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

4. RESULTS

4.1 Evaluation of DNA fragmentation in cerebellar granular cells of developing euthyroid rats

The TUNEL technique was applied to probe the apoptotic DNA fragmentation characteristic of cell death in the developing cerebellum of euthyroid rats at the postnatal day 2, 8, 12, 22, and 42. Examination of the layers of coronal sections reveal that the programmed cell death after birth is limited to the IGL of cerebellum although scattered apoptotic cells could be seen in the EGL of p8 and p12 (figure 2A). Based on a statistical evaluation, results show that apoptotic cells are absent at 2, 22 and 42 days postnatally (figure 2A-B). The peak of apoptosis was observed in the IGL at p8. There is about 50 % reduction in apoptotic cells at p12. In summary, as a results of postnatal cerebellum development, the number of apoptotic cells observed in the IGL increased significantly from the undetectable level at p2 reaching the highest level at p8, and gradually declined being undetectable at p22. Interestingly, DNA fragmentation was not observed in Purkinje cells layers (figure 2A-B, figure 3).

Figure 2A. Detection of DNA fragmentation of in the IGL of cerebellum of postnatal 8 days (p8), p12, and p22. Section of 12 mm were labeled for DNA fragmentation as described in the material and methods. At the age of 8 days, some apoptotic stained cells were detected in the euthyroid rats, whereas, more apoptotic cells were detected in the 8-day hypothyroid rats. At the age of 12 days, few apoptotic cells were found in the euthyroid rats. In contrary, lots of apoptotic cells were still stained in the 12-day hypothyroid rats. At the age of 22 days, none apoptosis were happened in the euthyoid cerebellum, relatively fewer apoptotic cells were detected in the 22-day hypothyroid rats. None apoptotic cells were Purkinje cells. Magnification, 10x except the last one of left column which is 5x.

Figure 2B. Coronal sections of euthyroid and hypothyroid cerebellar at p2 and p42 stained for apoptotic cells as described in the material and methods. Magnification, 10x.

4.2 Evaluation of DNA fragmentation in cerebellar granular cells of developing hypothyroid rats

The cerebella of hypothyroid rats were examined at postnatal day 2, 8, 12, 22, and 42 as described for euthyroid controls. Similar to the control, no apoptotic cells were detected in the cerebellum of p2 (figure 2B). By p8, a sharp increasing number of brown stained nuclei was observed, accounting for the highest number of apoptotic cells in the cerebellum of hypothyroid rats during postnatal maturation. Based on the number of stained nuclei per square mm, this increase was about four times higher when compared with that obtained with the euthyroid rats of this same developmental stage (figure 3). Most of these apoptotic nuclei were in the IGL, a few are seen in the EGL and ML. At p12, there is still a substantial number of stained nuclei in the IGL, but the apoptotic value is about 50% of that detected at p8 (figure 3). By p22, the labeling of apoptotic nuclei was further reduced to about 25% of the p8 value, contrary to the euthyroid age matched cerebellum, where apoptosis descended. Interestingly, the level of apoptotic activity in p22 of the hypothyroid cerebellum corresponds to that detected at the peak of apoptosis in control (p8). There was no evidence for DNA fragmentation in the cerebellum of hypothyroid rats at p42 (figure 2A-B). Noteworthy is the continuing appearance of the EGL as a result of hypothyroidism, since at p42 EGL in the euthyroid cerebellum is not evident (figure 2B).

Figure 3. Statistical evaluation of a number of apoptotic cells in cerebella of euthyroid and hypothyroid rats during development. Calculation was done as described in Materials and Methods. The bars represent the ± SD (n=20).