Three-Dimensional Imaging of Fibrinogen and Neurovascular Alterations in Alzheimer's Disease

Abstract

Cerebrovascular dysfunction is a hallmark of Alzheimer's disease (AD) that is linked to cognitive decline. However, blood-brain barrier (BBB) disruption in AD is focal and requires sensitive methods to detect extravasated blood proteins and vasculature in large brain volumes. Fibrinogen, a blood coagulation factor, is deposited in AD brains at sites of BBB disruption and cerebrovascular damage. This chapter presents the methodology of fibrinogen immunolabeling-enabled three-dimensional (3D) imaging of solvent-cleared organs (iDISCO) which, when combined with immunolabeling of amyloid β (Aβ) and vasculature, enables sensitive detection of focal BBB vascular abnormalities, and reveals the spatial distribution of Aβ plaques and fibrin deposits, in large tissue volumes from cleared human brains. Overall, fibrinogen iDISCO enables the investigation of neurovascular and neuroimmune mechanisms driving neurodegeneration in disease.

Keywords: 3D imaging; Alzheimer’s disease (AD); Biomarker; Blood-brain Barrier; Fibrinogen; Microscopy; Neurodegeneration; Neuroinflammation; Neurovascular; Tissue Clearing; iDISCO.

Fig 1. Fibrinogen iDISCO in AD Brain.

A–D: 3-D immunolabeling of cleared temporo-occipital brain tissue from three AD brains, stained for the vascular marker, CD31 (green), fibrinogen (red) and Aβ (blue). Arrows and asterisks indicate vessel-associated and parenchymal fibrinogen, respectively; # sign indicates intravascular fibrinogen. Magenta in (B) indicates Aβ-associated fibrinogen. E and F: Representative 3-D volume projections of iDISCO-cleared temporo-occipital AD brain tissue, stained for CD31, fibrinogen, and Aβ, showing examples of vascular tortuosity observed in AD brain. Vascular tortuosity was observed in 3 out of the 5 AD brains. Scale bars = 75 µm (A), 25 µm (B), 50 µm (C), and 25 µm (D–F). Images reprinted from Merlini et al [7].

Fig 2. Fibrinogen iDISCO in Non-demented Control Brain Tissue.

A–D: Representative 3-D volume projections of iDISCO-cleared temporo-occipital brain tissue from age-matched NDCTRL subjects stained for the vascular marker, CD31, fibrinogen, and Aβ. Images are representative from 3 NDCTRL brains. Vessel-associated fibrinogen deposits are present on the abluminal side of CD31-stained vessels. Arrows and asterisks indicate vessel-associated and parenchymal fibrinogen deposits, respectively; # sign indicates intravascular fibrinogen. Scale bars = 50 µm (A and B) and 25 µm (C and D). Images reprinted from Merlini et al. [7].