Gliovascular disruption and cognitive deficits in a mouse model with features of small vessel disease

Abstract

Cerebral small vessel disease (SVD) is a major cause of age-related cognitive impairment and dementia. The pathophysiology of SVD is not well understood and is hampered by a limited range of relevant animal models. Here, we describe gliovascular alterations and cognitive deficits in a mouse model of sustained cerebral hypoperfusion with features of SVD (microinfarcts, hemorrhage, white matter disruption) induced by bilateral common carotid stenosis. Multiple features of SVD were determined on T2-weighted and diffusion-tensor magnetic resonance imaging scans and confirmed by pathologic assessment. These features, which were absent in sham controls, included multiple T2-hyperintense infarcts and T2-hypointense hemosiderin-like regions in subcortical nuclei plus increased cerebral atrophy compared with controls. Fractional anisotropy was also significantly reduced in several white matter structures including the corpus callosum. Investigation of gliovascular changes revealed a marked increase in microvessel diameter, vascular wall disruption, fibrinoid necrosis, hemorrhage, and blood-brain barrier alterations. Widespread reactive gliosis, including displacement of the astrocytic water channel, aquaporin 4, was observed. Hypoperfused mice also demonstrated deficits in spatial working and reference memory tasks. Overall, gliovascular disruption is a prominent feature of this mouse, which could provide a useful model for early-phase testing of potential SVD treatment strategies.

Figure 1

Radiologic features observed on T2-weighted structural magnetic resonance imaging (MRI). Representative images show hyperintense signal in the cortex (A; thick arrow) and subcortex (thalamus) (A; small arrow), and hypointense signal in the subcortex (B; arrow) of 6-month hypoperfused mice (slice taken −1.2 to −2.4 mm from Bregma). Parallel pathologic assessment identified ischemic-like lesions associated with hyperintense features (C), and hemorrhagic lesions in the subcortex (D) associated with the hypointense signal.

Figure 2

Progression of pathology in response to cerebral hypoperfusion. Heat maps depicting lesion load in the hypoperfused mice after 1 (N=8 sham; N=13 hypoperfused) and 6 (N=11 sham; N=20 hypoperfused) months (A) are indicated by color ranging from low to high occurrence. This brain slice is shown as it contained the highest lesion load in the thalamus and cortex at 6 months after hypoperfusion. After 1 month of hypoperfusion, lesion load throughout the brain is minimal, exclusively ischemic and confined to the cortex (B). With increased duration of hypoperfusion to 6 months, cortical and subcortical lesions are evident throughout the brain (B). Subcortical hemorrhages represented the major lesion burden with a smaller load of cortical and subcortical ischemic lesions (B). Overall, there was significantly greater total lesion load in the 6-month compared with 1-month hypoperfused mice (C). Additionally, there was a significant decrease in total brain volume in 6-month hypoperfused mice, whereas the brain volume was not changed in the 1-month cohort (D). *P<0.05.

Figure 3

Pronounced vascular alterations and blood–brain barrier (BBB) breakdown. Collagen IV staining in sham and hypoperfused mice after 1 month (N= 6 sham; N=14 hypoperfused) or 6 months (N=14 sham; N=15 hypoperfused)(A). One-month hypoperfusion had no significant effect on density (B) or vessel width (C) in the subcortex (thalamus). In comparison, 6 months of hypoperfusion resulted in a significant increase in collagen IV density (A) and vessel width (B) within the thalamus. Evidence of fibrinoid necrosis is observed in hypoperfused mice (in 12 out of the 20) with vascular disruption (D) observed on hematoxylin and eosin (H&E) and Martius scarlet blue (MSB), with collagen accumulation, vessel wall enlargement, and fibrin deposition (red in MSB); surrounding hemosiderin blood products (Perls), parenchymal fibrogen accumulation, and inflammatory cells (ionized calcium binding adaptor molecule 1, Iba1) suggest BBB disruption and macrophage activation (features absent in 6-month sham mice, N=11). Quantification of Claudin-5 in vessel-enriched fractions by enzyme-linked Immunosorbent assay (ELISA) showed no changes in the levels of the protein after 1 month (E; N=8 sham; N=6 hypoperfused); in contrast, a significant decrease was found after 6 months in the hypoperfused group when compared with sham mice (F; N=10 sham; N=9 hypoperfused). *P<0.05.

Figure 4

Increased astrocyte number after long-term hypoperfusion. Representative images of glial fibrillary acidic protein (GFAP) (green) and collagen IV (red) double immunostaining in the thalamus of sham and hypoperfused mice after 1 month (N=8 sham; N=13 hypoperfused) and 6 months (N=11 sham; N=20 hypoperfused) (A). One month of hypoperfusion had no significant effect on astrocyte number (B), whereas at 6 months there was a significant increase in comparison with shams (C). **P<0.01.

Figure 5

Displacement of aquaporin 4 (AQP4) after long-term hypoperfusion. Representative images of AQP4 (green) and collagen IV (red) double immunostaining staining in the thalamus of sham and hypoperfused mice after 1 month (N=8 sham; N=13 hypoperfused) and 6 months (N=11 sham; N=20 hypoperfused) (A). One month of hypoperfusion had no significant effect on the percentage of AQP4 colocalized to vessels or to the parenchymal AQP4 levels ouwith the vessels (B and C); however, at 6 months there was evidence of a redistribution of AQP4, with a trend toward both a decrease in AQP4 colocalizing to vessels and an increase in parenchymal AQP4 outwith the vasculature (D and E). The percentage of vascular-associated AQP4 was negatively correlated (P<0.001) with parenchymal AQP4 levels (F).

Figure 6

Cognitive impairments after long-term hypoperfusion (N=14 per group). Spatial working memory was impaired in hypoperfused mice, performing significantly less novel arm entries and significantly greater revisiting errors (A). Analysis of spatial reference learning and memory in the water maze identified a significant deficit in latency and path length (B) in the hidden platform task. Data are shown from the first probe trial to examine long-term spatial reference memory (C and D). Sham mice showed better-than-chance performance when compared with hypoperfused mice who did not reach chance performance. Hypoperfused mice also conducted significantly less platform crossings (D) and spent less time (D) in the target area than sham mice. *P<0.05.