A simple automated approach to measure mouse whole gut transit

Abstract

Background:

Gastrointestinal (GI) motility is a complex physiological process critical for normal GI function. Disruption of GI motility occurs frequently in GI diseases and as side-effects of therapeutics. Whole gut transit measurements in mice, like carmine red leading-edge transit, form the cornerstone of in vivo preclinical GI motility studies.

Method:

We have developed an easily achievable, labor-saving method to measure whole gut transit time in mice. This approach uses inexpensive, commercially available materials to monitor pellet production over time via high definition cameras capturing timelapse video for offline analysis.

Key Result:

We describe the assembly of our automated gut transit setup and validate this approach by comparing conventional transit measurements with transit delay by loperamide. We demonstrate that compared to the control group, the loperamide group had slowed transit, evidenced by a decrease in total pellet production and prolonged whole gut transit time. The control group had an extended transit time compared with the results reported in the literature. Whole gut transit rates accelerated to times comparable to the literature by disrupting cages every 10–15 min to imitate the conventional approach, suggesting that disruption affects the assay and supports the use of an automated approach.

Conclusion & Inferences:

A novel automated, inexpensive, and easily assembled whole gut transit setup is labor-saving and allows minimal disruption to animal behavior compared to the conventional approach.

Introduction:

The GI tract requires highly coordinated transit to extract nutrients and package waste. Abnormalities in GI motility occur in 10–20% of the US adults and is a common side effect of medicines.¹ Thus, measurements of GI transit are essential in the diagnosis and treatment of GI motility disorders and drug discovery.^1,2

Preclinical studies in gut motility span from in vitro molecular, cellular, and tissue to in vivo transit measurements.^2,3 In vivo studies measure the regional transit times, like gastric emptying and intestinal or whole gut transit.^4,5 Studies also employed advanced imaging techniques to measure GI transit, including scintigraphy, ultrasound, CT, MRI, and radiography.^6–9 Still, an inexpensive and readily available dye transit measurement of “leading edge” whole gut transit remains the gold-standard.² This simple approach involves gastric gavage of dye, usually, carmine red, followed by visual observation of pellet output from singly housed mice, every 10–15 minutes for several hours, until the appearance of the red pellet. The interval between gavage and colored pellet output is the whole gut transit time. While this approach is robust and time-tested, there are several limitations. First, it is low throughput and labor-intensive, such that an experimenter often dedicates an entire day to a single experiment. Second, repeated observations make this testing disruptive for the animals, which may independently modify GI motility. Third, measurement frequency limits temporal resolution. Fourth, other valuable information, like the number of pellets per hour, can be overlooked when measuring only the time of appearance of the red pellet. Studies have automated whole gut transit measurements in mice, but this required animal acclimatization or custom cameras and light sources.^10,11

Here, we used inexpensive, commercially available materials to make an automated carmine red whole gut transit setup that uses time-lapse imaging and addresses the limitations of the manual approach. We validate our approach by measuring transit delay caused by loperamide. Additionally, we demonstrate that repeated disruption of mouse housing, typical of the conventional transit approach, can affect measured transit rates.

Methods:

All procedures and protocols were in accordance with the Mayo Clinic Institutional Animal Care and Use Committee (IACUC).

Animals:

Male and female wildtype C57/Bl6 (7–9 weeks old) mice were purchased from Jackson Laboratories, bred as wildtypes, maintained under specific pathogen-free conditions. The housing facility had a 12h:12h light-dark cycle (6:00 am on, 6:00 pm off), and we did our studies on non-fasted mice starting at 8:30 am ±30 min.

Reagents:

We purchased carmine (C1022), methylcellulose (274429), loperamide hydrochloride (L4762) from Sigma (St. Louis, MO). We prepared the gavage solution by dissolving methylcellulose (0.5% w/v) in hot distilled water and then mixing carmine (6% w/v) and stored it at 4°C until use.

Automated Whole Gut Transit Setup:

We assembled the automated whole gut transit setup (Figure 1a,b) using commercially available components in an under-counter two drawer shelf cabinet. We used modified cylindrical plastic jars with lids, consistent with AALAS guidelines for singly housed mice (4.87” diameter, Container and Packaging, B520) with bottoms cut-out and replaced with perforated aluminum sheets (1/4” diameter round opening pattern, 5/16” CTRS 48% open area) (Figure 1c,d), Teflon-coated funnels (Figure 1e), 35 mm collection plates (Corning #431111) (Figure 1b,f), 4) high-definition (HD) cameras (TENVIS JPT3815W-HD) (Figure 1b,h), and commercially-available software for time-lapse imaging (CandyLabs, http://www.candylabs.com/videovelocity). We placed the jars into a custom cabinet onto the top shelf, with 15 cm long Teflon-coated funnels under the jars, and collection plates filled with glycerol to improve visualization and decrease odor onto the bottom shelf (Figure 1f,h). To further decrease stickiness, we coated the surface of the chamber and funnel with silicone spray (Eco Guard Silicone Spray, Jaws Product). We used flexible LED strips to improve illumination when the cabinet doors were closed (Figure 1f,h). We placed the HD cameras on the lowest shelf underneath the collection plates and connected them to the local area network. We used Tenvis software to position and focus the cameras. We used VideoVelocity software to record time-lapse videos (1/10 sec), and offline image processing (playback 50x speed) (Figure 1i).

