The induction of severe hypothyroidism is essential to observe changes in gene expression by thyroid hormone. Moderate hypothyroidism leads to physiological changes aimed at maintaining T3 concentrations in neural tissue within normal levels. The most important mechanism concerns deiodinase type 2 (D2). D2 is a selenoenzyme that catalyzes the removal of the iodine atom in the 5' position of T4 to generate the active hormone, T3. D2 activity is inhibited by T4 through a mechanism involving increased degradation of the enzyme in proteasomes. In situations of low T4, the increased expression and activity of D2, with the concomitant increased efficiency of T4 to T3 conversion, tends to maintain T3 concentrations constant (30). Therefore, only under very severe hypothyroid conditions are T3 concentrations low in the brain, in contrast with other tissues, such as the liver or kidney.
Hypothyroidism can be induced in the rat by surgical or chemical means. Surgical thyroidectomy procedures are difficult in mice, owing to the anatomical features of the mouse thyroid gland, which is not easy to remove. In mice, as in rats, chemical thyroidectomy, combining two different antithyroid drugs gives satisfactory results.
220.127.116.11. Surgical Thyroidectomy
1. Anesthetize the rat.
2. 0pen a longitudinal incision in the neck.
3. Separate the borders of the skin and the neck muscles until the thyroid gland is exposed.
4. Pull off carefully each lobe of the gland.
5. Close the incision.
A number of drugs interfere with hormone synthesis in the thyroid gland. The most commonly used are sodium or potassium perchlorate, 1-methyl, 2-mercaptoimidazole (MMI) and 6-n-propylthiouracil (PTU). Perchlorate blocks the active transport of iodine to the thyroid gland carried out via the Na-I symporter. MMI and PTU block the intrathyroidal oxidation of iodine and, therefore, the iodination of thyroglobulin. PTU, in addition, inhibits the activity of type 1 deiodinase (D1), so that less T3 is formed from T4 in the liver and kidney. Since D2 is not inhibited by PTU, it is possible that D1 inhibition by PTU would spare T4 as a substrate for D2 in the brain. In our laboratory, we prefer the use of MMI for this reason and, also, because it is readily soluble in water. PTU, in addition to being less soluble, requiring alkalinization with NaOH, has a bitter taste and, therefore, is less tolerated by the animals. These drugs are given in the drinking water either alone or in combinations. In our laboratory, we use the following final concentrations: perchlorate: 10 g in 1 L of drinking water (1%); MMI: 200 mg in 1 L of water (0.02 %).
The most intense degree of postnatal hypothyroidism is achieved by giving MMI to the pregnant dams and then performing thyroidectomy of the new-borns. Taking into account the ontogenic dates for the T3 receptor (E14) and the thyroid gland (E18), and that hypothyroidism does not interfere with placental metabolism during the second half of pregnancy, we usually administer MMI continuously to the dams in the drinking water, starting around d 9 to 10 postconception. Newborns are then surgically thyroidectomized at P5. Since MMI crosses the placenta, the newborns develop goiter, which facilitates removal of the gland. With this protocol, a severe hypothyroidism is induced. The drawback is the high mortality, which is due not only to the surgical stress, but also to the fact that hypothyroidism is so profound that few pups survive after weaning. To ensure longer viabiliy, the pups need to be kept with their mothers. As an alternative to surgery, the combination of MMI and perchlorate works well also and results in lower mortality.
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