Chronic cerebral hypoperfusion induces post-stroke dementia following acute ischemic stroke in rats

Abstract

Background: Post-stroke dementia (PSD) is one of the major consequences after stroke. Chronic cerebral hypoperfusion (CCH) can induce vascular cognitive impairment and potentiate amyloid pathology. We investigated how CCH contributes to the development of PSD after stroke in the context of neuroinflammation and amyloid pathology.

Methods: We designed a unique animal model for PSD. We performed middle cerebral artery occlusion (MCAO) surgery in rats mimicking acute territorial infarct, which was followed by bilateral common carotid artery occlusion (BCCAo) surgery mimicking CCH. We performed behavioral tests including neurologic function test and water maze task and histological investigations including neuroinflammation, neuronal cell death, amyloid pathology, and aquaporin 4 (AQP4) distribution.

Results: Spatial memory was synergistically impaired when BCCAo was superimposed on MCAO. Neuroinflammation with astroglial or microglial activation and amyloid pathology were enhanced in the ipsilateral cortex, thalamus, and hippocampus when BCCAo was superimposed on MCAO. Glymphatic pathway-related AQP4 distribution changed from perivascular to parenchymal pattern.

Conclusions: Our experimental results suggest that CCH may contribute to the development of PSD by interfering with amyloid clearance through the glymphatic pathway and concomitant neuroinflammation. Therapeutic strategy to clear brain metabolic waste through the glymphatic pathway may be a promising approach to prevent PSD after stroke.

Keywords: Amyloid pathology; Animal model; Chronic cerebral hypoperfusion; Glymphatic pathway; Neuroinflammation; Post-stroke dementia.

Fig. 1

a The timeline of the experiment. b Allocation of rats into groups. c Representative images of cresyl violet staining and quantification of infarct volume in M + S and M + B groups. n = 10 in both groups. d Serial neurologic function test. n = 12 to 17 in each group. MCAO middle cerebral artery occlusion, BCCAo bilateral common carotid artery occlusion, S + S sham + sham, S + B sham + BCCAo, M + S MCAO + sham, M + B MCAO + BCCAo, mNSS modified neurologic severity scores, ns not significant

Fig. 2

Morris water maze task. a Search error, time latency, and path length. b Swimming speed. c Time staying in the target quadrant during probe trials. d Number of crossings over the removed platform during probe trials. n = 12 to 17 in each group; *p < 0.05, **p < 0.01, and ***p < 0.001 on post hoc analysis; MCAO middle cerebral artery occlusion, BCCAo bilateral common carotid artery occlusion, S + S sham + sham, S + B sham + BCCAo, M + S MCAO + sham, M + B MCAO + BCCAo, ns not significant

Fig. 3

Neuroinflammation measured by astroglial and microglial activation. a Glial fibrillary acidic protein (GFAP) immunoreactivity and its quantification. b Iba1 immunoreactivity and its quantification. n = 6 to 8 in each group; scale bar = 100 μm; *p < 0.05, **p < 0.01, and ***p < 0.001 on the post hoc analysis compared to S + S; MCAO middle cerebral artery occlusion, BCCAo bilateral common carotid artery occlusion, S + S sham + sham, S + B sham + BCCAo, M + S MCAO + sham, M + B MCAO + BCCAo

Fig. 4

Amyloid deposit in the cortex. a Amyloid plaques are indicated in 4G8 DAB staining (arrow heads) and double immunofluorescent labeled by neuron (NeuN) and amyloid deposit (4G8). b Quantification of amyloid plaque number and c area. n = 6 to 8 in each group; scale bar = 100 μm; *p < 0.05, **p < 0.001 on the post hoc analysis compared to S + S; †p < 0.05 on the post hoc analysis compared to ipsilateral M + S; ‡p < 0.05 on the post hoc analysis compared to contralateral M + B; MCAO middle cerebral artery occlusion, BCCAo bilateral common carotid artery occlusion, S + S sham + sham, S + B sham + BCCAo, M + S MCAO + sham, M + B MCAO + BCCAo

Fig. 5

Amyloid deposit in the thalamus. Amyloid plaques are indicated in 4G8 DAB staining (insets in the first column) and double immunofluorescent labeled by neuron (NeuN) and amyloid deposit (4G8). n = 12 in each group; scale bar = 100 μm; MCAO middle cerebral artery occlusion, BCCAo bilateral common carotid artery occlusion, S + S sham + sham, S + B sham + BCCAo, M + S MCAO + sham, M + B MCAO + BCCAo

Fig. 6

a Distribution patterns of AQP4 immunoreactivity. b Ratio of vascular and parenchymal AQP4. c AQP4/Col IV colocalization index. n = 5 to 7 in each group; scale bar = 50 μm; *p < 0.05, **p < 0.01, and ***p < 0.001 on post hoc analysis compared to S + S; vAQP4 vascular AQP4, pAQP4 parenchymal AQP4, Col IV collagen IV, MCAO middle cerebral artery occlusion, BCCAo bilateral common carotid artery occlusion, S + S sham + sham, S + B sham + BCCAo, M + S MCAO + sham, M + B MCAO + BCCAo

Fig. 7

a Hippocampus and its magnification in CA1 and CA3 regions. Scale bar = 100 μm. b Neuroinflammation measured by astroglial (GFAP) and microglial (Iba1) activation ant its quantification. n = 7 to 8 in each group; *p < 0.05 and **p < 0.01 on post hoc analysis compared to S + S. c Neuronal cell death measured by cresyl violet and its quantification. n = 8 to 10 in each group; *p < 0.05, **p < 0.01, and ***p < 0.001 on post hoc analysis compared to S + S. d Amyloid-positive degenerative neurons measured by 4G8 and its quantification. n = 6 to 8 in each group, †p < 0.001 on post hoc analysis compared to other groups; MCAO middle cerebral artery occlusion, BCCAo bilateral common carotid artery occlusion, S + S sham + sham, S + B sham + BCCAo, M + S MCAO + sham, M + B MCAO + BCCAo