Experiment 1. Dahl-S rat model of age-associated hypertension and cognitive impairment. Background: A premature increase in BP and aortic stiffening occur in Dahl salt-sensitive rats (Dahl-S) on a normal salt intake with advancing age; this is not observed in normotensive wild type control Sprague-Dawley (S-D) rats. Because a novel pro-hypertensive and pro-fibrotic steroid, marinobufagenin (MBG), is implicated in BP increases in Dahl-S, we hypothesized that hypertension, MBG increase and the arterial wall stiffening in aged Dahl-S may lead to the structural changes in the cerebral arteries and to a cognitive decline. Methods and Results: S-D (n=32) and Dahl-S (n=32) male rats were fed a normal salt diet for 12 months. Systolic BP (SBP), PWV, urine MBG, collagen in aorta and cerebral arteries were measured and brain MRI and angiography (MRA) scans were performed at 3, 6, 9 and 12-mo, while the Morris water maze (MWM) test was performed at 12-mo. We demonstrated for the first time that SBP and PWV increased with age in Dahl-S rats and were greater at 12 months vs. 3 months (SBP: 170+/-5 vs. 142+/-2 mmHg, P<0.01, paired t-test; PWV: 7.6+/-0.7 vs. 4.2+/-0.8 m/s, P<0.01, paired t-test; 12-mo vs. 3-mo Dahl-S rats). These cardiovascular changes were accompanied by an increase in urine MBG (2.5-fold), which highly correlated with PWV (R=0.54, P=0.01, Pearson correlation) and SBP (R=0.63, P=0.01, Pearson correlation). The above age-associates changes as well as an increase in the aortic wall collagen (20.8+/-1.7 vs. 12.1+/-2.3 %, P<0.01, paired t-test; 12-mo vs. 3-mo Dahl-S rats) were observed in Dahl rats only, and not in S-D rats. This indicates the fibrotic nature of the aortic stiffening and participation of MBG in the above changes. Notably, that unlike S-D rats, Dahl-S rats exhibited a special memory decline in the MWM test at 12-mo compared to 3-mo. Dahl-S rats have smaller internal volumes of the cerebral arteries (by MR angiography) as a proportion to the whole brain volume compared to S-D rats at all ages. Notably, sections of the posterior cerebral artery and the caudal communication artery, became unidentifiable or significantly smaller by 9-mo in the majority of Dahl-S rats; this was not observed in. S-D rats. The above changes in the 12-mo old Dahl-S rats occurred concomitantly with an impaired hippocampal-dependent spatial memory in the water maze test vs. 3-mo Dahl-S. Conclusions and Future Directions: Age-associated increase in SBP and aortic wall fibrosis, as measured by PWV, may contribute to hippocampal-dependent memory impairment. Because these cardiovascular changes occurred in the context of an increase in the pro-fibrotic factor MBG, MBG may be a link between central arterial wall stiffening, increased BP and cognitive decline in Dahl-S age-associated dementia. The nature of the structural changes in the large cerebral arteries in the aged Dahl-S rats as well as other brain parameters assessed by MRI will be investigated. The memory-improvement effect of treatments aimed at attenuating arterial wall fibrosis and hypertension, i.e., anti-MBG antibody or lisinopril, will be tested in the hypertensive Dahl-S rats. These preliminary findings indicate that in the animal models, an increase in central arterial stiffness due to age-associated cellular and molecular mechanisms may underlie and/or enhance neurodegeneration and AD development. Targeting cellular and molecular pro-fibrotic mechanisms using a treatment intended to decrease central arterial stiffening and tissue remodeling may be beneficial for prevention and relief of AD and dementia in animal models of vascular dementia and AD, and later can be used in clinical studies. Experiment 2. Background: APP/PS1 double mutant transgenic mice with overexpressed human AD genes (2xTg-AD; AD mice), exhibit early cognitive impairment and A pathologies as well as changes in hippocampal synaptic plasticity associated with AD development. These mice develop amyloid plaques and exhibit impaired learning development with an advancing age and are widely used to study AD and AD-related pathologies. Methods and Results: We used the AD male mice in a longitudinal and a cross-sectional study to investigate the relations between the stiffening of the aorta, reduced cerebral blood flow and cognitive impairment. We demonstrated that learning impairment was accompanied by the accumulation of small amyloid plaques in the brain cortex in 4-6 months old AD mice (n=11), which were not observed in age-matched wild type control animals (WT; n=12). Moreover, central aortic stiffness (as measured by pulse wave velocity: 3.5+/-0.4 m/s vs. 2.7+/-0.1 m/s; P<0.01 by unpaired t-test; AD vs. WT mice), cardiac concentric remodeling (LV internal diameter in diastole: 3.6+/-0.1 mm vs. 3.9+/-0.1 mm; LV internal diameter in systole: 2.5+/-0.1 mm vs. 3.0+/-0.1 mm; P<0.01 by unpaired t-test; AD vs. WT mice), and reduced cerebral blood flow (188+/-5 vs. 155+/-7 ml/100 g tissue/min; P<0.01 by unpaired t-test; AD vs. WT mice) were observed at 12 months. These were followed by the massive development of amyloid plaques in the cortex and hippocampus in 16-mo AD mice (n=14), which were absent in age-matched WT animals (n=18). Unexpectedly, no greater collagen abundance and no difference in aortic wall thickness or in aortic wall diameter were observed in the AD mice when compared with age-matched WT controls. Conclusions and Future Directions: 2xTg-AD mice, unlike WT, exhibit a relatively early onset of cardiovascular abnormalities that is evident only after the onset of amyloid accumulation and cognitive impairment. If and how amyloid accumulation in neurons adversely affects the cardiovascular system remain to be determined. Studies of the mechanistic basis of cardiovascular changes and structural differences in the cerebral vasculature (visualized by the magnetic resonance angiography) in AD mice and WT mice are in progress.
