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Novel cerebrovascular pathology in mice fed a high cholesterol diet Franciosi, Sonia; Gama Sosa, Miguel A; English, Daniel F; Oler, Elizabeth; Oung, Twethida; Janssen, William G; De Gasperi, Rita; Schmeidler, James; Dickstein, Dara L; Schmitz, Christoph; Gandy, Sam; Hof, Patrick R; Buxbaum, Joseph D; Elder, Gregory A Oct 24, 2009

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BioMed CentralssMolecular NeurodegenerationOpen AcceResearch articleNovel cerebrovascular pathology in mice fed a high cholesterol dietSonia Franciosi1,2,3, Miguel A Gama Sosa1,3, Daniel F English1,2, Elizabeth Oler4, Twethida Oung4, William GM Janssen4, Rita De Gasperi1,3, James Schmeidler1, Dara L Dickstein4,5, Christoph Schmitz4, Sam Gandy1,6,7, Patrick R Hof4,5,8, Joseph D Buxbaum1,2,4,9 and Gregory A Elder*1,6,7Address: 1Department of Psychiatry, Mount Sinai School of Medicine, One Gustave L Levy Place, New York, NY 10029, USA, 2Laboratory of Molecular Neuropsychiatry, Mount Sinai School of Medicine, One Gustave L Levy Place, New York, NY 10029, USA, 3Research and Development Service, James J Peters Department of Veterans Affairs Medical Center, 130 West Kingsbridge Road, Bronx, NY 10468, USA, 4Department of Neuroscience, Mount Sinai School of Medicine, One Gustave L Levy Place, New York, NY 10029, USA, 5Computational Neurobiology and Imaging Center, Mount Sinai School of Medicine, New York, NY 10029, USA, 6Department of Neurology, Mount Sinai School of Medicine, One Gustave L Levy Place, New York, NY 10029, USA, 7Neurology Service, James J Peters Department of Veterans Affairs Medical Center, 130 West Kingsbridge Road, Bronx, NY 10468, USA, 8Department of Geriatrics and Adult Development, Mount Sinai School of Medicine, One Gustave L Levy Place, New York, NY 10029, USA and 9Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, NY 10029, USAEmail: Sonia Franciosi - sfranciosi@cmmt.ubc.ca; Miguel A Gama Sosa - miguel.gama-sosa@mssm.edu; Daniel F English - englishdaniel@gmail.com; Elizabeth Oler - lizoler@gmail.com; Twethida Oung - twethida.oung@mssm.edu; William GM Janssen - bill.janssen@mssm.edu; Rita De Gasperi - rita.de-gasperi@mssm.edu; James Schmeidler - james.schmeidler@mssm.edu; Dara L Dickstein - dara.dickstein@mssm.edu; Christoph Schmitz - cs.999@gmx.net; Sam Gandy - samuel.gandy@mssm.edu; Patrick R Hof - patrick.hof@mssm.edu; Joseph D Buxbaum - joseph.buxbaum@mssm.edu; Gregory A Elder* - gregory.elder@mssm.edu* Corresponding author    AbstractBackground: Hypercholesterolemia causes atherosclerosis in medium to large sized arteries.Cholesterol is less known for affecting the microvasculature and has not been previously reportedto induce microvascular pathology in the central nervous system (CNS).Results: Mice with a null mutation in the low-density lipoprotein receptor (LDLR) gene as well asC57BL/6J mice fed a high cholesterol diet developed a distinct microvascular pathology in the CNSthat differs from cholesterol-induced atherosclerotic disease. Microvessel diameter was increasedbut microvascular density and length were not consistently affected. Degenerative changes andthickened vascular basement membranes were present ultrastructurally. The observed pathologyshares features with the microvascular pathology of Alzheimer's disease (AD), including thepresence of string-like vessels. Brain apolipoprotein E levels which have been previously found tobe elevated in LDLR-/- mice were also increased in C57BL/6J mice fed a high cholesterol diet.Conclusion: In addition to its effects as an inducer of atherosclerosis in medium to large sizedarteries, hypercholesterolemia also induces a microvascular pathology in the CNS that sharesfeatures of the vascular pathology found in AD. These observations suggest that high cholesterolmay induce microvascular disease in a range of CNS disorders including AD.Published: 24 October 2009Molecular Neurodegeneration 2009, 4:42 doi:10.1186/1750-1326-4-42Received: 5 July 2009Accepted: 24 October 2009This article is available from: http://www.molecularneurodegeneration.com/content/4/1/42© 2009 Franciosi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 11(page number not for citation purposes)Molecular Neurodegeneration 2009, 4:42 http://www.molecularneurodegeneration.com/content/4/1/42BackgroundCholesterol is essential for building and maintaining cellmembranes. Many steroid hormones also require choles-terol as a precursor. Yet, despite its role in essential bio-chemical processes and support of membrane structure,hypercholesterolemia is associated with negative healthoutcomes especially its association with vascular disease.In particular the level of serum low-density lipoproteincholesterol (LDL-C) is a key risk factor for atherosclerosisand lowering LDL-C significantly reduces the risk of coro-nary heart disease [1,2].Serum cholesterol has also been suggested as a risk factorfor or modulator of neurological diseases although theeffects appear complex and disease specific. In Alzheimer'sdisease (AD) much attention has focused on how choles-terol influences the enzymes which process the amyloidprecursor protein (APP) and in particular that high cellularcholesterol shifts APP processing towards production of theamyloid β peptide (Aβ), which in turn accumulates in neu-ritic plaques, while lower cellular cholesterol levels pro-mote α-secretase cleavage of APP and prevent Aβ formation[3]. In contrast, higher serum cholesterol levels have beensuggested to be associated with a lower risk of Parkinson'sdisease [4], while low serum LDL-C levels have been asso-ciated with worsening of