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Chronic vascular risk factors (cholesterol, homocysteine, ethanol) impair spatial memory, decline cholinergic neurons and induce blood–brain barrier leakage in rats in vivo

  • Daniela Ehrlich
    Affiliations
    Laboratory for Psychiatry and Exp. Alzheimer's Research, Department of Psychiatry and Psychotherapy, Anichstr. 35, 6020 Innsbruck, Austria
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  • Christian Humpel
    Correspondence
    Corresponding author at: Lab. Psychiatry and Exp. Alzheimer's Res., Innsbruck Medical University, Anichstr. 35, 6020 Innsbruck, Austria. Tel.: +43 512 504 23712; fax: +43 512 504 23713.
    Affiliations
    Laboratory for Psychiatry and Exp. Alzheimer's Research, Department of Psychiatry and Psychotherapy, Anichstr. 35, 6020 Innsbruck, Austria
    Search for articles by this author
Open AccessPublished:July 23, 2012DOI:https://doi.org/10.1016/j.jns.2012.07.002

      Abstract

      Epidemiological studies show that vascular risk factors (e.g. atherosclerosis, diabetes, homocysteine, hypertension or cholesterol) may play a role in the development of Alzheimer's disease. Animal models may help to discover the role of vascular risk factors on cognition. In the present project we treated male Sprague Dawley rats with a diet containing homocysteine (hyperhomocysteinemia) or cholesterol (hypercholesterolemia) for 5 months or exposed the rats to ethanol (20% in drinking water) or a combination of cholesterol+ethanol (mix) for 12 months. Our experiments show that all 3 treatments (homocysteine, cholesterol, ethanol) declined spatial memory in the 8-arm radial maze, reduced the number of cholinergic neurons and induced blood–brain barrier leakage in the cortex. Rats treated with cholesterol also displayed markedly enhanced inflammation in the cortex. 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. A combination of ethanol and cholesterol did not further potentiate the effects on spatial memory, cholinergic neurons and blood–brain barrier leakage. The data suggest that chronic mild vascular risk factors over months induce small lesions of the brain capillaries in the cortex, which may contribute to the development of vascular dementia or also Alzheimer's disease.

      Keywords

      1. Introduction

      1.1 Alzheimer's disease (AD)

      AD is characterized by cerebrovascular damage and neuronal dysfunction leading to progressive cognitive decline. The hallmark pathologies include beta-amyloid (Aβ) deposition in brain (plaques) and vessels (Aβ-angiopathy), neurofibrillary tangles containing hyperphosphorylated tau, blood–brain barrier leakage and increased microglial reactivity as well as inflammatory processes. Blood–brain barrier dysfunction is associated with a reduction of cerebral blood flow, hypoxia and accumulation of neurotoxic molecules in brain parenchyma [
      • Zlokovic B.V.
      Neurovascular pathways to neurodegeneration in Alzheimer's disease and other disorders.
      ,
      • Kalaria R.N.
      Vascular basis for brain degeneration: faltering controls and risk factors for dementia.
      ]. The amyloid hypothesis suggests that the accumulation of Aβ is the most important cascade for the development of AD [
      • Hardy J.
      • Selkoe D.J.
      The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics.
      ]. Aβ-peptides (40, 42 or 43 amino acids) originate from the membrane-associated amyloid precursor protein (APP) by cleavage with α-, β- and γ-secretases and a dysbalance in Aβ-production and clearance may trigger neurodegeneration [
      • Hardy J.
      • Selkoe D.J.
      The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics.
      ]. Vascular risk factors (e.g. cholesterol, homocysteine or ethanol) over long periods may cause such a dysfunctional Aβ-clearance as well as blood–brain barrier impairment and possibly initiate cerebrovascular dysfunction or inflammatory processes leading to the development of AD [
      • Shepardson N.E.
      • Shankar G.M.
      • Selkoe D.J.
      Cholesterol level and statin use in Alzheimer disease: II. Review of human trials and recommendations.
      ,
      • Brust J.C.
      Ethanol and cognition: indirect effects, neurotoxicity and neuroprotection: a review.
      ,
      • Zhuo J.M.
      • Wang H.
      • Praticò D.
      Is hyperhomocysteinemia an Alzheimer's disease (AD) risk factor, an AD marker, or neither?.
      ,
      • Dickstein D.L.
      • Walsh J.
      • Brautigam H.
      • Stockton Jr., S.D.
      • Gandy S.
      • Hof P.R.
      Role of vascular risk factors and vascular dysfunction in Alzheimer's disease.
      ,
      • Humpel C.
      Chronic mild cerebrovascular dysfunction as a cause for Alzheimer's disease?.
      ].

