a p p l i c at i o n N o t e

Pre-clinical Imaging
AuthorJeffrey D. Peterson, Ph.D.
PerkinElmer, Inc.
Waltham, MA USA

Imaging GastricEmptying withGastroSense 750

Abstract

The accurate quantitation of gastric emptying is crucial
for understanding the physiological, pathological andpharmacological mechanisms underlying gastric motility
both clinically and in animal studies. Current methods
used for determining gastric emptying rates in small animals
are either terminal or involve radioactive tracers, incurringhigh financial and time cost. We have developed a novel,near-infrared (NIR) fluorescent imaging agent, GastroSense™ 750, to monitor and quantitate gastric
emptying rates in murine models in vivo, non-invasively, and in real time. The imaging agent iscomprised of an acid-stable NIR fluorophore conjugated to a non-absorbable pharmacokinetic
modifier designed to be formulated in liquid or incorporated into a solid meal. Using female
8 week old BALB/c mice recipients, 0.25 nmol GastroSense 750 was orally gavaged in liquid
formulation (PBS) or fed in egg yolk, an experimental approach designed to detect changes ingastric emptying. In vivo 3D fluorescent imaging and quantitation using the FMT® 2500 Fluorescence
Molecular Tomography (FMT) system at multiple time points revealed a gastric half-life (t1/2) of
36 ±16 minutes for the liquid formulation and 63 ±18 minutes for the egg formulation. Clonidine(an a2 adrenergic agonist, 1 mg/kg) further inhibited gastric emptying time for the egg formulation,
halting the process completely between 2 and 4 h and ultimately delaying the completion of gastric
emptying to greater than 6-7 h. GastroSense 750 signal can also be quantitated as it transits into
the intestines, ultimately decreasing in intensity and volume as it is excreted. These findings revealthat GastroSense 750 and FMT 3D fluorescent imaging in vivo can be used successfully to quantitate
gastrointestinal transit of both liquid and solid diets in small animals.

Materials and Methods
Fluorescent agents for the quantitation of gastric
emptying
GastroSense 750, non-targeted physiologic agent, was used
to image the rate of gastric emptying. The imaging doses
were 0.25-1.0 nmoles in 150 mL saline administered via oral
gavage in all imaging studies.

As a validation of GastroSense transit through the gastrointestinal system, the agent was orally gavaged in multiple
mice, and tissue was excised for ex vivo fluorescence imaging.
Figure 2 shows the kinetics of localization of the PBSformulated agent in different gastrointestinal regions over
4 h. Ex vivo imaging (Figure 1C) shows that the GastroSense
empties from the stomach over 1 h, rapidly moves throughthe duodenum and jejunum, and more slowly transits
through the ileum, cecum, and colon.

Gastric emptying model
Eight week-old female BALB/c mice were purchased fromCharles River Laboratories (Wilmington, MA) and maintained in a pathogen-free animal facility with water andlow-fluorescence mouse chow (Harlan Tekland, Madison,WI). Handling of mice and experimental procedures werein accordance with PerkineElmer IACUC guidelines andapproved veterinarian requirements for animal care anduse. Mice were depilated (Nair® depilatory cream, Church& Dwight Co., Inc., Princeton, NJ) 24 h prior to initiation ofstudies. BALB/c mice were gavaged with agent (0.25 nmole)in PBS, final volume of 200 µL. The effect of solid food on
gastric emptying rates was assessed by feeding some micecooked egg yolk into which 25 µL of GastroSense 750(0.25 nmole) was pipetted.

GastroSense
Oral gavage

Gastric Emptying
Imaging

1-5 hBALB/c mice

0-60 minutes

Figure 1. Gastric emptying and intestinal imaging.

B. Transit Diagram

A. GI Diagram

In vivo FMT 2500 tomographic imaging and analysis

SMice were anesthetized by isoflurane inhalation. Depilatory
lotion was applied thickly on skin over the lower torso(front, back, and sides) of each mouse, rinsed off withwarm water, and re-applied until all fur had been removed.
Mice were then imaged using the FMT 2500 Quantitative
Pre-clinical Imaging System which collected both 2D surface
images as well as 3D tomographic imaging datasets.

StomachDuodenum
Jejunum
Ileum
Cecum

C

Ascending ColonFMT reconstruction and analysis

Descending Colon

The collected fluorescence data was reconstructed by FMT
2500 system software (TrueQuant™ v2.0) for the quantitationof three-dimensional fluorescence signal within the stomachand intestines. Three-dimensional regions of interest (ROI)
were drawn encompassing the relevant biology. A variety ofbackground thresholding levels were applied systematically,and results were compared to other independent readouts forvalidation.

0GastroSense validation
GastroSense 750 is a NIRF imaging agent designed tomonitor and quantitate gastric and intestinal emptying noninvasively in murine models in vivo as represented in Figure 1.
The fluorophore of this agent is acid-stable and conjugatedto a non-absorbable pharmacokinetic modifier designed to
be formulated in liquid or incorporated into a solid meal.

