This method could help answer key questions regarding gastrointestinal biology, such as how gene expression changes or environmental challenges alter gut function. The main advantage of this technique is that it takes less time and is easier to execute than traditional methods of measuring gut transit. On the day of the assay, larvae are fed food containing a fluorescent label, and as they eat that food, the label will accumulate in their gut.
After feeding, they are rinsed, separated from uneaten food and each larva is transferred into a well of a multi-well plate. The well has a conical bottom so that when fecal matter is voided, it falls to the center of the well. Now remember, there are many larvae in the multi-well plate that can be monitored simultaneously using a plate spectrophotometer, which measures the amount of voided fecal matter.
And as more is voided over time, the signal increases accordingly. In this way, we have a high throughput method for measuring gut transit. On the fourth day post-fertilization, sprinkle two milligrams of powdered larval fish food on top of the water in each Petri dish to feed the larvae.
Allow the larvae four hours to feed. After feeding, transfer all larvae to a large rinsing dish containing fresh embryo medium. Using a 50 milliliter serological pipette, gently vacuum as much of the leftover food as possible, being careful not to capture any larvae.
Transfer larvae to a new Petri dish containing 50 milliliters of fresh embryo medium. Repeat the feeding, rinsing, and transfer process for two additional days. On the sixth day post-fertilization, begin preparing the fluorescent food by adding 200 milligrams of dried food to a 10 centimeter watch glass.
Add 300 microliters of fluorescent label and 100 microliters of deionized water and mix briefly. Spread the resulting paste into a thin layer on the watch glass and allow it to dry at room temperature, in the dark, for at least eight hours. After this, scrape the dried mixture off the watch glass.
Crush it to a powder and store it at room temperature, in the dark. On the seventh day post-fertilization, feed the fluorescent food to the larvae by sprinkling two milligrams of it on top of the water in each Petri dish. Allow the larvae to feed for two hours.
Meanwhile, prepare the concentrated dosing solutions by dissolving each test compound in embryo medium to a concentration that is two times the target dose with a final volume of at least 2.5 milliliters. At the end of the feeding period, use a transfer pipette to move them to a large rinsing dish. Rinse all larvae previously described with more stringent vacuuming.
It's important to remove as much of the leftover fluorescent food as possible during the final rinse to reduce the possibility that it is inadvertently transferred to the 96-well plate. After each larva is rinsed, used a pipette to withdraw it along with 100 microliters of embryo medium from the rinsing dish. Dispense the larva and the entire 100 microliters of medium into a well of a 96-well polystyrene, conical-bottom, multi-well plate.
Once all the larvae have been transferred, add 100 microliters of the prepared dosing solutions to the appropriate wells. After all the dosing solutions have been added, load the plate into a plate spectrophotometer. Measure the fluorescence from the plate from the bottom, five times in immediate succession, without shaking the plate.
The lowest reading from each well will be designated as its initial fluorescence during data analysis. Incubate the plate at approximately 28 degrees Celsius. Repeat the measuring process, reading the fluorescence every 20 minutes for the first two hours post-dosing, and incubating between reads.
Measure the fluorescence every 30 minutes for the third and fourth hours post-dosing and then make hourly measurements for hours five through eight. Incubate the larvae at 28 degrees Celsius overnight. The next day, read the fluorescence at 24 hours post-dosing.
This protocol uses plate-based spectrophotometry to assess GI transit as a high-throughput replacement for fluorescent microscopy. Representative results comparing these two methods are generated by analyzing identically treated fish over a 24 hour period post-dosing. As seen here, the results are highly correlated, with a negative slope because microscopy measures the retained fluorescence signal and spectrophotometry measures the transmitted signal.
Analysis of compounds with disparate mechanisms reveals that when compared to vehicle treated controls, atropine and amitriptyline slow GI transit. Tegaserod and metoclopramide, on the other hand, are seen to accelerate transit time. Erythromycin, which was expected to accelerate transit time based on mammalian response, is instead seen to have no affect on zebra fish transit time.
Further analysis demonstrates that atropine slows GI transit, dose dependently, in zebra fish larvae. The lowest dose tested, 042 micromolar, had no significant effect. While the two higher doses are each seen to have significant impact.
This method has many applications, including detecting toxic GI effects from drugs or drug candidates, disease modeling, such as irritable bowel syndrome, discovery of novel therapies for such diseases, as well as the discovery of pro-kinetic compounds.
