This study leverages the innovative imaging capabilities of macrolaser light-sheet microscopy to elucidate the 3D spatial visualization of AD-associated neuropathologic networks in the transparent brains of 44-week-old 5xFAD mice. Brain samples from ten AD and seven control mice were prepared through a hydrophilic tissue-clearing pipeline and immunostained with thioflavin S (β-amyloid), anti-CD11b antibody (microglia), and anti-ACSA-2 antibody (astrocytes). The 5xFAD group exhibited significantly higher average total surface volumes of β-amyloid accumulation than the control group (AD, 898,634,368 µm3 [383,355,488-1,324,986,752]; control, 33,320,178 µm3 [11,156,785-65,390,988], p = 0.0006). Within the AD group, there was significant interindividual and interindividual variability concerning the number and surface volume of individual amyloid particles throughout the entire brain. In the context of neuroinflammation, the 5xFAD group showed significantly higher average total surface volumes of anti-ACSA-2-labeled astrocytes (AD, 59,064,360 µm3 [27,815,500-222,619,280]; control, 20,272,722 µm3 [9,317,288-27,223,352], p = 0.0047) and anti-CD11b labeled microglia (AD, 51,210,100 µm3 [15,309,118-135,532,144]; control, 23,461,593 µm3 [14,499,170-27,924,110], p = 0.0162) than the control group. Contrary to the long-standing finding that early-stage neuroinflammation precedes the subsequent later-stage of neurodegeneration, our data reveal that the second wave, late-stage active neuroinflammation persists in the aged AD brains, even as they continue to show signs of ongoing neurodegeneration and significant amyloid accumulation.
Keywords: 5xFAD; Alzheimer’s disease; astrogliosis; late-phase neuroinflammation; light-sheet microscopy; tissue clearing.