Spontaneous hemorrhagic stroke in a mouse model of cerebral amyloid angiopathy

Abstract

A high risk factor for spontaneous and often fatal lobar hemorrhage is cerebral amyloid angiopathy (CAA). We now report that CAA in an amyloid precursor protein transgenic mouse model (APP23 mice) leads to a loss of vascular smooth muscle cells, aneurysmal vasodilatation, and in rare cases, vessel obliteration and severe vasculitis. This weakening of the vessel wall is followed by rupture and bleedings that range from multiple, recurrent microhemorrhages to large hematomas. Our results demonstrate that, in APP transgenic mice, the extracellular deposition of neuron-derived beta-amyloid in the vessel wall is the cause of vessel wall disruption, which eventually leads to parenchymal hemorrhage. This first mouse model of CAA-associated hemorrhagic stroke will now allow development of diagnostic and therapeutic strategies.

Fig. 1.

Cerebral amyloid angiopathy in APP23 mice. Aβ staining reveals significant CAA (arrowheads) in neocortex (a) and thalamus (b) in aged 27-month-old APP23 mice. Within these regions, CAA showed a great variability (c–e), ranging from vessels with a thin rim of amyloid in the vessel wall (c; severity grade, 1), to vascular amyloid with amyloid infiltrating the surrounding neuropil (d; severity grade, 2), and to dyshoric amyloid with amyloid deposition within the vessel wall and with a thick and complete amyloid coat around the vessel wall (e; severity grade, 3). Scale bars:a, b, 100 μm; c–e, 10 μm.

Fig. 2.

Cerebrovascular amyloid in leptomeningeal vessels. Leptomeningeal vessels are the most consistent and the first to exhibit cerebrovascular amyloid in APP23 mice. Shown in a are leptomeningeal vessels at the surface of the cingulate cortex of a 19-month-old APP23 mouse. Note that the amyloid is mostly confined to the outer vessel wall (arrowhead), consistent with CAA in humans in which initial deposits are found in the outer basement membrane (Yamaguchi et al., 1992). b, 3D reconstruction of an Aβ-stained (orange pseudocolored) heavily affected leptomeningeal vessel in an aged APP23 mouse. Note that nearly the entire surface is covered by a thick amyloid coat. The reconstruction consists of 198 optical slices (<0.7 μm), with a sampling interval of 0.35 μm. Scale bars, 25 μm.

Fig. 3.

Age-related increase in CAA frequency and severity in APP23 mice. a, Number of amyloid affected vessels (CAA frequency) was quantified in systematically sampled sections through the neocortex of young (8 months), adult (19 months), and aged (27 months) APP23 mice. ANOVA revealed a significant affect of age (F_(2,38) = 41.6; p< 0.001). b, A grading score was then used to assess severity of affected vessels (for details, see Fig.1c–e and Materials and Methods). The mean CAA severity is indicated for the 19- and 27-month-old groups and revealed a significant age-related increase (t₍₂₉₎= 2.95; p < 0.01).

Fig. 4.

Cerebrovascular amyloid leads to smooth muscle cell loss. Confocal microscopy of double-immunolabeled vessels (green, smooth muscle actin; red, amyloid) in APP23 mice. a, Leptomeningeal vessel in an 8-month-old mouse shows no amyloid deposition and a complete layer of smooth muscle cells. b, Leptomeningeal vessel in a 19-month-old mouse shows focal disappearance of smooth muscle cells at the site of cerebrovascular amyloid (arrowheads).c, In 27-month-old mice, smooth muscle cells have greatly disappeared, and a thick sheet of amyloid covers the wall of a leptomeningeal vessel. d, e, Parenchyma in the neocortex of a 19-month-old mouse showing an unaffected (d) and an amyloid-laden vessel (e) in close anatomical proximity. Shown are superpositions of 0.9- to 5-μm-thick optical sections. Scale bars:a, 10 μm; b–e, 20 μm.

Fig. 5.

CAA-related hemorrhage in APP23 mice.a, Hemosiderin staining (blue) in the frontal cortex of a 27-month-old mouse indicative of an old hemorrhage. The section is counterstained with nuclear fast red. B, Perivascular hemosiderin-positive microglia (arrowhead) in close vicinity of a small vessel in a 27-month-old mouse.C, Hemosiderin-positive microglia surrounding an enlarged neocortical vessel of aneurysm-like appearance.d, e, Double-labeling for amyloid (brown) and hemosiderin (blue) localized bleedings to amyloid-laden vessels. f, Evidence for acute hematoma was assessed in H&E-stained sections. A significant hemorrhage (asterisk) in the frontal cortex of a 27-month-old APP23 mouse is shown. g, An adjacent section to f was stained with Berlin blue and revealed an old hemorrhage in the same region. Scale bars: a, 100 μm; b, c, f,g, 50 μm; d, e, 5 μm.

Fig. 6.

Age-related increase in hemorrhage in neocortex of APP23 mice. a, Frequency of perivascular hemosiderin-positive staining was assessed in systematically sampled sections through the neocortex. No evidence of old bleedings (hemosiderin) was found in the 8-month-old mice. From 19 to 27 months of age, there appears a striking increase in frequency of intracerebral hemorrhages (ANOVA;F(_2,38)= 26.1; p < 0.001). Because the sampling was done in the right hemisphere only and in every 10th section, total incidence of hemorrhages in the neocortex of 27-month-old mice can be estimated to be >100. b, Significant positive relationship between CAA score (frequency × severity) and hemorrhage number in neocortex of the 27-month-old mice (p < 0.01). Similar positive correlations were found between CAA frequency and hemorrhage and between CAA severity and hemorrhage (for bothR² = 0.44).c, In contrast, no relationship between neocortical amyloid plaque load and hemorrhages was found (p > 0.05).

Fig. 7.

Vasculitis in aged APP23 mice with severe CAA.a, H&E staining of two vessels affected by a chronic lymphocytic vasculitis. Lymphocytic infiltrates are seen throughout the entire vessel walls. The vessel wall on the left appears thickened and the lumen is obliterated. There is severe amyloid deposition in the right vessel wall (arrowhead). b, Double-staining for H&E and for Congo red (green-yellow birefringence) reveals amyloid deposits in a vessel heavily affected by a lymphocytic vasculitis. Scale bars, 50 μm.