Cerebral venous congestion exacerbates cerebral microhemorrhages in mice

Abstract

Cerebral microhemorrhages (CMHs; microbleeds), which are small focal intracerebral hemorrhages, importantly contribute to the pathogenesis of cognitive decline and dementia in older adults. Although recently it has been increasingly recognized that the venous side of the cerebral circulation likely plays a fundamental role in the pathogenesis of a wide spectrum of cerebrovascular and brain disorders, its role in the pathogenesis of CMHs has never been studied. The present study was designed to experimentally test the hypothesis that venous congestion can exacerbate the genesis of CMHs. Increased cerebral venous pressure was induced by internal and external jugular vein ligation (JVL) in C57BL/6 mice in which systemic hypertension was induced by treatment with angiotensin II plus L-NAME. Histological analysis (diaminobenzidine staining) showed that mice with JVL developed multiple CMHs. CMHs in mice with JVL were often localized adjacent to veins and venules and their morphology was consistent with venous origin of the bleeds. In brains of mice with JVL, a higher total count of CMHs was observed compared to control mice. CMHs were distributed widely in the brain of mice with JVL, including the cortical gray matter, brain stem, the basal ganglia, subcortical white matter, cerebellum, and the hippocampi. In mice with JVL, there were more CMHs predominantly in cerebral cortex, brain stem, and cerebellum than in control mice. CMH burden, defined as total CMH volume, also significantly increased in mice with JVL. Thus, cerebral venous congestion can exacerbate CMHs. These observations have relevance to the pathogenesis of cognitive impairment associated with right heart failure as well as elevated cerebral venous pressure due to jugular venous reflux in older adults.

Keywords: Cerebral circulation; Heart failure; ICH; Intracerebral hemorrhage; Microbleed; VCI; Vascular cognitive impairment; Vascular contributions to cognitive impairment and dementia (VCID); Vein; Venous congestion.

Fig. 1

Cerebral venous congestion induced by jugular vein ligation (JVL) exacerbates CMHs. A–H Representative images of larger, confluent hemorrhages (A,B), perivascular (C,D), capillary (E,F), and peri-venular (G,H) CMHs stained by diaminobenzidine in brains of hypertensive mice with JVL. Note in (G) the origin of the hemorrhage at the daughter branch of a venule. Brightfield images of CMHs were captured using a 10 × objective and used for quantification. The images were batch-processed for color deconvolution, thresholding, and area measurement. Scale: 100 µm. I Total number of CMHs throughout the entire brains of control and JVL mice. Data are means ± SEM (n = 6 for control and n = 3 for JVL). ***p < 0.0001 vs. control. J CMH volume distribution in different brain areas (logarithmic scale). Control: sham-operated control mice (plus treatment with Ang-II + L-NAME); JVL: mice with jugular vein ligation (plus treatment with Ang-II + L-NAME)

Fig. 2

Effects of jugular vein ligation (JVL) on distribution of CMHs by location. A–F Representative images of CMHs stained by diaminobenzidine in the cortex (A), white matter (B), basal ganglia (C), brain stem (D), cerebellum (E), and hippocampus (F) of hypertensive control and JVL mice. Scale: 100 µm. G Bar graphs showing the distribution of CMHs by location. Total CMHs counts (G) and total CMH volumes (H) per region per animal were averaged. I Average individual CMH volumes. Data are mean ± SEM (n = 6 for control and n = 3 for JVL). *p < 0.05; **p < 0.01 vs. control. Control: sham-operated control mice (plus treatment with Ang-II + L-NAME); JVL: mice with jugular vein ligation (plus treatment with Ang-II + L-NAME)