Synopsis

The purpose of the present study was to evaluate key molecular and cellular features of Graves’ orbitopathy by simultaneous monitoring of alterations in morphology, inflammatory patterns, and tissue remodeling. Beyond anatomical ¹H MRI, we employed T2 mapping for visualization of edema, chemical exchange saturation transfer for detection of hyaluronan, and ¹⁹F MRI for tracking of in situ labeled immune cells after intravenous injection of perfluorcarbons. In particular, ¹⁹F MRI allowed a sensitive demarcation of inflammatory foci in the orbit, even when other markers indicated only weak or no signs of tissue alterations.

Introduction

Graves’ disease is an autoimmune condition of the thyroid gland caused by thyrotropin receptor (TSHR) stimulating autoantibodies, leading to overproduction of thyroid hormones. A frequent complication is Graves’ orbitopathy (GO), also known as thyroid eye disease, where inflammation of the orbit associated with increased adipogenesis and deposition of hyaluronan results in detrimental remodeling of the orbital soft tissue (1,2) – in the worst case, it can lead to blindness.

Treatment of GO patients remains difficult, since current diagnostics are limited in encompassing the complex phenotype and the highly variable pathogenesis of the disease. Beyond careful ophthalmological examination when clinical symptoms occur, modern imaging modalities are mainly confined to visualize the expansion of pathologically affected orbital tissue (3,4). However, patient-specific therapy would greatly benefit from additional knowledge about the degree of the underlying inflammation and a more detailed characterization of the ongoing remodeling processes. Thus, the present study aimed at developing an MRI-based approach which takes account of the complex pathogenesis of GO and addresses several key characteristics of the disease in one imaging session.

Materials and Methods

Results and Discussion

In T2-weighted ¹H MRI scans acquired with an in-plane resolution of 50x50 µm², the anatomy of the murine orbit could be visualized in great detail. Anatomically corresponding ¹H and ¹⁹F MR images from GO mice demonstrated a marked accumulation of ¹⁹F signal around the eyeballs (Figure 1). Besides the predominant peri-orbital localization of infiltrated PFCs, retro-orbital regions around the optic nerve were affected too (Figure 2). In contrast, healthy Ctrls exhibited only minor ¹⁹F signals in the orbit (Figure 1+2). In parallel, T2 mapping indicated onset of edema in peri-orbital tissue and adjacent ocular glands (P=0.038/0.017; Figure 2, Table 1).

Accompanying remodeling processes in the orbital extracellular matrix were assessed by exploiting the strong susceptibility of CEST signals to alterations in tissue GAG/hyaluronan content (7). Orbits of GO mice clearly exhibited enhanced CEST contrast as compared to Ctrl mice (Table 1; P=0.031). Concomitanty, a moderate expansion of retrobulbar fat (P=0.029) was apparent, but no signs for extraocular myopathy were detectable (Table 1). Overall, the MR data reveal characteristics of GO in mice to parallel those in human patients, recapitulating orbital inflammation and expansion of the orbital tissues due to edema, adipogenesis, and deposition of hyaluronan. ¹⁹F MRI provided the highest significance level to discriminate between healthy and diseased mice (P=0.006, Figure 3), and furthermore showed the best correlation with clinical eye signs (P=0.0007). Linear regression of the significant different MRI parameters, however, revealed only weak or no correlations indicating a primarily independent development/progression of the disease characteristics. This assumption is further supported by the occurrence of ¹⁹F signals at sites that were not characterized by increased T2, CEST contrast or adipose tissue expansion.

Infiltration patterns of inflammatory cells were corroborated by examination of excised tissue after fixation: 3D high resolution ¹H/¹⁹F MRI (isotropic voxel size 40 µm) confirmed the prevalent PFC deposition in the peri-orbital tissue of GO mice. Further histological analysis of orbital tissue with regard to immune cell infiltration, fibrosis, and adipogenesis mainly supported the in vivo findings (Figure 3). Quantification of these data revealed an elevated abundance of macrophages, increased collagen deposition, and enlargement of brown/beige fat depots in orbits of GO compared to Ctrl mice. The differences and significance levels for the individual parameters were similar to the MRI data (Figure 4).

Conclusion

Acknowledgements

References

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