amyotrophic lateral sclerosis [5].Curiously, despite hypercholesterolemia's well-establishedrole in promoting ischemic heart disease, serum cholesterolis not a strong risk factor for ischemic stroke [2,6] or vascu-lar dementia [7].How cholesterol modulates susceptibility to neurologicaldiseases is incompletely understood. Hypercholestero-lemia is best known for producing atherosclerosis in rela-tively large arteries such as the aorta or coronary arteries[8]. In contrast, hypercholesterolemia is not normallythought of as affecting microvessels pathologically eventhough a substantial literature exists showing that highcholesterol adversely affects the physiological functioningof the microvasculature including microvessels in thebrain [9].Here we report that mice fed a high cholesterol dietdevelop a vascular pathology that affects the CNS microv-asculature. This pathology is distinctive from cholesterol-induced atherosclerotic disease and shares some featuresof the microvascular pathology associated with AD [10].These findings have implications for the role that choles-terol may play in inducing vascular disease in a variety ofneurological diseases including AD.ResultsExperimental design for manipulation of plasma standard low cholesterol rodent chow diet (~4-6% fat,0.04% or less cholesterol) also have only moderatelyincreased plasma cholesterol levels with a slightlyincreased susceptibility to atherosclerosis [11-13]. How-ever, by increasing dietary cholesterol to 0.15% or greater[14], plasma cholesterol in LDLR-/- mice increases dra-matically and within 16 weeks of dietary modification themice develop extensive atherosclerotic lesions in the aor-tic root [15,16].We have previously utilized the ability to modulateplasma cholesterol levels in LDLR-/- mice to test theeffects of relatively short term modulation of plasma cho-lesterol (16 weeks on diet) on brain Aβ production andbehavior in LDLR-/- mice [17,18]. Hypercholesterolemiahas a well-established role in promoting atherosclerosis,for example in the coronary arteries. Less is known abouthow cholesterol affects the brain vasculature and indeedserum cholesterol is not regarded as a major risk factor forischemic stroke [2,6] or vascular dementia [7]. We there-fore determined whether short term (16 weeks) or longterm (10 months) modulation of dietary cholesterolinduces brain vascular pathology. Beginning at twomonths of age, we fed C57BL/6J wild type mice andLDLR-/- mice either low (LCD, standard laboratory chow)or high cholesterol (HCD, 0.15% cholesterol) diets for 16weeks or 10 months and examined the brains for vascularpathology at the end of treatment.Plasma cholesterol levels in C57BL6/J and LDLR-/- mice fed low- or igh-cholest rol dietsFigure 1Plasma cholesterol levels in C57BL6/J and LDLR-/- mice fed low- or high-cholesterol diets. Two-month old C57BL/6J wild type (C57BL) or LDLR-/-mice were fed either low (LCD) or high (HCD) cholesterol diets for 4 or 10 months. Plasma cholesterol was determined at baseline (Pre-diet, n = 11/group), as well as after 4 (n = 11/group; except n = 12, LDLR-HCD) or 10 (n = 6, C57BL-LCD; n = 6, C57BL-HCD; n = 5, LDLR-LCD; and n = 2, LDLR-HCD) months. Asterisks indicate groups different from C57BL-LCD at each Page 2 of 11(page number not for citation purposes)cholesterol in LDLR-/- miceC57BL/6J mice fed a high cholesterol diet develop at mostmild elevations in plasma cholesterol. LDLR-/- mice fed atime point (p < 0.05). Results are discussed further in the text.Molecular Neurodegeneration 2009, 4:42 http://www.molecularneurodegeneration.com/content/4/1/42To verify the effects of dietary treatment, we measuredtotal plasma cholesterol pre- and post-treatment (Figure1). As in our previous studies [17,18], we found that base-line plasma cholesterol at two months of age was mod-estly elevated in LDLR-/- mice (~160 mg/dl) compared toC57BL/6J mice (~100 mg/dl, p = 0.0008, unpaired Stu-dent's t test). Following 16 weeks of dietary treatment,plasma cholesterol was ~825 mg/dl in the LDLR-/- micefed the HCD compared to ~280 in LDLR-/- mice contin-ued on the LCD. Plasma cholesterol in C57BL/6J miceranged from 188 mg/dl in mice fed the LCD diet to 216mg/dl in mice fed the HCD. A two-way ANOVA revealedthe expected effects of both diet (F1,41 = 12.89, p = 0.0009)and genotype (F1,41 = 19.28, p < 0.0001) and a significantinteraction of diet and genotype (F1,41 = 10.54, p =0.0023) with plasma cholesterol increased in LDLR-HCDmice compared to all of the other groups (p < 0.001,Tukey HSD). Despite 16 weeks on the HCD, plasma cho-lesterol in C57BL/6J mice was unchanged compared toC57BL/6J mice on the LCD.Comparing pre- to post-16-week treatment values, arepeated measures two-way ANOVA revealed effects ofgroup (F3,41 = 17.43, p < 0.0001) and time (F1,41 = 38.31,p < 0.0001), as well as a significant interaction effect (F3,41= 11.33, p < 0.0001) with comparisons between groupsshowing that only in the LDLR-HCD group was plasmacholesterol significantly changed from baseline (p <0.001, Tukey HSD).In the cohort of animals that was treated for 10 months,plasma cholesterol was higher than 1,600 mg/dl in theLDLR-/- mice fed the HCD compared to ~630 mg/dl inLDLR-/- mice continued on the LCD. Two-way ANOVArevealed effects of both diet (F1,16 = 226.7, p < 0.0001) andgenotype (F1,16 = 795.2, p < 0.0001) and a significantinteraction of diet and genotype (F1,16 = 210.1, p <0.0001) with plasma cholesterol increased in LDLR-HCDcompared to other groups (p = 0.04 vs. C57BL-LCD andC57BL-HCD; p = 0.06 vs. LDLR-L, unpaired t test withWelch correction). LDLR-LCD was also increased vs. bothC57BL/6J groups (p < 