Experiment 1. Dahl-S rat model of age-associated vascular dementia. Background: A premature increase in aortic stiffening and BP, accompanied by spatial cognitive impairment, occur in Dahl salt-sensitive rats (Dahl-S) on a normal salt intake with advancing age. Because a novel pro-hypertensive and pro-fibrotic steroid, marinobufagenin (MBG), is implicated in BP increases in Dahl-S, we hypothesized that the changes in the levels of the pro-fibrotic factors, including MBG and angiotensin II, which stimulates MBG, will cause the arterial wall stiffening, which will lead to the structural changes in the cerebral arteries and neurons following by cognitive decline. We posited that pharmacologic interventions targeting age-dependent pro-fibrotic processes within the arterial wall to reduce central arterial stiffness, will ameliorate brain microvascular disease, cognitive impairment and dementia. Methods: Dahl-S (n=20) male rats were fed a normal salt diet for 12 months; 10 of them were continuously treated with an ACE inhibitor Lisinopril (15 mg/kg/day) administered through their water for 6 months, and 10 Dahl-S rats remained on regular water as a control (2 non-treated control animals died before the end of the experiment due to hypertension). Systolic BP (SBP), PWV, urine MBG, brain MRI scans, open field and attention tests were performed at 6 (baseline), 9 and 12 months, while Morris water maze (MWM) test and brain histochemistry were performed at 12-mo. Results and conclusions: We demonstrated that (i) greater PWV, a marker for central arterial stiffness, was associated with a decreased hippocampal perfusion and a decreased neuronal mass (NAA). Higher hippocampal perfusion was associates with the lower anxiety level. This finding suggests that central arterial stiffness may be a potential therapeutic target for the prevention and treatment of dementia. (ii) Next, we demonstrated that Lisinopril treatment was associated with a decrease in PWV and SBP, and that MBG level was stable for the duration of the study in the treated rats whereas control animals exhibited increase in MBG and higher SBP and PWV. (iii) In hypertensive Dahl-S rats, Lisinopril treatment improved spatial hippocampal memory, which was associated with a decrease of distance traveled to find a hidden platform in Morris water maze and with a better path efficiency vs. non-treated controls. (Path efficiency is a ratio of the optimal path to the actual path taken by the rat to quantify the efficiency of the strategy pursued in reaching the hidden platform). (iv) Preliminary results in attention test in the operant chambers suggest that Dahl-S rats treated with Lisinopril for 6 months demonstrated increased attention during a challenge with auditory and visual distractions (p=0.04). Further analysis will be done to assess impulsivity and compulsivity in this attention test. (v) In addition, we found, that Lisinopril-treated animals demonstrates stabilization of hippocampal CBF and NAA while untreated animals exhibited trend to decline of these values with aging. Increased performance for treated animals in MWM points to spatial hippocampal memory benefits of treatment that support trends seen for hippocampal CBF and NAA. These findings are consistent with the preliminary brain vessels density histochemical data, obtained by a green light sheet microscopy. Dahl-S rats treated with Lisinopril, exhibited an increase in blood vessel density vs. non-treated controls, which can be translated that a higher blood supply to the brain is resulted after anti-hypertensive and anti-fibrotic treatment. This study is in progress; after the finalizing of the database analyses, the linear mixed effect model analysis will be performed. 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 and cognitive decline in Dahl-S age-associated dementia. The memory-improvement effect of Lisinopril treatment aimed at attenuating arterial wall fibrosis and hypertension, was tested in the hypertensive Dahl-S rats. 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. We will add more behavioral tests to analyze different memory components, biochemical tests to estimate blood-brain barrier integrity, and MRI/NMR analyses to the Dahl-S model to assess the effect of anti-fibrotic treatments, i.e., a monoclonal anti-MBG antibody to reduce aortic fibrosis and PWV, and to investigate the effect of this treatment on cognitive functions. 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 translated 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 amyloid-beta 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.
