An Improved Graves’ Disease Model Established by Using in Vivo Electroporation Exhibited Long-Term Immunity to Hyperthyroidism in BALB/c Mice

Abstract

In Graves' disease, the overstimulation of the thyroid gland and hyperthyroidism are caused by autoantibodies directed against the TSH receptor (TSHR) that mimics the action of TSH. The establishment of an animal model is an important step to study the pathophysiology of autoimmune hyperthyroidism and for immunological analysis. In this study, we adopted the technique of electroporation (EP) for genetic immunization to achieve considerable enhancement of in vivo human TSHR (hTSHR) expression and efficient induction of hyperthyroidism in mice. In a preliminary study using beta-galactosidase (beta-gal) expression vectors, beta-gal introduced into the muscle by EP showed over 40-fold higher enzymatic activity than that introduced via previous direct gene transfer methods. The sustained hTSHR mRNA expression derived from cDNA transferred by EP was detectable in muscle tissue for at least 2 wk by RT-PCR. Based on these results, we induced hyperthyroidism via two expression vectors inserted with hTSHR or hTSHR289His cDNA. Consequently, 12.0-31.8% BALB/c mice immunized with hTSHR and 79.2-95.7% immunized with hTSHR289His showed high total T(4) levels due to the TSHR-stimulating antibody after three to four times repeated immunization by EP, and thyroid follicles of which were hyperplastic and had highly irregular epithelium. Moreover, TSHR-stimulating antibody surprisingly persisted more than 8 months after the last immunization. These results demonstrate that genetic immunization by in vivo EP is more efficient than previous procedures, and that it is useful for delineating the pathophysiology of Graves' disease.

Cutting Edge: Retrobulbar Inflammation, Adipogenesis, and Acute Orbital Congestion in a Preclinical Female Mouse Model of Graves' Orbitopathy Induced by Thyrotropin Receptor Plasmid-in Vivo Electroporation

“…Accordingly, other reported mouse models of GO have proved difficult to replicate (9 -11). In the course of our studies on experimental GD (12,13), we recently expanded the human (h) TSHR Asubunit plasmid-in vivo electroporation model (14) to report histologic signs of orbital fibrosis in female BALB/c mice undergoing experimental hyperthyroidism (15). Upon observing orbital fibrosis, we modified our protocol to develop an improved mouse model with all the features of orbital inflammation, adipogenesis, and eyelid chemosis.…”

Immune Response of Mice Transgenic for Human Histocompatibility Leukocyte Antigen-DR to Human Thyrotropin Receptor-Extracellular Domain

“…Models of GD in mice have been developed in several laboratories through immunization using fibroblasts transformed with cDNA for TSH-R and major histocompatibility complex class II protein (9), plasmids expressing hTSH-R (10), adenovirus expressing hTSH-R (11) or hTSH-R-A subunit (12), or cDNA delivered by electroporation (13). Adenovirus immunization is reported to develop a strong T-cell response (reviewed in Ref.…”

TSH Receptor Cleavage Into Subunits and Shedding of the A-Subunit; A Molecular and Clinical Perspective

“…In contrast, immunization with the free A-subunit induced TSAb and hyperthyroidism in most mice (47) (Figure 5). The greater efficacy of the free A-subunit vs the TSH holoreceptor in inducing experimental Graves' disease has been confirmed in other studies (125)(126)(127), and this modification is now used by most investigators (for example, see Refs. 128 -133).…”