      1.2 Vascular dementia (vaD)

      AD and vaD share many risk factors suggesting a related pathogenesis [
      • De la Torre J.C.
      Alzheimer's disease as a vascular disorder.
      ]. The differentiation of AD from vaD is very difficult, because many symptoms of both diseases are overlapping. Thus, the differentiation is based on evidence of cerebrovascular dysfunction in vaD [
      • Humpel C.
      Chronic mild cerebrovascular dysfunction as a cause for Alzheimer's disease?.
      ]. Indeed, cerebral vessel pathology results in blood–brain barrier leakage and such multiple cortical infarcts (silent strokes) may play a major role in development of vaD. This is accompanied by ischemic changes with cerebral hypoperfusion and oxidative stress. Clinical symptoms are multifaceted depending on location and size of the stroke lesions which are often asymptomatic for a long period. Vascular risk factors, such as cholesterol, homocysteine and ethanol may play an important role in the development of vaD [
      • Mostofsky E.
      • Burger M.R.
      • Schlaug G.
      • Mukamal K.J.
      • Rosamond W.D.
      • Mittleman M.A.
      Alcohol and acute ischemic stroke onset: the stroke onset study.
      ] which is suggested by the fact that vaD is potentially preventable by life style modification and counteracting vascular risk factors [
      • Lee A.Y.
      Vascular dementia.
      ].

      1.2.1 The cerebrovascular system

      In contrast to leaky vessels in peripheral organs [
      • Mann G.E.
      • Zlokovic B.V.
      • Yudilevich D.L.
      Evidence for a lactate transport system in the sarcolemmal membrane of the perfused rabbit heart: kinetics of unidirectional influx, carrier specificity and effects of glucagon.
      ], the blood–brain barrier restricts entry of polar molecules into the brain. Nutrients such as vitamins, glucose, and amino acids cross the blood–brain barrier by using specific transporters [
      • Zlokovic B.V.
      • Apuzzo M.L.
      Cellular and molecular neurosurgery: pathways from concept to reality—part I: target disorders and concept approaches to gene therapy of the central nervous system.
      ]. Peptides in general poorly cross the blood–brain barrier [
      • Zlokovic B.V.
      • Segal M.B.
      • Begley D.J.
      • Davson H.
      • Rakić L.
      Permeability of the blood–cerebrospinal fluid and blood–brain barriers to thyrotropin-releasing hormone.
      ,
      • Zlokovic B.V.
      • Begley D.J.
      • Chain-Eliash D.G.
      Blood–brain barrier permeability to leucine-enkephalin, D-alanine2-D-leucine5-enkephalin and their N-terminal amino acid (tyrosine).
      ], but they can be transported into the brain via specific receptors expressed in brain endothelium under physiological or pathological conditions [
      • Zlokovic B.V.
      • Mackic J.B.
      • Djuricic B.
      • Davson H.
      Kinetic analysis of leucine-enkephalin cellular uptake at the luminal side of the blood–brain barrier of an in situ perfused guinea-pig brain.
      ,
      • Zlokovic B.V.
      • Hyman S.
      • McComb J.G.
      • Lipovac M.N.
      • Tang G.
      • Davson H.
      Kinetics of arginine-vasopressin uptake at the blood–brain barrier.
      ]. Intact neurovascular functions are necessary for proper neuronal structure and function. Thus, pericyte deficiency leads to microvascular degeneration and brain accumulation of toxic substances (e.g. Aβ) preceding neuronal degenerative changes, learning and memory impairment and neuroinflammatory response [
      • Bell R.D.
      • Winkler E.A.
      • Singh I.
      • Sagare A.P.
      • Deane R.
      • Wu Z.
      • et al.
      Apolipoprotein E controls cerebrovascular integrity via cyclophilin A.
      ,
      • Bell R.D.
      • Winkler E.A.
      • Sagare A.P.
      • Singh I.
      • LaRue B.
      • Deane R.
      • et al.
      Pericytes control key neurovascular functions and neuronal phenotype in the adult brain and during brain aging.
      ].