0.0007). Interestingly, 10 monthson a HCD resulted in no change in cholesterol levels inC57BL/6J mice with plasma cholesterol averaging 145mg/dl in mice fed the LCD and 165 mg/dl in mice fed theHCD (p = 0.4117).Microvascular pathology in mice fed a high cholesterol dietWe examined the brains of six month old or one-year oldmice fed HCD or LCD diets for 4 or 10 months respec-tively using collagen IV immunostaining to visualize thevasculature. At both ages, microvessels in C57BL-HCD,LDLR-LCD, and LDLR-HCD exhibited pathologicalchanges. Examples of pathological vessels in the hippoc-ampus are shown in Figure 2 for mice fed these diets for10 months. Microvessels in the C57BL-LCD mice exhib-ited generally smooth contours and were regular inappearance. In contrast, mice in the high cholesterol-fedgroups (C57BL-HCD and LDLR-HCD) exhibited a mix-ture of microvessels that were often thinner and irregularwhile other microvessels appeared enlarged. In additionmicrovessels in C57BL-HCD and LDLR-HCD mice exhib-Abnormal vascular morphologies in C57BL/6J and LDLR-/- mice fed a high- cholester l dietFigur  3Abnormal vascular morphologies in C57BL/6J and LDLR-/- mice fed a high- cholesterol diet. Anti-collagen IV immunoperoxidase-stained microvessels in the hippocam-pus of one year old C57BL/6J (A, B, C), or LDLR-/- mice (D, E, F) fed a HCD for 10 months. Degenerating vascular seg-ments are indicated by arrowheads in panels A and F. Typical string vessels are indicated by arrows in panels D and E. Pan-Vascular pathology in C57BL/6J and LDLR-/- mice fed a high-cholesterol dietFigure 2Vascular pathology in C57BL/6J and LDLR-/- mice fed a high-cholesterol diet. Anti-collagen IV immunoperoxi-dase-stained microvessels in the hippocampus of one-year old C57BL/6J (A-D), or LDLR-/- mice (E-H) fed low-choles-terol (A, B, E, F), or high-cholesterol (C, D, G, H) diets for 10 months. Mice in the high-cholesterol fed groups (C57BL-HCD and LDLR-HCD) exhibit a mixture of microvessels that are often thinner and irregular (see especially panels C, D) Page 3 of 11(page number not for citation purposes)els B and C show dysmorphic and abnormally twisted ves-sels. Scale bar = 50 μm.while other microvessels appear enlarged (see especially pan-els F, H). Scale bar = 50 μm.Molecular Neurodegeneration 2009, 4:42 http://www.molecularneurodegeneration.com/content/4/1/42ited a variety of abnormal morphologies including string-like vessels while other vessels displayed a kinked ortwisted morphology (Figure 3). These changes were foundin both cortical and subcortical regions and, whereaspresent in 6-month old mice fed the experimental dietsfor four months, they were more apparent in one-year oldmice that had received the diets for 10 months. The thin-ning and irregularity as well as the strings and twistedmorphologies are suggestive of a degenerative process andare highly reminiscent of the vascular pathology seen inAD [10,19].To quantify the number of pathological microvessels inthe hippocampus of C57BL/6J and LDLR-/- mice fed lowor high cholesterol diets for 10 months, microvessels wereselected using a systematic-random stereologic samplingmethodology and visually classified as normal, irregular,or abnormally dilated, tortuous, or string-like. As shownin Figure 4, compared to C57BL-LCD mice, there weremore irregular as well as pathologic vessels in C57BL-HCD, LDLR-LCD, and LDLR-HCD mice.Increased microvessel diameter in mice fed a high cholesterol dietTo determine whether quantitative vascular parameterswere altered, we performed stereologic assessments meas-uring vascular length, length density, and diameter on col-lagen IV-immunostained vessels in the hippocampus ofanimals fed the diets for 10 months. The most consistentchange quantitatively was that microvessel diameter wasincreased in C57BL-HCD, as well as LDLR-/- fed either theLCD or HCD. As shown in Figure 5, mean diametersincreased from 6.7 ± 0.24 μm in C57BL-LCD to 7.8 ± 0.12μm in C57BL-HCD, 7.7 ± 0.05 μm in LDLR-LCD and 8.1± 0.31 μm in LDLR-HCD (F1,20 = 14.00, p = 0.0013 fordiet; F1,20 = 9.319, p = 0.0063 for genotype; F1,20 = 3.554,p = 0.074 for interaction; p < 0.015, C57BL-HCD, LDLR-LCD and LDLR-HCD vs. C57BL-LCD, Tukey HSD; no sig-nificant differences between C57BL-HCD, LDLR-LCD andLDLR-HCD groups). Compared to controls where micro-vessel length density was 1,142 ± 86 mm/mm3 (± SEM),the length density was reduced by 21% in C57BL-HCD(909 ± 97), by 23% in LDLR-LCD (879 ± 26) and by 15%in LDLR-HCD (971 ± 22) with a two-way ANOVA reveal-ing an interaction effect of diet and genotype (F1,20 =0.9222, p = 0.34 for diet; F1,20 = 2.612, p = 0.12 for geno-type; F1,20 = 6.435, p = 0.02 for interaction), although theonly significant group difference was that LDLR-LCD wasreduced compared to C57BL-LCD (p = 0.038, TukeyHSD). There were no differences between the groups intotal microvessel length (p > 0.05 all group comparisons,Tukey HSD) although a two-way ANOVA revealed aninteraction effect of diet and genotype (F1,20 = 0.1576, p =0.6956 for diet; F1,20 = 2.461, p = 0.1324 for genotype;F1,20 = 4.562, p = 0.0452 for interaction). Thus the moststriking change quantitatively was that hypercholestero-Quantification of pathological microvessels in mice fed low- or high- holesterol dietsFigure 4Quantification of pathological microvessels in mice fed low- or high-cholesterol diets. The number of irregu-lar and pathological microvessels was assessed in the hippoc-ampus of C57BL/6J and LDLR-/- mice fed low- or high-cholesterol diets for 10 months (n = 5 per group). Microves-sels were classified as normal, irregular, abnormally dilated, tortuous, or string-like. An