Hypercholesterolemia in rats impairs the cholinergic system and leads to memory deficits

Abstract

Alzheimer's disease (AD) is a chronic brain disorder characterized by cognitive impairment, cholinergic dysfunction, inflammation, tau and beta-amyloid pathology and vascular damage. Recent studies have shown, that high cholesterol levels are linked to the pathology of AD. The aim of our present work was to study the effects of hypercholesterolemia in adult rats. Five months after 5% cholesterol-enriched diet plasma cholesterol levels and total weight were significantly enhanced compared to controls. Spatial memory was studied in an 8-arm radial maze and cholesterol-treated rats showed an impaired learning and long-term memory. Hypercholesterolemia significantly reduced the number of cholinergic neurons in the basal nucleus of Meynert and decreased acetylcholine levels in the cortex. Nerve growth factor was only slightly enhanced in the cortex of cholesterol-treated animals. Levels of amyloid precursor protein, beta-amyloid(1-42), as well as tau and phospho-tau 181 were significantly enhanced in the cortex of cholesterol-fed rats. Hypercholesterolemia markedly increased several cerebral inflammatory markers and enhanced microglial CD11b-like immunoreactivity. Vascular density, stained by RECA-1 was not changed. However, cholesterol induced cortical microbleedings illustrated by intensive anti-rat IgG-positive spots in the cortex. In conclusion, our data demonstrate that hypercholesterolemia in rats caused memory impairment, cholinergic dysfunction, inflammation, enhanced cortical beta-amyloid and tau and microbleedings, all indications, which resemble an AD-like pathology.

Fig. 1

Hypercholesterolemia increases plasma cholesterol levels and decreases spatial memory. Sprague Dawley rats were treated with control or high cholesterol diet for 5 months. (A) Measurement of plasma cholesterol levels by HPLC and UV detection demonstrated increased plasma cholesterol concentrations in cholesterol-treated animals (Chol) compared to controls. (B) After 2 months cholesterol-fed rats (■) exhibited significantly enhanced gain in weight compared to controls (●). (C) Cholesterol-treated animals showed no differences in (1) the time needed to find all 4 baits (filled bar), (2) the number of total visits (open bar) and (3) the length of time in the visited arm (hatched bar). (D) Hypercholesterolemic rats (■) showed an impaired ability of spatial learning and storage of long-term memory compared to controls (●). Statistical analysis for plasma cholesterol and absolute weight gain was performed by one way ANOVA with a Fisher LSD posthoc test. Behavioral testings were statistically analyzed within each group by using one-way ANOVA with repeated measures and between control and cholesterol-fed animals by using student T-test. *** p < 0.001; ** p < 0.01; * p < 0.05; ns, not significant.

Fig. 2

Hypercholesterolemia impairs the cholinergic activity in the brain. (A) The number of choline acetyltransferase (ChAT) positive neurons in the basal nucleus of Meynert was counted between Bregma −0.8 mm and −3.1 mm (B) and revealed a decreased number of ChAT-positive neurons in cholesterol-fed rats (■) (D) compared to control animals (●) (C). Hypercholesterolemic rats (E) exhibited a decreased number of ChAT-positive neurons in the nBM (hatched bar), compared to controls (filled bar). (F) The level of cortical acetylcholine was diminished in cholesterol-fed rats (hatched bar), compared to controls (filled bar). Statistical analysis was performed by one way ANOVA with a Fisher LSD posthoc test. *** p < 0.001; ** p < 0.01; ns, not significant. Scale bar in A = 2300 μm (A), 500 μm (C, D).

Fig. 3

Hypercholesterolemia affects APP and beta-amyloid in rats. (A) Western Blot analysis was performed for amyloid precursor protein (APP) and beta-amyloid (Aβ) in controls (−) and cholesterol-fed (Chol) animals. (A) Standards (Std) for APP (100 ng) and beta-amyloid (100 ng) were clearly visualized at ~ 90 kDa (APP) or ~4kDa (Aβ). (A) Cortical APP was detected in controls and rats fed with high cholesterol diet, while beta-amyloid was not detectable. (B) Densitometric analysis (corrected for total actin levels) exhibited an enhanced concentration of APP protein in cholesterol-treated animals (hatched bar) compared to controls (filled bar). Immunohistochemical detection of beta-amyloid-like immunoreactivity in the cortex was visualized by fluorescence microscopy (C, D, Alexa-488, green; counterstained against nuclear DAPI, blue). Hypercholesterolemic rats (D) exhibited an enhanced beta-amyloid-positive staining compared to control animals (C). Statistical analysis was performed by one way ANOVA with a Fisher PLSD posthoc test. Values are given as mean ± S.E.M. optical units. * p < 0.05. Scale bar in D = 230 μm (C, D).

Fig. 4

Immunohistochemical staining in the cortex for microglial CD11b and anti-rat IgG of controls and cholesterol-fed rats. Controls (A) and hypercholesterolemic (B) animals were immunohistochemically stained for the microglial marker CD11b. (C) Quantitative analysis revealed an increased number of CD11b-positive spots in the cortex of cholesterol-fed rats. Microbleedings in cortex of control (D) and cholesterol-fed animals (E, F) were immunohistochemically visualized by anti-rat IgG staining. A magnification of an anti-rat IgG-positive spot is shown in F. (G) The number of anti-rat IgG-positive spots in the cortex was significantly enhanced in cholesterol-treated animals (hatched bar) compared to controls (filled bar). Statistical analysis was performed by one way ANOVA with a Fisher LSD posthoc test. **p < 0.01; * p < 0.05; ns, not significant. Scale bar in A = 200 μm (A , B); 800 μm (D, E); 360 μm (F).