Visualization of Unconjugated Bilirubin In Vivo with a Novel Approach Using Chemical Exchange Saturation Transfer Magnetic Resonance Imaging in a Rat Model

Unconjugated bilirubin (UCB) visualization is valuable for early bilirubin encephalopathy (BE) diagnosis and management. UCB neurotoxicity is a challenge, necessitating improved imaging modalities for precise localization and characterization. This study developed a noninvasive method for UCB imaging in the brain using chemical exchange saturation transfer (CEST) magnetic resonance imaging, which visualizes UCB distribution through amide–bulk water proton exchange, a process termed bilirubin CEST (Bil-CEST) imaging. Bil-CEST imaging parameters were initially optimized; the exchange rate of the amide protons of UCB was calculated. Bil-CEST imaging characteristics and specificity were assessed using in vitro images of UCB solutions under different conditions and images of other brain metabolites. Bil-CEST maps of the rat brain were collected at the baseline and dynamically, postinjection of the UCB solution or vehicle into lateral ventricles of Sprague–Dawley rats. The model was validated using a water maze and pathological staining. In vitro, the Bil-CEST effect was observed at approximately 5.5 ppm downfield from bulk water. This effect was proportional to the UCB concentration and B1 amplitude. In vivo, Bil-CEST imaging revealed a progressive enhancement following a lateral ventricular UCB injection. Conversely, no significant imaging changes were observed in the vehicle group. Compared with the vehicle group, the UCB group had more hippocampal neuronal apoptosis and worse cognitive function. These findings highlight the utility of Bil-CEST in direct UCB imaging, indicating its potential as a clinically valuable biomarker for BE diagnosis and management.