212Time (h)34C. Ex Vivo Tissue: Planar Imaging of GastroSense Transit

5 min

Results

Intestine
Imaging

30 min1h2h3hImaging the stomach. GastroSense was administered by oral gavage in
PBS (Liquid) and gastrointestinal tissue was excised to determine agent
localization. A. Schematic diagram of the different regions of thegastrointestinal tract. B. Diagrammatic representation of GastroSense
localization over time based on two studies. C. Ex vivo planar NIR
images of excised gastrointestinal tissue from representative mice at
5 timepoints.
Figure 2. Gastrointestinal transit kinetics.

In vivo imaging of gastric emptying
Gastric emptying can be assessed non-invasively by imaging
the stomach region over time, from immediately after oral
gavage of GastroSense through 1.5 h. Figure 3 (left panels)
shows 3D FMT images of representative mice to illustrate
the difference in gastric emptying rates (GER) of fluorescencedepending on whether the agent is administered with solid
food (egg yolk) (GER = 47 ±11%/h) or as a liquid formulation(GER = 167 ±28%/h). Allowing access to solid chow after
liquid formulation gavage has an appreciable effect on theemptying rate (GER = 78 ±9%/h). It is interesting to note thatplanar imaging (Figure 3, right panels), which only qualitatively
detects fluorescence at the surface of the animal, was relativelyineffective in monitoring gastric emptying. Because of thedepth of the stomach, there was a dramatic loss of sensitivityof detection with only 50-70% decrease in stomach fluorescence.
FMT quantitation of the kinetics of change in these small
groups of mice (Figure 4A) show the low variability andaltered kinetics with the different GastroSense imaging
Tomographic Imaging (3D)

approaches. Comparable delays were achieved with theaddition of solid food substances either admixed in egg orby allowing access to mouse chow, and this was a measurable
and statistically-significant change (Figure 4B).
It is important to note, however, that the gastrointestinal
tract contains significant pockets and regions of air throughout
the lumen and that this air can cause an artifactual
enhancement of fluorescent signal during in vivo imaging.
Furthermore, the formulation of GastroSense and/or thevolume of agent administered can lead to introductionof additional air artifacts. Indeed, the admixture of
GastroSense in egg yolk appeared to increase the amountof gastrointestinal air. Similarly, gavaging of small volumesof Gastrosense (i.e. 100 µL or less) permits significant
retention of air in the stomach, enhancing the apparent
stomach fluorescence. However, the pattern of changes offluorescence will not be altered, and normalization to starting
stomach signal allows comparison of pattern changes
between different formulations or changes that occurwith drug treatment.

Planar Imaging (2D, surface)

A. GastroSense/PBS in Fasted Animals

5 min

30 min60 min90 min

5 min

30 min60 min90 min60 min90 min

5 min

30 min60 min90 min60 min90 min

5 min

30 min60 min90 minB. GastroSense/PBS in Solid Chow-Fed Animals

5 min

30 minC. GastroSense/egg in Fasted Animals

5 min

30 minImaging the stomach.
GastroSense was administeredby oral gavage in PBS orprovided to animals mixed inegg yolk as food. Mice were
either fasted for the remainderof the study, or they wereallowed free access to standard
mouse chow. A time course offluorescence tomographic
datasets were acquired byimaging of the gastric region
over time, comparing
tomographic imaging (leftpanels) to surface planar
imaging (right panels).
Improved sensitivity ofdetection is achieved by deep
tissue tomographic imaging,with surface imaging signal
dropping off much more
rapidly.

Figure 3. Imaging gastric emptying.

Quantitating Fluorescence. FMT tomographic imaging datasets forstudy groups represented by mice shown in Figure 3 were used toquantitate fluorescence. Every mouse was imaged repeatedly to capture
7 time points over the span of 3 h. This provided real-time, quantitative
data regarding the rate of gastric emptying. Results in pmol were adjusted
to reflect the % of fluorescence at the starting time.

Figure 4. FMT quantitation of gastric emptying.

3the first 90 minutes, after which gastric emptying nearly
halted for 2 h. Gastric clearance increased as drug effects
appeared to decline 4 h post dosing.
In vivo imaging of gastrointestinal transit

Inhibiting Gastric Transit. Mice were treated by i.p. injection withclonidine (1 mg/kg), a gastric emptying inhibitor. Fifteen minutes after
treatment, GastroSense, formulated in egg yolk, was consumed by mice,and animals (n = 6) were imaged over time. Fluorescence tomographic
datasets were analyzed by placing three dimensional ROIs for thestomach, cecum, and intestinal region. Intestine signal quantitation wasdetermined by subtraction of cecum fluorescence artifacts at each timepoint. All fluorescence was thresholded minimally, at 10% of the initial
fluorescence concentration.
Figure 5. Inhibiting gastric motility with clonidine.