Figure 1. Automated whole gut transit setup.

a) Conceptual design, b) Images of the assembled system and components. Components include chamber from c) top and d) oblique view, and e) funnel oblique view. Assembly in the under-counter cabinet, f) on two shelves, and h) closing doors. i) Timelapse images from a typical experiment (numbers denote the sequence of pellets, *red pellet [267 min]).

Experimental Design:

We used three groups: 1) control (300 μL carmine), 2) loperamide-treated (300 μL carmine+0.3mg/kg loperamide), 3) conventional (300 μL carmine) visually examined every 10 min. We started the video recording and digitally stamped when we placed mice in the chamber after gavage. For group 3, to imitate the traditional approach, thirty minutes after gavage, we opened cabinet doors until the end of measurements, and we checked the mice every 10 minutes by pulling out the shelf, opening chamber lids, and tapping the chambers while opening. We monitored pellet production until the detection of a red pellet for each mouse in the experiment group or up to 600 min. We repeated this protocol for all groups and mice every other day for three averaged measurements (Mon-Wed-Fri). At the end of the experiments, we extracted the soiled chambers and funnels from setup and placed them into 10% hypochlorite/water solution for 10 min and then washed with soap.

Data Analysis:

Offline analysis started at the time stamp (T₀) and times and color of all pellets (T_pellet#), including the first red pellet (T_transit), and averaged (Mean±SD) T_pellet# and T_transit values from individual mice. We excluded the individual results from mice that had a red pellet above the metal sheet at the experiment end. We used one-way ANOVA, Tukey’s post hoc analysis (GraphPad Prism 7).

Results:

Our automated carmine red whole gut transit method (Figure 1) allowed us to examine several animals in parallel and to collect pellets produced during 10-hour experiments without disruption of animal behavior (Figure 1i). Time-lapse high definition videos enabled us to measure the number of pellets per unit of time and to distinguish pellet color, which we confirmed visually at the end of the experiment (Figure 1i).

We were able to monitor all pellets and red pellet production in all groups (Figure 2a–c). We found that the control group (Figure 2a), produced 5.9±3.1 pellets in 2 hours and 7.5±2.9 pellets in 4 hours (Figure 2d,e) and had a whole gut transit time of 372±79 min (Figure 2f). We used loperamide, a μ-opioid receptor agonist, and a well-established anti-diarrheal medicine that slows gut transit in humans and mice to evaluate the validity of the automated transit chamber approach.^12–14 Loperamide (Figure 2b) indeed decreased the pellet production to 2.8±1.3 at 2 hours and 3.4±1.3 at 4 hours (Figure 2d,e) and extended the whole gut transit time to 474±34 min (Figure 2b,f).

Figure 2. Automated setup validation.

Pellet production for a) control, b) loperamide, and c) conventional groups (each line is a single mouse averaged over three measurements, ■ carmine pellet time). d) Average pellets over the first 2 hours and e) 4 hours pellet production. f) Average red pellet appearance time (N=7 control, N=6 loperamide, and conventional, one-way ANOVA, *p<0.05)

We noticed that the whole gut transit measured by this approach resulted in transit times that are longer than typically reported in the literature using a conventional manual approach (130–250 mins^15–22). We hypothesized that this might be due to disruptions during repeated measurements. We used our approach but manually examined the animals for pellet production every 10 min, emulating the conventional approach (Figure 2c). We found that compared to the controls, this approach resulted in an increase in pellet production 10.6±3.4 at 2 hours and 14.1±4.7 at 4 hours (Figure 2d,e) and speeding of the whole gut transit time to 168±30 min (Figure 2f).

Discussion:

Preclinical in vivo GI transit measurement in rodents is essential for studies in GI physiology and pathophysiology, for determination of the effectiveness of medications that target GI motility, and medication side effects. The current gold-standard for whole gut transit measurements in rodents is the carmine red dye transit, where mice receive a gavaged meal of non-nutrient viscous with non-absorbable red dye. Investigators then intermittently examine pellet production until the appearance of the first red pellet. This approach is labor-intensive for the experimenter, disruptive to the animals, does not capture all of the available data, and suffers from low temporal resolution. In this study, we used inexpensive, commercially available materials to develop an automated approach to conduct whole gut transit and monitor overall pellet production in time by using time-lapse imaging. Our setup addresses the limitations of the current approach by decreasing labor, animal disruption, and increasing temporal resolution.

We validated our approach by comparing the transits of a control group and a group treated with a well-established transit-delaying μ-opioid agonist medication, loperamide (Imodium).¹² Acute loperamide administration indeed slowed whole gut transit and decreased the number of produced pellets. Interestingly, we found that our control transit group had slower whole gut transit times than many reported in the literature.^15–22 We hypothesized that the faster transit times reported elsewhere might be due to the disruption of animal behavior during measurements. So, we compared the whole gut transit times of our control group and a group for which we used a more traditional approach, manually observing pellet production every 10 minutes. We found that the conventional approach had substantially accelerated whole gut transit times, confirming that experimental conditions contribute to whole gut transit time.

In addition to the measurement of whole gut transit time, we were able to track pellet numbers over time. There are additional possibilities for this setup that we have yet to explore. For example, since we visualize all pellets, we could integrate other quantifiable measures, like pellet shape, size, and weight. We conduct our experiments in daylight in the open, but since rodents are nocturnal and prefer dark enclosed locations, our current settings are suboptimal. This simple automated setup can be further modified to perform experiments at night and in the dark.

In conclusion, we have developed a simple approach to automate whole gut transit studies in rodents that is time-saving, reproducible, and allows for minimal disruptions to the study animals.