tissue. Measurement of theratios of fluorescence intensity in lungs of mice with induced
asthma yielded signal intensity >3.5-fold higher than thatin lungs of control mice (Figure 3b). Notably, the individual
fluorescence ratios for each excised lung correlated wellwith the noninvasive quantitation performed by the FMT
2500LX system (Figure 3c), supporting the contention
that tomographic assessment was solely quantitating lungfluorescence. This confirms that the noninvasive tomographic
assessment of asthmatic mice can be used to accurately
assess deep tissue changes in protease activity associatedwith asthma, in agreement with BAL eosinophil counts(Figure 2b).

Conclusion
PerkinElmer’s Fluorescence Molecular Tomography (FMT)system offers a noninvasive alternative to invasive andterminal assessments of mouse pulmonary inflammation.3
At the core of this technology is the FMT 2500LX system,
which uses trans-illumination (that is, passing near-infrared
light through the imaging subject) from multiple viewpoints.
The FMT system provides a rich dataset to mathematicallymodel and reconstruct tomographic quantitative distributionof all sources of fluorescence within the subject. Pairing thistechnology with PerkinElmer’s ProSense 680 near-infrared
fluorescence agent allows the accurate detection and quantitation of protease activity associated with eosinophils;4 theProSense agent is optically silent and fluoresces only when
activated by disease-related proteases such as cathepsins.
The near-infrared fluorochrome allows maximal tissue
penetration and minimal absorption by physiologicalabsorbers such as hemoglobin or water.

2

These studies demonstrate the utility of the FMT 2500LX
system’s quantitative tomography in combination witha near-infrared, protease-activatable fluorescence agent(ProSense 680), for the noninvasive in vivo imaging andquantitation of pulmonary inflammation. After inducing
asthma by ovalbumin in BALB/c mice, we effectively imaged
the ProSense 680 agent to detect eosinophil protease signalthat can be tomographically quantitated by the FMT 2500LXsystem. Correlation of noninvasive tomographic quantitation
with invasive assessment of lung inflammation established
the accuracy of the FMT 2500LX system’s quantitation ofdeep tissue inflammation. PerkinElmer’s FMT 2500LX system
also offers the potential for repeated noninvasive imaging
and multiplex detection of multiple biological activities,
allowing researchers to better study the progression andalteration of processes associated with disease progression
and therapeutic intervention.

A. Quantitation of ProSense Activation In Vivo

B. Bronchoalveotar Lavage Cell Counts

C. Ex vivo 2D planar imaging of representative lungs

Figure 2. Assessment of asthma severity using the PerkinElmer FMT 2500LX quantitative tomography system (noninvasive) and bronchoalveolar lavage cellcounts (terminal). (A) Quantitation of tomographic images shows high amounts of ProSense 680 fluorescence within the lung region of asthmatic mice, withminimal fluorescence measured within that of control mice as assessed by the FMT system. Whole body 2D planar imaging detected no differences between
asthma and control mice. Error bars, ± s.e.m. (B) BAL assessment indicates large numbers of eosinophils infiltrating the lungs of asthmatic mice compared tothose of control mice, consistent with in vivo imaging results. The experiment is representative of five separate studies; n = 5 mice per group. Error bars, ± s.e.m.
(C) Non-quantitative 2D planar imaging of representative lungs excised from asthmatic and control mice is consistent with in vivo imaging results.

A. Tomographic Quantitation

B. Tomography Correlation to Tissue Fluorescence

C. Tissue Fluorescence(individual lung lobes)

Figure 3. Ex vivo correlation between noninvasive quantitative tomography and ex vivo tissue assessment. (A) Noninvasive FMT 2500LX tomographic quantitationshows clear quantitative differences between cohorts of mice with differing levels of disease. The results shown are representative of five separate studies; n = 5
mice per group. (B) Comparison of non-invasive FMT system assessment and ex vivo tissue assessment of asthma yields a good correlation between the tworeadouts. The FMT system yields greater sensitivity in discriminating between full asthma, moderate asthma, and controls, whereas ex vivo tissue assessment(ratio to mean control tissue fluorescence) was relatively insensitive at detecting moderate asthma relative to negative controls, yet detected significant differences
between moderate and full asthma. (C) Excised lung tissues (separated into individual lung lobes) provided images revealing significant asthma-related increases inlung fluorescence intensity and distribution patterns differing with asthma severity.

3References
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July 2012

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