In vivo Detection of Macromolecule Free Radicals in Mouse Sepsis-Associated Encephalopathy Using a New MRI and Immunospin Trapping Strategy

Hanrui Liu,1 Chengyong Ma,2 Huayan Xu,1 Huan Zhang,3 Rong Xu,1 Kun Zhang,1 Ran Sun,1 Kuan Li,1 Qihong Wu,1 Lingyi Wen,1 Lizhi Zhang,2 Yingkun Guo1 1West China Second University Hospital, Sichuan University, Chengdu, Sichuan, People’s Republic of China; 2West China Hospital, Sichuan University, Chengdu, People’s Republic of China; 3College of Chemistry and Materials Science, Northwest University, Xi’an, People’s Republic of ChinaCorrespondence: Yingkun Guo, Development and Related Diseases of Women and Children Key Laboratory, West China Second University Hospital, Sichuan University, No. 20, Section 3, Renmin South Road, Chengdu, Sichuan, 610041, People’s Republic of China, Tel +86+18180609256, Email gykpanda@163.com Lizhi Zhang, Department of Radiology, West China Hospital of Sichuan University, No. 37, Guoxue Road, Chengdu, Sichuan, 610041, People’s Republic of China, Email littche_2000@163.comIntroduction: Free radicals in oxidative stress are known to play a pathogenic role in sepsis. A major clinical challenge associated with sepsis is sepsis-associated encephalopathy (SAE). The rapid increase of free radicals in the brain promotes SAE progression. Here, macromolecule free radicals in the mouse brain were uniquely detected by immunospin trapping (IST) and magnetic resonance imaging (MRI).Methods: The new strategy uses spin trapping agent DEPMPO-biotin to capture macromolecule free radicals in lesions and form biotin-DEPMPO-radical adducts. Then, a targeting MRI probe, avidin-BSA@Gd-ESIO, was used to detect the radical adducts through the highly specific binding of avidin and biotin. The avidin-BSA@Gd-ESIO probe was synthesized and systematically characterized. The detection capability of the new strategy was evaluated in vitro and in vivo using a confocal microscope and a 7T MRI, respectively.Results: In reactive oxygen species (ROS)–induced microglial cells, the accumulation of the avidin-BSA@Gd-ESIO probe in the DEPMPO-biotin-treated group was significantly higher than that of control groups. In vivo MRI T1 signal intensities were significantly higher within the hippocampus, striatum, and medial cortex of the brain in mice with a mild or severe degree of sepsis compared with the sham control group. Histological analysis validated that the distribution of the avidin-BSA@Gd-ESIO probe in brain tissue slices was consistent with the MRI images. The fluorescence signals of ROS and avidin-BSA@Gd-ESIO probe were overlapped and visualized using immunofluorescent staining. By evaluating the T1 signal changes over time in different areas of the brain, we estimated the optimal MRI detection time to be 30 minutes after the probe administration.Discussion: This method can be applied specifically to assess the level of macromolecular free radicals in vivo in a simple and stable manner, providing a pathway for a more comprehensive understanding of the role of free radicals in SAE.Graphical-Abstract: Keywords: macromolecule radical, ESIO, spin trapping, sepsis-associated encephalopathy