average of 57 ± 4.6 microvessels were scored per animal. Abnormally dilated, tortuous and string vessels were summed as ''pathologic''. In panel (A), the average number of irregular, pathological or irregular + path-ological microvessels per hippocampus is presented. In panel (B), the number of irregular + pathological vessels divided by the total number of vessels counted is presented. Compari-sons were made between each experimental group and C57BL-LCD mice using unpaired t tests with the test of sig-nificance according to the Holm procedure. Asterisks indi-Quantification of microvascular parameters in mice fed low- or high- holesterol dietsFigure 5Quantification of microvascular parameters in mice fed low- or high-cholesterol diets. Vascular parameters were assessed stereologically in the hippocampus of C57BL/6J and LDLR-/- mice fed low- or high-cholesterol diets for 10 months. Quantitative analyses were performed on collagen IV immunoperoxidase-stained sections (n = 6 per group). Data are presented as total vascular density (A), total vessel length per hippocampus (B), and average microvessel diame-ter (C). Asterisks indicate values that are statistically differ-Page 4 of 11(page number not for citation purposes)cate: * p = 0.01-0.05; ** p = 0.001-0.01; *** p < 0.001. ent from C57BL-LCD (p < 0.05, Tukey HSD).Molecular Neurodegeneration 2009, 4:42 http://www.molecularneurodegeneration.com/content/4/1/42lemic LDLR-/- mice as well as C57BL/6J mice fed a HCDexhibit increased microvessel diameters.Ultrastructural analysis of microvascular pathologyTo determine the ultrastructural basis of the pathology weexamined the microvasculature by electron microscopy.We examined animals after 16 weeks of dietary treatment,as at that time, at the light microscopy level, microvascularpathology was already evident. Figures 6 and 7 showexamples of microvessels in the entorhinal cortex fromeach group. Microvessels in the C57BL-LCD mice exhib-ited classic neurovascular ultrastructure with circularlumens, intact endothelial cells and smooth capillarywalls (Figure 6A). In contrast, microvessels in C57BL-HCD, LDLR-LCD, and LDLR-HCD groups showed vary-ing degrees of pathology. The endothelial cell nuclearchromatin often appeared amorphous (Figure 6B) with insome cases the endothelial cell nuclei distorted and some-times swollen (Figure 6C). In addition, luminal circularitywas frequently lost (Figure 6D) and there were varyingdegrees of capillary wall degeneration (Figure 6E).Figures 7A and 7B show examples of advanced degenera-tive changes in microvessels from an LDLR-HCD mouse.Accompanying these changes, the vascular basal laminae(Figure 7C) were often thickened and expanded perivas-cular spaces containing amorphous debris were some-times apparent (Figure 7C, D). Similar changes were alsoapparent in C57BL-HCD mice (Figures 6E, 7D).ApoE protein levels in brain are increased in C57BL/6J mice fed a high cholesterol dietPlasma ApoE is elevated in LDLR-/- mice and ApoE levelsrise in the presence of hypercholesterolemia [12]. We [17]Ultrastructural analysis of the cerebral microvasculature in mice fed low- or high- cholesterol dietsFigur  6Ultrastructural analysis of the cerebral microvascula-ture in mice fed low- or high- cholesterol diets. Trans-versely sectioned capillaries are shown from the entorhinal cortex of C57BL/6J or LDLR-/- mice fed low- (LCD) or high- (HCD) cholesterol diets for 4 months. The microvessel in panel A from a C57BL/6J mouse fed a LCD exhibits a circular lumen, an intact endothelial cell, and smooth capillary walls. Note the altered chromatin structure in the endothelial cell nucleus in panel B, and the swollen endothelial cell nucleus in panel C. A normal endothelial cell nucleus is indicated by an asterisk in A. Arrowheads indicate an expanded extravascu-lar space containing amorphous debris (D), or luminal degen-eration (E) in an LDLR-/- mouse fed a LCD (D), or a C57BL/Ultrastructural analysis of microvascular pathology in LDLR-/- and C57BL/6J miceFigure 7Ultrastructural analysis of microvascular pathology in LDLR-/- and C57BL/6J mice. Additional examples of microvascular pathology from LDLR-HCD (A, B, E, F), LDLR-LCD (C), and C57BL-HCD (D) are shown. In A and B, note the severely degenerating microvessels in an LDLR mouse fed a high cholesterol diet. In C, note the thickened basal laminae (white asterisk), expanded perivascular space (black asterisk), and degenerating capillary wall (arrowhead) in an LDLR-/- mouse fed a LCD. In D, an arrow indicates an expanded perivascular space filled with amorphous debris in a C57BL/6J mouse fed a HCD. Examples of degeneration in the capillary wall in LDLR-HCD mice are indicated by arrow-heads in panels E and F. Scale bar in B = 2 μm for panels A Page 5 of 11(page number not for citation purposes)6J mouse fed a HCD (E). Scale bar = 1 μm. and B; scale bar in D = 500 nm for panels C-F.Molecular Neurodegeneration 2009, 4:42 http://www.molecularneurodegeneration.com/content/4/1/42and others [20] have reported that ApoE levels are ele-vated in the brain of LDLR-/- mice. To determine whetherApoE levels might be modulated in brain in C57BL/6Jmice fed a HCD as well, we determined by Western blot-ting levels of ApoE in the cortex of C57BL/6J mice fed aHCD for 16 weeks. As shown in Figure 8, compared to lab-oratory chow-fed mice, ApoE levels were increasedapproximately 5-fold in mice fed the HCD (p = 0.0002,unpaired Student's t test). Thus brain ApoE levels are dra-matically increased in C57BL/6J mice fed a HCD diet.DiscussionHere we describe a novel cerebrovascular pathology asso-ciated with feeding a high cholesterol diet to