An In Ovo Model for Testing Insulin-mimetic Compounds

The overall goal of the presented modified chick embryo model is to characterize substances with insulin-mimetic properties in a living organism. This method can help answer key questions in the field of diabetics research such as finding herbal extracts with insulin-mimetic properties that reduce blood glucose levels without side effects. The main advantage of this technique is that the efficacy of compounds can be tested in a living organism with adequate throughput rates, and that permission by an ethics committee is not necessary.

Visual demonstration of this method is critical as the preparation and cutting steps are difficult to learn, because the vessels are not easily prepared, or often cut completely and get lost. Check eggs for fertilization after incubation at 38-degrees Celsius with an average humidity of 40 to 60 percent for up to 11 days. To do this, dip the fringe of the candling lamp into an ink pad, then candle the pointed side of the egg.

To identify the air bladder, check for differences in brightness on the top of the egg. The air bladder appears as a round, more transparent region, while the albumin appears darker. Mark the location of the air bladder by pressing the lamp slightly against the egg with the previously applied ink.

Exclude non-fertilized eggs, which do not show a difference in brightness between the albumin and air bladder. Prepare the insulin-mimetic, or insulin, by diluting to the desired concentration in Hank's Balanced Salt Solution. To apply the selected compound, use a pointed pair of tweezers to carefully peck the eggshell in the marked area of the air bladder to form a hole equal to the needle syringe diameter.

Use a sterile one-milliliter syringe to inject 300 microliters of the buffer solution containing the substance of interest. To determine the blood glucose reducing effect of a substance, place the eggs back into the incubator and measure blood glucose after 60, 120, and 180 minutes. At the appropriate time point, carefully remove the eggshell above the air bladder and equilibrate the eggshell membrane by adding enough PBS buffer to cover the whole membrane.

After pouring away excess PBS, use a pointed pair of tweezers to carefully pull off the eggshell membrane. Use a micro scissor to cut and remove the chorioallantoic membrane while avoiding any large vessels in the membrane. Locate the large vessel originating from the abdomen of the embryo.

Lift this vessel out of the albumin with a closed micro scissor and place it on a plastic pH strip. Move the pH strip under the vessel and pull back the micro scissor carefully. It is essential preparing the large blood vessel originating from the abdomen, as smaller ones may rip apart rendering blood collection impossible.

Remove any liquid surrounding the vessel with a pipette to avoid dilution of the blood. Then, use filter paper to dry the vessel and pH strip. For blood collection, it is important not to completely cut the vessel as it will get lost.

Next, use a micro scissor to cut the blood vessel carefully on one side. Do not cut more than halfway through the vessel to avoid cutting through the vessel entirely. Collect the blood leaking onto the pH strip with a pipette.

10 microliters of blood are required to determine glucose levels using the blood glucose meter. After application of the selected compound for 24 hours, equilibrate the eggshell membrane with PBS as before for at least 30 seconds. Remove excess PBS buffer by pouring it away.

Then, pull off the eggshell membrane carefully with a pointed pair of tweezers. Check the blood vessels for lesions, and confirm vitality by observing movement of the chick embryo. Extracts prepared from combretum indicum, or the Rangoon creeper, and a non-disclosed extract labeled extract 0845 were used for these experiments.

As indicated in this figure, the extract from Rangoon creeper did not result in a significant blood glucose reduction in ovo. However, the blood glucose concentration was successfully reduced with extract 0845 with a significant effect observed when the incubation time was prolonged. Compared to untreated eggs, treatment of eggs with a toxic herbal extract on day 10 for 24 hours led to severe lesions of blood vessels in the chorioallantoic membrane.

Lesions were also apparent on the treated embryo compared to normal control. Once mastered, two eggs can be analyzed for glucose levels and toxicity in five minutes if performed properly. After watching this video, you should have a good understanding of how to prepare blood vessels and collect blood in the chicken embryo model to quantitate the blood glucose reducing effects of natural or synthetic compounds.