Evaluation of 99mTc-rhAnnexin V-128 SPECT/CT as a diagnostic tool for early stages of interstitial lung disease associated with systemic sclerosis

Abstract

Background: Given the need for early detection of organ involvement in systemic sclerosis, we evaluated 99mTc-rhAnnexin V-128 for the detection of early stages of interstitial lung disease (ILD) in respective animal models using single photon emission computed tomography (SPECT/CT). Methods: In bleomycin (BLM)-challenged mice, fos-related antigen 2 (Fra-2) transgenic (tg) mice and respective controls, lung injury was evaluated by analysis of hematoxylin and eosin (HE) and Sirius red staining, with semi-quantification of fibrosis by the Ashcroft score. Apoptotic cells were identified by TUNEL assay, cleaved caspase 3 staining and double staining with specific cell markers. To detect early stages of lung remodeling by visualization of apoptosis, mice were injected intravenously with 99mTc-rhAnnexin V-128 and imaged by small animal SPECT/CT. For confirmation, biodistribution and ex vivo autoradiography studies were performed. Results: In BLM-induced lung fibrosis, inflammatory infiltrates occurred as early as day 3 with peak at day 7, whereas pulmonary fibrosis developed from day 7 and was most pronounced at day 21. In accordance, the number of apoptotic cells was highest at day 3 compared with saline controls and then decreased over time. Epithelial cells (E-cadherin+) and inflammatory cells (CD45+) were the primary cells undergoing apoptosis in the earliest remodeling stages of experimental ILD. This was also true in the pathophysiologically different Fra-2 tg mice, where apoptosis of CD45+ cells occurred in the inflammatory stage. In accordance with the findings on tissue level, at day 3 in the BLM and at week 16 in the Fra-2 tg model, biodistribution and/or ex vivo autoradiography showed increased pulmonary uptake of 99mTc-rhAnnexin V-128 compared with controls. However, accumulation of the radiotracer and thus the signal intensity in lungs was too low to allow the differentiation of healthy and injured lungs in vivo. Conclusion: At the tissue level, 99mTc-rhAnnexin V-128 successfully demonstrated early stages of ILD in two animal models by detection of apoptotic epithelial and/or inflammatory cells. In vivo, however, we did not detect early lung injury. It remains to be investigated whether the same applies to human ILD.