A New Rat Model of Chronic Cerebral Hypoperfusion Resulting in Early-Stage Vascular Cognitive Impairment

Abstract

Objective: Currently, most models of vascular cognitive impairment are established by occluding the carotid arteries uni- or bilaterally to reduce the cerebral blood flow mimicking chronic cerebral hypoxia. Due to the sudden blood flow interruption, a gradual narrowing of the carotid artery cannot be completely imitated. This paper aims to establish a bilateral carotid stenosis model with mild cognitive dysfunction and mild white matter changes to simulate patients with vascular predementia.

Methods: Aged Wistar rats (18 months old) underwent either bilateral common carotid artery stenosis (BCAS) or occlusion (BCAO) surgery or a sham operation (control group). The cerebral blood flow in the frontal cortex was measured using Doppler flowmetry. Thirty days after surgery, cognitive function impairments were determined with the Morris water maze; cerebral magnetic resonance imaging was performed to detect changes in fractional anisotropy to assess white matter injuries, and histological studies were performed.

Results: The aged rats in the BCAS group showed a more gradual cerebral blood flow reduction and a lower mortality rate (11%) compared to rats in the BCAO group. The water maze test revealed a more marginal impairment affecting spatial learning and memory in rats with BCAS than in rats with BCAO. Diffusion tensor imaging detected white matter injuries in the hippocampus and cerebral cortex of BCAS rats. Particularly, a small portion of nerve fibers of the lateral somatosensory cortex was significantly different between rats of the BCAO and BCAS groups. In the BCAS group, the microscopic structure of the hippocampal CA1 region changed slightly after 30 days and sustained a slight mitochondrial crista crack. Fluorescence staining indicated that the number of GFAP-positive cells was increased in rat brains of the BCAS group, and this phenomenon was even more pronounced in the BCAO group. The hnRNPA2/B1 and GABA_AR-α1 expression levels were significantly decreased in the hippocampus of rats with BCAS compared to those of controls.

Conclusion: Severe bilateral carotid stenosis induced mild cognitive dysfunction and slight structural changes in the brains of aged rats. Thus, a chronic cerebral hypoperfusion model was successfully established.

Keywords: anisotropy; bilateral carotid artery stenosis; chronic cerebral hypoxia; cognitive function; vascular cognitive impairment.

FIGURE 1

Schematic diagram of common carotid artery stenosis operation. (A) Operative procedure of carotid artery stenosis and anatomical structure around the common carotid artery. (B) Common carotid artery after constriction. a. : artery.

FIGURE 2

The survival rates (A) and CBF values (B) in each group after surgery. The data are presented as the mean ± SD and expressed as percentages of the preoperative value. *P < 0.05 and **P < 0.01 compared with the control group using Dunnett’s multiple comparison test. CBF, cerebral blood flow; BCAS, bilateral common carotid artery stenosis; BCAO, bilateral common carotid artery occlusion; SD, standard deviation.

FIGURE 3

(A) Voxel-based statistical analysis of the diffusion tensor imaging parameter FA. (B,C) The change of white matter neural fiber (B) and fiber length in corpus callosum. The significant main effects of the phenotype are shown for the mean diffusivity. Results are displayed on an anatomical template with a visualization threshold of false discovery rate with P < 0.005 and a minimum cluster size of 20 voxels. The color scale indicates f-values, with yellow indicating a larger difference between the conditions investigated. Control-BCAS means that the FA value in BCAS rats is lower than in controls. BCAS-BCAO means that the FA value in BCAS rats is higher than in BCAO rats. The strongest effects according to the voxel-based analysis are displayed in the graphs. Data are expressed as the mean ± SD (n = 6/group). ^∗P < 0.05 compared with the control group; ^#P < 0.05 compared with the BCAS group. FA, fractional anisotropy; BCAS, bilateral common carotid artery stenosis; BCAO, bilateral common carotid artery occlusion; SD, standard deviation.

FIGURE 4

Electron microscopy data for the hippocampal CA1 area in the three experimental groups. (A) Red circles indicate mitochondria. The average area (B) and total number of mitochondria (C) in the hippocampal CA1 area 30 days after surgery are depicted. (D) The number of injured mitochondria (mitochondrion with crista broken) in the control and BCAS groups. Data are expressed as the mean ± SD (n = 6/group). *P < 0.05 compared with the control group, ^#P < 0.05 compared with the BCAS group; BCAS, bilateral common carotid artery stenosis; BCAO, bilateral common carotid artery occlusion; SD, standard deviation.

FIGURE 5

Expression of hnRNPA2/B1 at 30 days after surgery. Protein detected by immunofluorescence in the hippocampal CA1 region (A and C). Hippocampal hnRNPA2/B1 expression detected by western blotting (B and D). Data are expressed as the mean ± SD (n = 6/group). ^∗P < 0.05 compared with the control group; ^#P < 0.05 compared with the BCAS group; BCAS, bilateral common carotid artery stenosis; BCAO, bilateral common carotid artery occlusion; hnRNPA2/B1, heterogeneous nuclear ribonucleoprotein A2/B1; SD, standard deviation; MAP2, Microtubule associated protein 2; DAPI, 4’,6-diamidino-2-phenylindole.

FIGURE 6

GABA_AR-α1 expression in the hippocampal CA1 area at 30 days after surgery. Protein detected by immunofluorescence (A and C). GABA_AR-α1 expression in the rat hippocampus detected by western blotting (B and D). Data are expressed as the mean ± SD (n = 6/group). ^∗P < 0.05 compared with the control group; ^#P < 0.05 compared with the BCAS group. BCAS, bilateral common carotid artery stenosis; BCAO, bilateral common carotid artery occlusion; GABA_AR-α1, gamma-aminobutyric acid receptor subunit α1; SD, standard deviation.

FIGURE 7

Astrocyte proliferation and neuronal apoptosis in the hippocampal CA1 region of BCAS and BCAO rats (A). GFAP and NeuN expression in the hippocampal CA1 area 30 days after surgery (B and C). The endpoint, the process length and average area of astrocytes. (D–G) Data are expressed as the mean ± SD (n = 6/group). ^∗P < 0.05 compared with the control group; ^#P < 0.05 compared with the BCAS group. BCAS, bilateral common carotid artery stenosis; BCAO, bilateral common carotid artery occlusion; SD, standard deviation; GFAP, glial fibrillary acidic protein; DAPI, 4’,6-diamidino-2-phenylindole.