APOPTOSIS IN THE DEVELOPING CEREBELLUM OF THE THYROID HORMONE DEFICIENT RAT

Qianxun Xiao and Vera M. Nikodem

Mechanism of Gene Regulation Section, Genetics and Biochemistry Branch, National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, 10 Center Dr. MSC 1766, Bethesda, MD 20892-1766

Received 8/20/98 Accepted 9/7/98

3. MATERIALS AND METHODS

3.1 Animals and tissue preparation

Hypothyroid rats (Charles River)were generated using protocol previously described (23,24). In order to induce fetal and neonatal hypothyroidism, pregnant rats were drinking 0.02% methyl mercaptoimidazol (MMI) solution (Sigma Chemical Co., St. Louis, MO) ad libitum from gestation day 10. This treatment continued until the day of brain samples collection. These protocol produced severe hypothyroidism in the offspring, as shown by increased serum TSH level (figure 1), reduced body growth, and the delays in developmental landmarks, such as the eye opening. Animals were handled in a humane way, following the NIH guideline for the use and care of laboratory animals. Two groups, hypothyroid and euthyroid, were raised side by side. Whole brains of rats at the postnatal (p) day 2, p8, p12, p22, and p42 were collected from both groups. The entire brains were frozen in isopentane on dry ice and stored at -80° C until sectioning. A serials coronal sections from cerebellum, 12 mm thick, were made in a cryostat at -25° C (25).

Figure 1. Serum thyroid stimulating hormone (TSH) level of the postnatal 2 days (p2), p12. P22 and p42 from the both hypothyroidism and euthyroid rats were determined by radioimmunoassay (RIA). Columns value represent the mean ± SD (n=4).

3.2 In situ apoptosis detection

In situ apoptosis detection was performed using the Apop Tag TM kit (Oncor, Gaithersburg, MD) following the manufacture protocol. This procedure detects the DNA using the terminal deoxynucleotidyl transferase (TdT) mediated d-uridine 5’-triphosphate (UTP) nick end labeling (TUNEL) technique (26). Briefly, sections were fixed in 10% buffered formalin in a coplin jar for 10 min at room temperature. Washed in 2 changes of PBS for 5 min each. Post-fixed in ethanol - acetic acid (2:1) for 5 min at -20° C, washed in 2 changes of PBS for 5 min each wash. Then, the sections were quenched in 2.0% hydrogen peroxide in PBS for 5 min at room temperature and rinsed two times with PBS. Two drops of 1x equilibration buffer was applied and incubated 10 min at room temperature. The equilibration buffer was removed and 54 m l working strength TdT enzyme was added, then incubated at 37° C for 1 hour with a plastic coverslip over the specimen. The slides were washed with pre-warmed stop/wash buffer and incubated for 30 min at 37° C. Two drops Anti-Digoxigenin-Peroxidase was applied incubating 30 min at room temperature and washed again in PBS. Then, the sections were subjected to color development in 0.05% DAB (Sigma Chemical Co., St. Louis, MO) solution for 10 min. Cell death was quantified according to Neveu and Arenas (27) counting the number of brown stained nuclei in a total of 20 randomly chosen fields contained in four different sections. The values with standard deviations (SD) show the number of dying cells per square millimeter. As a negative control, adjacent sections were processed following the identical procedure, except that an equal volume of water was substituted for the omitted TdT. In these control section, no nuclei were stained, confirming the specificity of the labeling.

3.3 Detection of thyroid stimulating hormone (TSH)

TSH level in serum of euthyroid and hypothyroid rats was determined by radioimmunoassay and performed by CORNING Hazleton Inc., Vienna, VA 22182.