Adult newts, or axolotls, or Pleurodeles at various stages of larval growth are usually used for these studies. The limbs are amputated, and then the animals are treated with retinoids in various ways. One method of administration is simply to add 10-60 mg retinyl palmitate (Sigma, complexed with corn starch) to 1 L of water in which the animals are kept. The water turns cloudy because of the corn starch and should be changed every day. Treatment times vary from 1-14 d (22). It is also possible to treat animals in this way with tRA and other retinoids. In this case the appropriate amount of retinoid is used dissolved in DMSO (e.g., 300 pL of a 1 mg/mL solution of tRA), and this is put into the water. The retinoid immediately precipitates out, being insoluble in water, forming a yellow scum on the surface. Nevertheless, this method works perfectly well.
It is also common to administer retinoids by ip injection (e.g., 33). Animals are anesthetized and placed on their backs on a wet towel. A 27-gage needle is used to make a puncture through the skin and abdominal muscles into the ip cavity of each animal, a few millimeters anterior to one hindlimb and lateral to the midline. The volume of solution containing the desired dose to tRA is then injected into the abdomen with a Hamilton microsyringe using the previously made puncture hole. tRA percipitates at the injection site, being visible through the skin as a yellow mass, and gradually dissipates over the next 24-48 h. Animals are returned to their water as soon as possible. Control animals are injected with an equal amount of DMSO. This method tends to induce some mortality among the animals, since DMSO injected into the peritoneum is very unpleasant for the liver.
A very reliable and nontoxic method of administration is to use retinoids in Silastic as described above. Pieces of a uniform size of Silastic, e.g., 1 mm3 containing tRA at the appropriate dose, e.g., 100 mg/mL, are prepared. Animals are anesthetized and a tunnel made in the limb under the skin by inserting the points of a pair of watchmaker forceps through the skin and pushing them along the limb between the skin and the underlying musculature. In this way, the piece of Silastic can be placed directly adjacent to the regeneration blastema on either the dorsal, ventral, anterior, or posterior side. Animals are returned to the water, and the Silastic remains in place for several weeks. With a colored compound, such as tRA, one can see the color gradually disappear from the Silastic. The diffusion characteristics of tRA from Silastic have been described above (26). Only very rarely are the blocks ejected from the limb. One example of this we have found is when the Siastic contains retinal (22). Uniquely, this retinoid induced the epidermis to eject the block.
Finally, gastric intubation has been used to administer retinoids to adult newts (34). In this case, the retinoid solution in DMSO is squirted into the stomach of the anesthetized animal through a thin plastic tube attached to a syringe.
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