To assess the ability of GastroSense to detect drug-induced
alterations in gastric motility, we used clonidine, an a2
adrenergic agonist that affects gastric fundus contractility
and delays stomach emptying. Mice were treated with clonidine 15 minutes prior to feeding with a GastroSense/egg
formulation (Figure 5). Mice were imaged repeatedly overa 3 h period to quantitate stomach fluorescence. A subtle
decrease in the rate of gastric emptying was evident over

GastroSense, upon emptying from the stomach, enters theintestines and provides a tool for quantitating intestinal transit.
Figure 6 shows the quantitative results of non-invasive tracking
of stomach emptying in the context of intestinal transit
kinetics. Measurement of signal in the cecum, as comparedto the intestines (minus the cecum), show that the cecum
contributes approximately 50% of the signal generated in
the entire intestine. Interestingly, the volume of fluorescencein the cecum is substantially lower, suggesting that the signal
is approximately 3-4X brighter than signal in the rest of theintestines. Based on ex vivo tissue studies showing an eventransit through the tissue, we believe that the cecum signal
is overestimated by 3-4X, presumably due to air or gas
within the cecum.
Gastrointestinal transit can also be assessed using theGastroSense/egg yolk formulation (Figure 7). As shown in
Figure 4A, stomach emptying is delayed as compared toformulation of the agent in PBS. It is interesting to note thatthe total intestine and cecum signal is approximately 4-fold
reduced (compare Figure 7A to Figure 6A), although it isunclear whether the longer residence time in the stomach
increases degradation of this agent. However, clearly theintestinal transit rate (ITR) after 2 h is still approximately
13-fold slower for egg-formulate GastroSense (ITR = 4.2%/h)than that of GastroSense/ PBS (ITR = 54%/h). Even
representing rates as a percentage of the signal seen at
120 minutes, there is still a 3-fold difference in ITR (24%/hfor egg yolk formulation vs. 72%/h for the PBS formulation).

Imaging Gastric Transit. GastroSense, formulated inPBS, was administered by oral gavage, and mice (n = 6)
were imaged over time. Fluorescence tomographic
datasets were analyzed by placing three dimensional
ROIs for the stomach, cecum, and intestinal region.
Intestine signal quantitation was determined bysubtraction of cecum fluorescence artifacts at each timepoint. All fluorescence was thresholded minimally, at
10% of the initial fluorescence concentration.

Figure 6. Gastric transit (GastroSense/PBS).

4

Imaging Gastric Transit. GastroSense, formulated in egg yolk, was consumed by
mice, and animals (n = 6) were imaged over time. Fluorescence tomographic
datasets were analyzed by placing three dimensional ROIs for the stomach, cecum,and intestinal region. Intestine signal quantitation was determined by subtraction ofcecum fluorescence artifacts at each time point. All fluorescence was thresholded
minimally, at 10% of the initial fluorescence concentration.
Figure 7. Gastric transit (GastroSense/egg yolk).

5 min

30 min60 min90 min

120 min180 min

240 min

300 min

Fluorescence volume can be assessed
quantitatively, but also represented as fluorescenceisosurfaces (i.e. 3-dimensional images of all
fluorescence greater than a given cut-off
threshold) as shown in Figure 8. Although thephysics of light propagation through tissue place
limitations on the generation of images with highanatomical accuracy (particularly in large regionsof continuous, overlapping signal), you cansee clear regional differences in fluorescenceacross the intestines. We are currently validating
an approach for using such data as a tool forintestinal co-registration with other agentsthat detect inflammatory pathology within
the intestines, as this may be a useful tool for
improving the sensitivity of disease detection bylimiting the analysis to GI tissue. Based on very
preliminary studies, intra-rectal administration may
be an alternative for the specific localization of thedescending colon (which shows poor retention of
GastroSense signal when given orally).

Fluorescence Volume Assessment. GastroSense,formulated in egg yolk, was consumed by mice, andanimals (n = 6) were imaged over time. Fluorescencedatasets were represented as isosurfaces forfluorescence > 10% of the mean stomach signal. ROIs
for the stomach and intestinal regions. Intestine
signal quantitation was determined by subtractionof cecum fluorescence artifacts at each time point.

≥120 nM

Figure 8. Fluorescence volume images (GastroSense/egg yolk).

Table 1. Summary guidelines for GastroSense application.		

Imaging Time

Oral Agent (PBS Formulation)
Gastric Emptying
Intestinal Transit
Effect of Drug Treatment
Intestinal Contrast AgentInflammatory Bowel Disease

0-2 h
1-4 h
0-5 h
~2 h
0-5 h decreased signal, faster transit

Oral Agent (egg yolk formulation)
Gastric Emptying
Intestinal Transit
Effect of Drug Treatment on Food Transit

0-2 h
1-4 h0-5 h

Intrarectal Agent (PBS formulation)
Descending Colon Contrast Agent,
Combined with Disease-Imaging Agents

Immediate Imaging

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Dec. 2012