C57BL/6Jwild type or LDLR-/- mice. The pathology includes a vari-ety of abnormal vascular morphologies including twistedvessels and string vessels. Stereologic assessments con-firmed a visual impression that microvessels are larger inhigh cholesterol fed mice and revealed a tendency towardsa decreased vascular density in mice fed high cholesteroldiets for 10 months. At the ultrastructural level, microves-sels showed varying degrees of endothelial cell pathologyincluding altered nuclear chromatin structure and nuclearswelling as well as degenerative changes in the luminalwall. There was also thickening of the vascular basal lam-inae and expanded perivascular spaces often filled withamorphous debris. All of these features are indicative of adegenerative process and have not been previouslydescribed as being part of the spectrum of cholesterolrelated vascular pathology.LDLR-/- mice spontaneously develop moderatelyincreased total plasma cholesterol levels and increasedLDL-C when fed a standard low cholesterol rodent chowdiet (~4-6% fat, 0.04% or less cholesterol). When fed ahigh cholesterol diet, LDLR-/- mice develop massivelyincreased both total plasma cholesterol as well as LDL-C[14]. On a standard lab chow diet, nearly all plasma cho-lesterol in C57BL/6J mice is HDL-C [21]. However, whenfed a high cholesterol diet such as that used here, most ofthe plasma cholesterol in C57BL/6J mice becomes LDL-Cand the ratio of LDL-C to HDL-C becomes inverted com-pared to mice on a lab chow diet [22]. Thus, although wedo not have LDL-C and LDL-H levels available on themice in the present study, we suspect that the pathologyobserved in C57BL/6J fed a high cholesterol diet likelyreflects increased LDL-C despite normal total plasma cho-lesterol levels.Hypercholesterolemia is best known for its role in athero-sclerotic vascular disease, a process that is most apparentin larger arteries such as the aorta or the coronary arteries.Cholesterol is generally not thought to cause pathology inthe microvasculature. However, despite the lack ofreported morphological changes a substantial literaturehas shown that high cholesterol/high fat diets inducemicrovascular dysfunction [23-29]. In arterioles, this dys-function takes the form of impaired responses to stimulithat induce vasodilation, while in postcapillary venules itis manifested as increased leukocyte and platelet adhe-sion. For example in hypercholesterolemic LDLR-/- micefollowing ischemia/reperfusion injury, larger numbers ofadherent leukocytes are seen in postcapillary venules andvascular permeability is increased [27]. A low-gradeinflammatory response occurs in association with thesefunctional changes [9]. In experimental animals, func-tional changes can be seen in the microvasculature beforearterial lesions develop and occur in multiple tissuesincluding the brain [9,25,26]. This report is to our knowl-edge the first to describe pathologic changes in the micro-vasculature associated with a high cholesterol diet.Along with cholesterol, hypertension, and diabetes arealso potent cardiovascular risk factors for atherosclerosisin medium to large sized arteries and both are associatedExpression of ApoE in brain of C57BL/6J mice fed a high-cho-lesterol dietFigu e 8Expression of ApoE in brain of C57BL/6J mice fed a high-cholesterol diet. Shown are levels of ApoE deter-mined by quantitative Western blotting on brain homoge-nates from C57BL/6J fed a HCD (n = 5) diet for 16 weeks beginning at 2 months of age compared to C57BL/6J main-tained on a LCD (n = 9). Data are shown plotted as individual values (A) or as summed values ± SEM (B). Asterisk indicates Page 6 of 11(page number not for citation purposes)with microvascular pathology. In hypertension, themicrovascular pathology takes the form of a hyaline thick-p = 0.0002 vs. control (unpaired Student's t test).Molecular Neurodegeneration 2009, 4:42 http://www.molecularneurodegeneration.com/content/4/1/42ening of arteriolar walls referred to hyaline arteriosclerosisor a hyperplastic change in which a laminated thickeningof the vessel walls occurs due to smooth muscle cell pro-liferation as well as thickening and reduplication of thebasement membrane, a process referred to as hyperplasticarteriosclerosis [8]. Both conditions can produce progres-sive luminal narrowing. However, the changes describedhere do not resemble either form of hypertension relatedvascular pathology.Diabetes may also be associated with hyaline arteriolo-sclerosis [30]. However, diabetes produces its own distinc-tive microangiopathy, one of the most consistent featuresof which involves diffuse thickening of the vascular base-ment membrane [30]. This form of microangiopathy isseen in capillaries in a variety of tissues and is regarded asunderlying the development of diabetic nephropathy,retinopathy, and some types of peripheral neuropathy.The pathology described here shares some features withdiabetic microangiopathy, in terms of thickened vascularmembranes and indeed C57BL/6 mice fed a high-fatWestern diet develop hyperglycemia and insulin resist-ance [31-33], raising the possibility that diet inducedinsulin resistance might be contributing to the vascularpathology observed here. Arguing against this possibility,however, is the fact that C57BL/6 mice develop insulinresistance consistently only on diets containing more than20% fat [31-33] and do not develop insulin resistance onlower-fat atherogenic diets [33]. C57BL/6 mice fed a 1%cholesterol-enriched diet without increased fat (4.4%),similar to that used here also do not become insulin resist-ant [34]. LDLR-/- mice fed a high-fructose diet in additiondo not become