A male rat model integrating hypertension, chronic cerebral hypoperfusion, and aging recapitulates multimodal cerebral small vessel disease phenotypes.

Cerebral small vessel disease (CSVD) usually develops under the combined influence of vascular risk factors, chronic perfusion insufficiency, and age-related brain vulnerability; however, animal models driven by a single insult do not fully reproduce this multifactorial clinical context. We established a composite CSVD rat model by combining spontaneous hypertension, bilateral common carotid artery stenosis (BCAS)-induced chronic cerebral hypoperfusion, and D-galactose (D-gal)-induced aging-related stress. Fourteen-week-old male Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) were assigned to the WKY, SHR, SHR+BCAS, and SHR+BCAS+D-gal groups. Spatial learning and memory, exploratory behavior, spontaneous locomotion, and motor coordination were assessed using the Morris water maze, open field test, and balance beam test. T2-weighted magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI) were used to quantify regional brain volumes and white matter microstructural integrity. Luxol fast blue (LFB) staining and transmission electron microscopy (TEM) were performed to examine corpus callosum myelin pathology, and enzyme-linked immunosorbent assay (ELISA) was used to assess molecular markers related to the blood-brain barrier (BBB), neuroinflammation, oxidative stress, myelin injury, axonal injury, and neuronal damage. Compared with WKY rats, SHR rats showed early spatial memory impairment, behavioral abnormalities, and structural brain changes. Superimposition of BCAS and D-gal progressively aggravated these abnormalities. SHR+BCAS+D-gal rats exhibited the longest escape latency, reduced target-quadrant dwell time and platform crossings, decreased locomotor and exploratory activity, prolonged balance beam crossing time, increased foot slips, and greater waiting/stopping behavior. MRI revealed ventricular enlargement and reduced hippocampal, cortical, and corpus callosum volumes. DTI showed lower fractional anisotropy in the genu of the corpus callosum, cingulum, dorsal hippocampus, and thalamic core region. LFB staining showed reduced myelin staining and disorganized fiber arrangement, whereas TEM revealed myelin lamellar loosening, irregularity, and deformation in the corpus callosum. Molecular analyses showed decreased Claudin-5, ZO-1, MBP, and SOD, and increased Iba1, GFAP, MDA, corpus callosum NfL, serum NfL, and serum NSE, especially in the SHR+BCAS+D-gal group. In male rats, the SHR+BCAS+D-gal composite model recapitulates the multifactorial background, chronic progression, and multimodal neurological impairment of clinical CSVD at behavioral, neuroimaging, histopathological, and molecular levels. This male rat model may provide a useful experimental platform for mechanistic and interventional studies targeting BBB protection, neuroinflammation, oxidative stress, and white matter repair.