Alternate Immersion in Glucose to Produce Prolonged Hyperglycemia in Zebrafish

This protocol induces hyperglycemia in zebra fish in a rise and fall pattern that mimics the hyperglycemia that is seen in type two diabetes. The main advantage of this technique is that it is a noninvasive technique that makes it possible to ensure that hyperglycemia is the cause of any alterations observed in the hyperglycemic fish. This protocol could potentially be used to explore therapeutic avenues or pharmaceuticals that target complications of hyperglycemia.

This protocol has a lot of steps, but when the steps are followed correctly and with care, it should quickly become second nature. Remember however, to always treat the animals gently and humanely throughout the entire protocol. Begin by setting up six tanks, two for each experimental group.

Use two liter tanks, if the total number of fish is less than 20 and four liter tanks if the total number of fish is more than 20. Label one of the two tanks, housing tank and the other solution tank. Keep the tanks in a water bath at 28 to 29 degrees Celsius to maintain water temperature.

On day one, place the fish into their respective treatment solutions for 24 hours. On day two, transfer the fish from their treatment solutions to water for 24 hours. On day three, transfer the fish from water to treatment solutions.

Continue this alternating exposure for the remainder of the experiment. Transferring water treated control fish from water-to-water daily. Ensure that the fish are fed and transferred within the same two-hour window each day throughout the duration of the experiment.

Transfer fish in each treatment group from the housing tank to the corresponding solution tank using a standard fish net. Place the tank containing the fish back in the water bath and replace the air stone and tank lid. This tank is now the housing tank and the tank that previously held the fish is now the solution tank.

Discard the old solution and clean the tank along with the tank lids, air lines, air stones and nets to prevent buildup of glucose and mannitol. Use water and a dedicated sponge for each treatment condition to properly clean the tanks. Dry the newly cleaned solution tanks with a paper towel and prepare the solutions for the following day using this tank.

Ensure the other items are dried and separated by appropriate treatment groups. To prepare the sugar solutions, fill each solution tank with two or four liters of system water. Measure the correct amount of glucose and mannitol using a top-loading scale and separate weigh boats for each chemical.

Add the weighed glucose or mannitol aliquot to the appropriate clean solution tank. Stir the solutions with separate glass stir rods until the sugars are completely dissolved. Then return the solution tanks to the water bath and cover them with their corresponding lids.

To prepare the water solution, fill experimental tanks with system water Return these solution tanks to the water bath and cover them with their corresponding lids. Blood sugar values were significantly elevated after both four-week and eight-week glucose treatments. With hyperglycemia defined as three times the control averages from both water treated and mannitol treated groups.

Retinal tissue collected after four weeks of hyperglycemia had an increase in glial fibrillary acidic protein or GFAP levels. GFAP expression is observed in Mueller glial cells in the retina, which are altered in diabetic retinopathy. This increase in GFAP was associated with an increase in nuclear factor kappaB levels.

Suggesting that the induced hyperglycemia triggers an inflammatory response and reactive gliosis. ERG recordings after four weeks of treatment identified a decreased response in glucose treated retinas compared to mannitol treated controls. Amplitudes of both photoreceptor and bipolar cell components were decreased in hyperglycemic fish.

This procedure can be supplemented by other tests of hyperglycemia. For instance, a memory assay can be used to look at cognitive deficits or record visual based responses such as the optomotor response to assess vision-based complications. Once this technique was established, our lab was able to use it to study hyperglycemia induced complications in the zebra fish model.

We observed these complications relatively quickly, after four weeks of treatment.