Cerebral β-Amyloidosis in Mice Investigated by Ultramicroscopy

<div><p>Alzheimer´s disease (AD) is the most common neurodegenerative disorder. AD neuropathology is characterized by intracellular neurofibrillary tangles and extracellular β-amyloid deposits in the brain. To elucidate the complexity of AD pathogenesis a variety of transgenic mouse models have been generated. An ideal imaging system for monitoring β-amyloid plaque deposition in the brain of these animals should allow 3D-reconstructions of β-amyloid plaques via a single scan of an uncropped brain. Ultramicroscopy makes this possible by replacing mechanical slicing in standard histology by optical sectioning. It allows a time efficient analysis of the amyloid plaque distribution in the entire mouse brain with 3D cellular resolution. We herein labeled β-amyloid deposits in a transgenic mouse model of cerebral β-amyloidosis (APPPS1 transgenic mice) with two intraperitoneal injections of the amyloid-binding fluorescent dye methoxy-X04. Upon postmortem analysis the total number of β-amyloid plaques, the β-amyloid load (volume percent) and the amyloid plaque size distributions were measured in the frontal cortex of two age groups (2.5 versus 7-8.5 month old mice). Applying ultramicroscopy we found in a proof-of-principle study that the number of β-amyloid plaques increases with age. In our experiments we further observed an increase of large plaques in the older age group of mice. We demonstrate that ultramicroscopy is a fast, and accurate analysis technique for studying β-amyloid lesions in transgenic mice allowing the 3D staging of β-amyloid plaque development. This in turn is the basis to study neural network degeneration upon cerebral β-amyloidosis and to assess Aβ -targeting therapeutics.</p></div

Location of measured test-cubes.

<p>β-Amyloid plaques (yellow dots) in the right hemisphere of the frontal cortex in the APPPS1 mouse model, side view: example from the young (2.7 month-old) group (A) and the adult (7.8 month-old) group (B). Positioning of the six cubed-shaped areas (purple color) in the frontal cortex for measuring the β-amyloid plaque volumes by applying a threshold segmentation technique. C-D) Top view of the frontal cortex: example from the young group (C) and old group (D). After segmentation amyloid plaque volumes of the six cubed shaped areas are represented in various colors. Scale bar 500 μm.</p

UM´s example images of APPPS1.

<p>Cross sections in the orthogonal directions are used to analyze the β-amyloid plaque distribution in the entire mouse brain of APPPS1. The β-amyloid plaques appear as bright dots in the neocortex (example of an animal of the young group is shown). A) Transversal plane (x,y); B) transversal plane (x,y); C) Computed sagittal plane (yz); D) computed coronal plane (xz). ctx: cortex; cb: cerebellum; hc: hippocampus, bs: brainstem; ob: olfactory bulb.</p

Determined Plaque diameters compared to a previous study.

<p>Determined Plaque diameters compared to a previous study.</p

Histograms of plaque diameters.

<p>The plaque sizes were binned into size groups of 10 μm for the young and the adult mice. In total, the number of β-amyloid plaques in all size classes was increased in the adult group. All plaques within the young group and the adult group, respectively were counted for histogram generation.</p

Quantification of the number of amyloid plaques per mm³.

<p>The total β-amyloid plaque number per mm^<b>3</b> was obtained from six sample cubes, acquired within the frontal cortex of young (2.5 months) and adult (7–8.5 months) APPPS1 tg mice. The total number of plaque per mm^<b>3</b> increases with age.</p

Comparison between a young (2.3-month-old) and adult (7.5-month-old) APPPS1 tg brain.

<p>3D reconstructions demonstrate an overall age-related increase in β-amyloid plaques. The maximum intensity projections of reconstructed images show a higher plaque density with increasing age. Brain from a mouse at 2.3 months of age viewed from the top (A), from the right side (B) and left side of (C). D) Brain from a mouse at 7.5 months of age viewed from the top (D), from the right side (E) of and left side (F). Scale bar: 1mm.</p

Quantification of the 3D β-amyloid plaque load.

<p>The β-amyloid plaque load (volume %) was obtained from six sample cubes, acquired within the frontal cortex of young (2.5 months) and adult (7–8.5 months) APPPS1 tg mice. The β-amyloid plaque load in the adult group is significantly higher compared to the young group (p<0.001, t-test).</p

Variations in the fraction of plaques for different diameters.

<p>In the β-amyloid plaque diameter categories 40–50μm plaques were relatively more frequent in the adult animals. However, in the diameter group of 20–30 μm plaques were relatively more frequent in the young animals. For the β-amyloid plaque diameter categories 0–20 μm and 30–40 μm no significant differences could be shown. It cannot by excluded that plaques > 50 μm may be due to agglomeration of smaller plaques or artefacts.</p

Ultramicroscopy setup.

<p>Principle of standard UM consisting of a slit aperture and a single cylindrical lens on each illumination side. The transparent brain is illuminated perpendicular to the observation pathway by a laser forming a thin sheet of light. The emitted fluorescence light is projected to a camera target by an objective, while a matched optical filter blocks the excitation light. By moving the specimen through the light sheet a stack of images is recorded. From these data a 3D-reconstruction is calculated by software. 1) Optical system (OS), which generates a magnified parallel beam. This beam is divided into two equal parts using a beam splitter. 2) Rectangular-slit aperture, 3) cylindrical lens, 4) clamp, 5) brain, 6) Sy: computer-controlled elevation (y-direction), 7) Sz: computer-controlled linear stage (z-direction), 8) immersion cap, 9) objective, 10) tube lens(es), 11) camera target, 12) computer with imaging software, 13) computer with 3D reconstruction software.</p