An improved method for the establishment of a model of Graves' disease in BALB/c mice

Abstract

The present study aimed to develop a stable Graves' disease (GD) model in BALB/c mice by immunization and electroporation (EP). A total of 90 mice were divided into experimental (n=50), control (n=20) and blank (n=20) groups. The recombinant plasmid pcDNA3.1/thyroid‑stimulating hormone (TSH) receptor 268 was constructed and injected into the bilateral gastrocnemius of experimental group mice at weeks 1, 4, 7 and 10. Equal volumes of saline were injected into the control and blank groups at the same time. The experimental and control groups were subjected to EP at the same time and location to enhance immunization. The levels of total serum thyroxine (T4) and serum TSH were examined by radioimmunoassay and immunoradiometric assay, respectively. The levels of serum thyrotropin receptor N‑terminal (TRAb N) and C‑terminal (TRAb C) antibodies were assessed by ELISA. Whole body pertechnetate (99mTcO4‑) imaging was performed. Mouse weight and thyroid morphology and pathology were analyzed. The GD BALB/c mouse model was successfully established, with a positive rate of 79.17% (38/48). T4 levels increased from baseline levels of 12.05±4.23 to 52.51±23.58 ng/ml by week 12 (P<0.0001). TSH levels decreased from baseline levels of 5.53±2.78 to 1.43±0.89 µIU/ml by week 12 (P<0.0001). TRAb N antibody levels increased from baseline levels of 0.006±0.002 to 0.278±0.106 mIU/ml by week 12 (P<0.0001). TRAb C antibody levels increased from baseline levels of 11.111±2.808 to 46.701±26.436 arbitrary units/ml by week 12 (P<0.0001). At week 21, TSH levels remained reduced compared with pre‑immunization levels (P<0.0001). Although T4, and TRAb N and C levels decreased, they remained increased compared with preimmunization levels (P<0.0001, P<0.0001, P=0.001). There were no significant alterations in antibody levels between the control and blank groups. Following four immunizations, the uptake of 99mTcO4‑ by the thyroid was significantly increased in the experimental group. The mean weight of the experimental mice was significantly reduced compared with the control and blank groups (all P<0.0001). Furthermore, the thyroid glands of the immunized mice were enlarged and exhibited lymphocyte infiltration, fewer colloid nodules and an increased height of epithelial cells. In conclusion, by injecting recombinant plasmid pcDNA3.1/TSHR268 and EP, a GD mouse model was successfully established.

Figure 1.

Immunization elevates total serum T₄ levels. Prior to immunization, mean levels of T₄ in the experimental, blank and control groups were similar. Following immunization, mean levels of T₄ in experimental mice increased gradually. Upon cessation of immunization, T₄ levels in the experimental group decreased slightly; however, they remained significantly elevated compared with pre-immunization levels. There were no significant alterations in T₄ levels in the control and blank groups during the experimental process. Data are presented as the mean ± standard deviation. *P<0.05. T₄, thyroxine.

Figure 2.

Immunization reduces serum TSH levels. Prior to immunization, mean levels of serum TSH in the experimental, blank and control groups were similar. Following the first immunization, mean levels of TSH in experimental mice reduced until the fourth immunization at week 12. TSH levels fluctuated following cessation of immunization; however, they remained reduced compared with pre-immunization levels. There were no significant alterations in serum TSH levels in the control and blank groups. Data are presented as the mean ± standard deviation. *P<0.05 vs. control. TSH, thyroid-stimulating hormone.

Figure 3.

Immunization elevates TRAb N and C levels. (A) Mean levels of TRAb N, the thyroid-stimulating hormone receptor-stimulating antibody, markedly increased in the experimental group compared with the control and blank groups following immunization at week six. (B) Mean levels of TRAb C increased synchronously with TRAb N; however, at a more gradual rate. Following the third immunization at week nine, TRAb N and TRAb C levels peaked. Upon cessation of immunization, mean levels of TRAb N and C decreased; however, they remained significantly increased compared with pre-immunization levels. There were no significant alterations in the control and blank groups during the experimental process. Data are presented as the mean ± standard deviation. *P<0.05 vs. control. TRAb N, thyroid-stimulating hormone receptor N-terminal; TRAb C, thyroid-stimulating hormone receptor C-terminal.

Figure 4.

^99mTcO₄⁻ radioactive isotope imaging. Prior to immunization at week 0, the uptake of ^99mTcO₄⁻ by the thyroid was similar between the experimental, blank and control group mice. Following four immunizations (at week 12), increased uptake of ^99mTcO₄⁻ by the thyroid was observed in the experimental group. At week 21, this uptake remained increased compared with pre-immunization levels. There were no significant alterations in ^99mTcO₄⁻ uptake in the control and blank groups. ^99mTcO₄⁻, pertechnetate.

Figure 5.

Thyroid morphology analysis. Representative images of the thyroids of control and immunized BALB/c mice. Following immunization, the thyroid glands from hyperthyroid mice were enlarged compared with the thyroid glands from control mice, a feature that may have been induced by the thyroptrophin receptor antibody.

Figure 6.

Thyroid pathology analysis. Frozen sections of the thyroid glands from control and immunized mice were stained with hematoxylin and eosin. Examination of the thyroid tissue via microscopy revealed lymphocyte infiltration, fewer colloid nodules and an increase in the height of epithelial cells in experimental compared with control mice. Magnification, ×200.