Biomarkers.

Background: Macro laser light-sheet illuminating microscopy (Macro-LSFM), allied with tissue clearing technologies, herald a transformative paradigm in biomedical imaging, allowing 3D visualization of neuropathologic networks in a transparent intact mouse brain. Moreover, although traditional focus of AD diagnostic has been on CNS pathology, emerging research points to peripheral amyloid-beta deposition, specifically in the eyeball, as an avenue for investigation. Coupled with conventional [F]flutemetamol PET/CT imaging, this study leverages the innovative imaging capabilities of Macro-LSFM with hydrophilic tissue-clearing technique to elucidate the 3D spatial distribution of AD-associated neuroinflammation and neuropathologic change both in the brain and eyeball of old transgenic AD mouse.

Method: For thirteen 5xFAD (43 weeks) and seven control mice, we applied a hydrophilic tissue clearing pipelines to extracted brain and eyeball samples, encompassing fixation, clearing, permeabilization, and volume immunostaining using fluorochrome-conjugated Thioflavin S for β-amyloid (488 nm), anti-CD11b for microglia (561 nm), and anti-ACSA-2 for astrocytes (647 nm). Macro-LSFM imaging was performed using a Lightsheet Z.1 microscope (Objective 5x NA 0.16, Zoom 0.75x, single-mode illumination). 3D volumetric surface models for amyloid, astrocytes, and microglia mapping were generated using Imaris v9.6 software with a minimum surface grain size of 4.0 μm.

Result: In Macro-LSFM, AD showed significantly higher total surface volumes of amyloid accumulation than control (AD, 898634368 µm³ [383355488-1324986752]; Control, 33320178 µm³ [11156785-65390988], p=0.0006). Eyeballs of AD also showed higher amyloid deposits than control (AD, 51091002 µm³ [1002006-97460784]; Control, 229293 µm³ [8466-3501462], p=0.0066), showing strong positive correlation of amyloid accumulation between brain and eyeball (R=0.8, p=0.001). AD showed higher anti-ACSA-2 specific astrocyte (AD, 59064360 µm³ [27815500-222619280]; Control 20272722 µm³ [9317288-27223352], p=0.0047) and anti-CD11b specific microglial expression (AD, 51210100 µm³ [15309118-135532144]; Control, 23461593 µm³ [14499170-27924110], p=0.0162) than control.

Conclusion: Our synergistic amyloid PET/LSFM approach revealed that active neuroinflammation persists in old AD brain, marked by substantial amyloid deposition, challenging the traditional sequence of early neuroinflammation leading to late-stage neurodegeneration. Our novel observation of correlated ocular amyloid deposition introduces the eye as a promising, accessible site for the early diagnosis of AD pathology.