insulin resistant, despite the diet elevatingplasma cholesterol levels to those as high as seen withhigh-fat diets [32]. Thus, wild type C57BL/6 mice fed thetype of high-cholesterol diet used in the present study(0.15% cholesterol/4.3% fat) do not typically developinsulin resistance, making it unlikely that the vascularpathology observed here can be ascribed to insulin resist-ance.Interestingly, some of the pathological alterationsdescribed bear a resemblance to the vascular pathologyassociated with AD. Whereas it seems that no simple cor-relation exists between serum cholesterol levels and therisk of developing AD [7,35,36], elevated midlife choles-terol has been associated with an increased risk of AD [7].In addition, higher total serum cholesterol and LDL-C cor-relate with a more rapid cognitive decline in patients withAD [37]. Dyslipidemia is also a component of the meta-bolic syndrome along with obesity, hypertension, andhyperglycemia. There has been recently much interest inthe role that the individual components of the metabolicthat age-related cognitive impairment is more likely todevelop when the metabolic syndrome is present [41].AD is accompanied by vascular pathology. In the mostrecognized form of this pathology, cerebral amyloid angi-opathy, amyloid is deposited the walls of blood vesselswith leptomeningeal and neocortical arteries and arteri-oles being most affected [42]. Vascular pathology, how-ever, also occurs in the microvasculature leading to adecreased density and fragmentation of microvasculature[10,19]. Microvessels appear less branched and thinatrophic vessels known as string vessels appear whileother vessels become kinked and looped. The cause andrelationship of vascular pathology to cognitive decline inAD remains unclear although patients with Down syn-drome display a similar vascular pathology that is presentin young cases devoid of neuritic plaques and neurofibril-lary tangles [19], arguing that vascular changes may pre-cede the development of these lesions. Alterations of thevascular basement membranes, in particular thickening ofbasement membranes, have been suggested to be an earlyfeature of the microvascular pathology in AD [43]. Simi-larities between the cholesterol related pathologydescribed here and the microvascular pathology of ADinclude the presence of string vessels, tortuous and loopedvessels, and thickened basement membranes. The pathol-ogy observed here however differs from AD by the pres-ence of increased microvessel diameters.How a high-cholesterol diet induces microvascularpathology in brain is not known. The generation of reac-tive oxygen species, in particular superoxide is thought tobe a major factor in cholesterol's effects on dysfunction ofthe microvasculature [9]. Upregulation of cell adhesionmolecules including intercellular adhesion molecule-1(ICAM-1) and P-selectin on the endothelium along withthe release of additional cytokines from circulating lym-phocytes likely underlie the increased leukocyte and plate-let adhesion, and create a generally proinflammatory state[9]. This effect is seen in experimental animals in ranges ofplasma cholesterol that are only modestly elevated.Increased proinflammatory cytokines, microglial reac-tion, and astrogliosis have also been seen in the brains ofLDLR-/- mice following high-cholesterol/high-fat diets[44]. However, based on immunohistochemical stainingwe have not seen any obvious microglial or astroglial reac-tion in LDLR-/- mice fed a selective high-cholesterol diet(unpublished observations). Clearly further studies willbe needed to delineate the molecular mechanisms under-lying the microvascular pathology induced by a high-cho-lesterol diet.ApoE plays a significant role in modulating cholesterolPage 7 of 11(page number not for citation purposes)syndrome may play in the development of AD as well asother dementias [38-40], with some studies suggestingtransport in the periphery as well as the brain where it isknown to affect for example amyloid deposition [45]. Pre-Molecular Neurodegeneration 2009, 4:42 http://www.molecularneurodegeneration.com/content/4/1/42viously we [17] and others [20] have reported that ApoElevels are elevated in the brain of LDLR-/- mice. Here weshow that ApoE levels in brain are also increased inC57BL/6J mice fed a HCD. Altered ApoE levels in brainmay in addition modify responses to disease processes inthe CNS.ConclusionFuture studies will be necessary to elucidate the exactmechanisms that underlie the cerebral microvascularpathology associated with elevated cholesterol. However,collectively, these studies show that mice fed a high-cho-lesterol diet develop a distinctive CNS microvasculaturepathology. These findings have implications for the rolethat cholesterol related vascular disease might play in neu-rological diseases including AD.Materials and methodsMiceMale LDLR-/- mice were purchased from Jackson Labora-tories (Bar Harbor, MA; Ldlr KO stock # 002207 strainname B6.12957-Ldlrtm1Her). These mice were originallygenerated using a 129 ES cell line and have been back-crossed for 10 generations onto the C57BL/6J back-ground. Age-matched male C57BL/6J wild type mice alsoobtained from Jackson Laboratories were used as controls.Animals were housed on 12 h light/dark cycles. All proto-cols were approved by the Mount Sinai School of Medi-cine Institutional Animal Care and Use Committee andwere in conformance with the National Institutes ofHealth "Guide for the Care and Use of Laboratory Ani-mals".Dietary manipulationsLDLR-/- and C57BL/6J control mice were maintained ona standard low-cholesterol rodent chow diet containing0.02% cholesterol and 6% fat (Lab Diet 5K52; Purina, St.Louis, MO) until two months of age. At two months, micewere randomly assigned to receive a low-cholesterol orhigh-cholesterol diet. Those that received a high-choles-terol diet were fed D12102N base diet (Research Diets,New Brunswick, NJ) supplemented with 0.15% choles-terol with a constant proportion of fat (4.3%) and otherconstituents. Mice not assigned to the high-cholesteroldiet were continued on the standard low cholesterol labo-ratory chow diet. Mice were fed their respective diets andhad access to water ad libitum for periods of 4 or 10months.Measurement of serum cholesterolAt the initiation of dietary manipulations and at the ter-mination of the study, blood samples were taken from theretro-orbital sinus and total plasma cholesterol levels wereTissue processingMice were anaesthetized with a mixture of ketamine (100mg/kg) and xylazine (10 mg/kg) and then sacrificed bytranscardial perfusion with cold 1% paraformaldehyde in0.1 M PBS pH 7.4 (phosphate buffered saline) for 1 min,followed by cold 4% paraformaldehyde in 0.1 M PBS pH7.4 for 10 min. After perfusion, brains were removed andpostfixed in 4% paraformaldehyde for 48 hrs and thentransferred to 0.1 M PBS, and stored at 4°C until section-ing. Series of 50 μm-thick coronal sections were cut usinga Vibratome.Histology and immunohistochemistryImmunoperoxidase staining was performed on free-float-ing sections using an antigen retrieval method that utilizesa pepsin digestion treatment which has been previouslydescribed [46]. Prior to pepsin treatment, sections wereincubated in distilled water for 5 min at 37°C and thentransferred to 1 mg/ml pepsin (Dako, Carpinteria, CA) in0.2 N HCl. Sections were incubated in the pepsin solutionat 37°C for 10 min. After washing in PBS for 15 min at27°C followed by three 10-min washes at room tempera-ture, sections were processed for immunohistochemistry.Sections were pretreated with 10% methanol/1% hydro-gen peroxide in PBS for 10 min and then blocked withTris-buffered saline (TBS; 50 mM Tris-HCl, 0.15 M NaCl,pH 7.6, 0.15 M NaCl)/0.1% Triton X-100/5% goat serum(TBS-TGS) for 1.5 h. After a wash in PBS, sections wereincubated with rabbit polyclonal anti-collagen IV antise-rum (1:500; Chemicon, Temecula, CA) in TBS-TGS atroom temperature overnight. Sections incubated withoutprimary antibody served as controls. Following a wash inPBS, sections were incubated with goat anti-rabbit horse-radish peroxidase (HRP)-conjugated secondary antibody(1:500, Santa Cruz Biotechnology, Santa Cruz, CA) for 2h in TBS-TGS. Staining was visualized using 3,3'-diami-nobenzidine in 50 mM Tris-imidazole buffer (pH 7.6).After being mounted on slides, sections were dried over-night and counterstained with 0.5% cresyl violet for 6 minfollowed by dehydration through a graded series of etha-nol solutions (70, 85, 90, 100% for 2 min each). Slideswere then treated with Americlear (Fisher, Tustin, CA) for2 min, followed by xylene for 10 min and coverslippedwith Cytoseal 60 (Richard-Allan Scientific, Kalamazoo,MI). Sections were photographed using an OptonicsMicroFire true color microscope 1600 × 1200 digital CCDcamera (Optronics, Goleta, CA). Digital images were colorbalanced using Adobe Photoshop 7.0 (Adobe Systems,San Jose, CA).Stereologic determination of hippocampal microvascular density, length, and diameterFor analysis of microvascular parameters, every 6th coro-Page 8 of 11(page number not for citation purposes)determined using the Infinity Cholesterol Reagent kit(Thermotrace, Arlington, TX) according to the manufac-turer's instructions.nal section throughout the hippocampus from LDLR-/-and C57BL6/J mice fed high- and low-cholesterol diets for10 months was stained with the anti-collagen IV antise-Molecular Neurodegeneration 2009, 4:42 http://www.molecularneurodegeneration.com/content/4/1/42rum and counterstained with cresyl violet. The hippocam-pus was delineated using a stereology workstation,consisting of a modified Olympus BX50 light microscopewith a PlanApo objective 2.5× (numerical aperture [N.A.],0.04) to delineate brain regions and a UPlanApo objective20× (N.A., 0.8; Olympus, Tokyo, Japan). For counting, amotorized specimen stage for automatic sampling (LudlElectronics; Hawthorne, NY), CCD color video camera(HV-C20AMP; Hitachi, Tokyo, Japan), and stereologysoftware (StereoInvestigator; MBF Bioscience, Williston,VT) were utilized. Vessel density and length were deter-mined using the "Space Balls" method as previouslydescribed [47,48]. Hemispheres with a radius of 30 μmwere placed in a systematic-random manner within thesections. The vessel density and length was obtained fromthe total number of intersections between the hemi-spheres and vessels (at least 300-400 hits per brain half)as described previously [48]. Vessel diameter was deter-mined by using the "Fast Measure Line" tool of the stere-ology software. Diameter was taken as the shortestdiameter of the outer wall of vessels coming into focusduring the Space Ball analysis. Large vessels with a diame-ter > 30 μm were not considered as representative of themicrovasculature and were not included in the analysisfor vessel length and density. The number of irregular andpathological microvessels was determined by randomlyselecting microvessels using the Space Balls software.Based on visual inspection, an observer blinded to thegenotype of the animal, classified each sampled vessel as"normal", "irregular", or abnormally "dilated", "tortu-ous", or "string-like". Abnormally dilated, tortuous, andstring vessels were summed as "pathologic".Electron microscopyElectron microscopy was performed as described previ-ously [49] using the cryofixation/cryosubstitution embed-ding technique [50]. Three mice from each of the LDLR-/- fed high- or low- cholesterol diets and correspondinggenotype controls (C57BL/6J) fed high- or low- choles-terol diets for four months (n = 12 mice total) were anaes-thetized and perfused as described above with 4%paraformaldehyde containing 0.125% glutaraldehyde.Tissue was removed and postfixed in the same solutionovernight. Brains were then removed and 250 μm-thickcoronal sections were cut using a Vibratome and theentorhinal region of the cortex was dissected out. Freezesubstitution and low-temperature embedding of the spec-imens was performed as described elsewhere [50-52]. Theslices were cryoprotected by immersion in 4% D-glucose,followed by increasing concentrations of glycerol (from10% to 30% in phosphate buffer, v/v). Sections wereplunged rapidly into liquid propane cooled by liquidnitrogen (-190°C) in a Universal Cryofixation Systemmethanol (-90°C, 24 h) in a cryosubstitution AFS unit(Leica, Vienna, Austria). The temperature was raised from-90°C to -45°C in steps of 4°C/h. After washing withanhydrous methanol, the samples were infiltrated withLowicryl HM20 resin (Electron Microscopy Sciences, FortWashington, PA) at -45°C. Polymerization with ultravio-let light (360 nm) was performed for 48 hrs at -45°C, fol-lowed by 24 h at 0°C. Ultrathin 70 nm sections were cutwith a diamond knife on a Reichert-Jung ultramicrotomeand mounted on nickel grids using a Coat-Quick adhesivepen (Electron Microscopy Sciences). Grids were examinedon a Joel 1200 EX electron microscope (Tokyo, Japan) andimaged with an advantage CCD camera (Advanced Micro-scopy Techniques, Danvers, MA). Images were adjustedfor brightness and contrast using Adobe Photoshop 7.0.Determination of apolipoprotein E levels in brainMice were sacrificed with carbon dioxide and the brainsremoved and regionally dissected. Apolipoprotein E (ApoE) levels in brain were determined by quantitative West-ern blotting with normalization to α-tubulin as previ-ously described [17] using pooled samples of anterior andposterior neocortex.Statistical analysisAll data are presented as mean ± the standard error of themean (S.E.M.). Equality of variance was assessed using theLevene test. Comparisons were made using two-way uni-variate or repeated measures analysis of variance(ANOVA) as well as unpaired t tests. When multiple com-parisons were made for all pairs among the four groups,the Tukey HSD procedure was used if the Levene test wasnot significant (p > 0.05). Otherwise, comparisons for allpairs of groups or selected comparisons were made usingunpaired t tests with significance determined according tothe procedure of Holm [53] to correct for multiple com-parisons. If the Levene statistic was significant (p < 0.05)and groups were of unequal sizes, the unpaired t testsemployed the Welch correction for unequal variances. Sta-tistical tests were performed using the program GraphPadPrism 4.0 (GraphPad Software, San Diego, CA) or SPSS16.0 (SPSS, Chicago, IL).AbbreviationsAβ: amyloid β peptide; AD: Alzheimer's disease; APP:amyloid precursor protein; ANOVA: analysis of variance;ApoE: apolipoprotein E; CNS: central nervous system;H&E: hematoxylin and eosin; HCD: high-cholesterol diet;ICAM-1: intracellular adhesion molecule 1; LCD: low-cholesterol diet; LDL-C: low-density lipoprotein choles-terol; LDLR: low density lipoprotein receptor; NFT: neu-rofibrillary tangle; NP: neuritic plaque; PBS: phosphatebuffered saline; SEM: standard error of the mean; TBS:Page 9 of 11(page number not for citation purposes)KF80 (Reichert-Jung, Vienna, Austria). The samples wereimmersed in 0.5% uranyl acetate dissolved in anhydrousTris-buffered saline; TGS: Triton X-goat serum.Molecular Neurodegeneration 2009, 4:42 http://www.molecularneurodegeneration.com/content/4/1/42Competing interestsThe authors declare that they have no competing interests.Authors' contributionsSF participated in the design of the experiments, carriedout the majority of the experimental work, and partici-pated in the data analysis and writing of the manuscript.MAGS, EO, TO, RDG, and DLD participated in differentaspects of the collection of the morphological data.WGMJ conducted the EM studies. DFE measured the apol-ipoprotein E levels. JS participated in the statistical analy-sis and CS in the analysis of quantitative vascularparameters. SG participated in the interpretation of theresults and writing of the manuscript. PRH supervised thecollection and analysis of the morphological data andparticipated in the writing of the manuscript. JDB partici-pated in designing the experiments and drafting the man-uscript. GAE participated in experimental design, dataanalysis, and writing of the manuscript. All authors readand approved the final manuscript.AcknowledgementsWe thank Bridget Wicinski for expert technical assistance. This work was supported by NIH grants AG02219 and AG05138.References1. Grundy SM, Cleeman JI, Merz CN, Brewer HB Jr, Clark LT, Hunning-hake DB, Pasternak RC, Smith SC Jr, Stone NJ: Implications ofrecent clinical trials for the National Cholesterol EducationProgram Adult Treatment Panel III guidelines.  Circulation2004, 110(2):227-239.2. Lewington S, Whitlock G, Clarke R, Sherliker P, Emberson J, HalseyJ, Qizilbash N, Peto R, Collins R: Blood cholesterol and vascularmortality by age, sex, and blood pressure: a meta-analysis ofindividual data from 61 prospective studies with 55,000 vas-cular deaths.  Lancet 2007, 370(9602):1829-1839.3. Wolozin B: Cholesterol and the biology of Alzheimer's dis-ease.  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