      1.3 Methodological aspects

      1.3.1 Treatment of rats

      Male Sprague Dawley rats (aged 6 months) were housed at the Animal Department of the Medical University Innsbruck and had free access to food and tap water with a 12/12 h light–dark circle. All animal experiments were approved by the Austrian Ministry of Science. The diet of the control animals contained following ingredients: 450 g/kg cornstarch, 140 g/kg casein, 155 g/kg maltodextrin, 100 g/kg sucrose, 40 g/kg soybean oil, 50 g/kg fiber, 35 g/kg mineral mix, 1.8 g/kg L-cystine, 1.4 g/kg choline chloride, 0.008 g/kg butylhydroxytoluol, 10 g/kg vitamin mix (without folic acid), 1 g/kg chocolate aroma and 0.002 g/kg folic acid (Ssniff special diet GmbH; Soest Germany). The animals of the cholesterol group were fed for 5 months (n=10) or 12 months (n=12) with additional 50 g/kg cholesterol [
      • Ullrich C.
      • Pirchl M.
      • Humpel C.
      Hypercholesterolemia in rats impairs the cholinergic system and leads to memory deficits.
      ]. The animals of the homocysteine group were fed with additional 3 g / kg dl-homocysteine for 5 months (n=9) or 15 months (n=10) [
      • Pirchl M.
      • Ullrich C.
      • Humpel C.
      Differential effects of short- and long-term hyperhomocysteinemia on cholinergic neurons, spatial memory and microbleedings in vivo in rats.
      ]. The animals of the ethanol group were treated with 20% ethanol in drinking water ad libitum for 12 months (n=12) [
      • Daniela Ehrlich
      • Michael Pirchl
      • Humpel Christian
      Effects of long-term moderate ethanol and cholesterol on cognition, cholinergic neurons, inflammation and vascular impairment in rats.
      ]. The animals of the cholesterol-homocysteine mix group were treated with a combined diet of 3 g ⁄ kg dl-homocysteine and 5% cholesterol for 5 months and the animals of the cholesterol ethanol mix group (n=12) were fed with a diet containing 5% cholesterol and 20% ethanol in drinking water [
      • Pirchl M.
      • Ullrich C.
      • Humpel C.
      Differential effects of short- and long-term hyperhomocysteinemia on cholinergic neurons, spatial memory and microbleedings in vivo in rats.
      ,
      • Daniela Ehrlich
      • Michael Pirchl
      • Humpel Christian
      Effects of long-term moderate ethanol and cholesterol on cognition, cholinergic neurons, inflammation and vascular impairment in rats.
      ].

      1.3.2 Cognition in the partially baited eight-arm radial maze

      Spatial learning and long-term memory performance was assessed in the partially baited eight-arm radial maze (Fig. 1A ), a well-established method to explore learning and memory in a controlled environment, as recently described by us in detail [
      • Pirchl M.
      • Ullrich C.
      • Humpel C.
      Differential effects of short- and long-term hyperhomocysteinemia on cholinergic neurons, spatial memory and microbleedings in vivo in rats.
      ]. The maze consists of eight identical arms with side panels and sunk-in-food cups at the end radiating from a circular platform. For spatial navigation small high contrast visual cues (triangle, vertical bars, cross and squares) were placed above the doors of four arms and on the corresponding walls. Four arms were baited with food pellets (chocolate cereals) and the trial ended when all baits were found or after 10 min exceeded. The task for the animals was to find all baits in respective arms without visiting unbaited arms. The animals were restricted to food before the learning sessions were started to increase motivation. In a shaping session the rats were habituated to the testing procedures to decrease stress. In five training sessions (with each five trials) the animals had to learn the task. After three weeks of the last session the retention (five trials) was performed to investigate long-term memory performance. Memory errors were quantified according to Jarrard et al.'s definition [
      • Jarrard L.E.
      • Okaichi H.
      • Steward O.
      • Goldschmidt R.B.
      On the role of hippocampal connections in the performance of place and cue tasks: comparisons with damage to hippocampus.
      ]. The whole experiments was automatically controlled and monitored by a computer with MAZESOFT Software (Version 8.1.9).
      Figure thumbnail gr1
      Fig. 1(A) Spatial learning and long-term memory performance was assessed in the partially baited eight-arm radial maze. For spatial navigation small high contrast visual cues (triangle, vertical bars, cross and squares) were placed above the doors of four arms and on the corresponding walls. Four arms were baited with food pellets and the trial ended when all baits were found or after 10 min exceeded. (B) Rat brains were sectioned into 60 μm slices which were stained for the enzyme choline-acetyltransferase (ChAT). The number of ChAT-positive neurons located in the nucleus basalis of Meynert (small insert) was counted under the microscope. (C) Inflammatory markers were measured by multiplex ELISA. For the ELISA cortex extracts were added to pre-spotted plates, immune-detection was performed and the luminescent signal was evaluated by the Searchlight imaging and analysis system. (D) Blood–brain barrier leakage was assessed by using immunohistochemistry against rat immunoglobulin G (IgG). Rat IgG-positive spots (small insert) in the cortex were counted under the microscope at a 40× magnification.
      B, adapted from Pirchl et al. [
      • Pirchl M.
      • Ullrich C.
      • Humpel C.
      Differential effects of short- and long-term hyperhomocysteinemia on cholinergic neurons, spatial memory and microbleedings in vivo in rats.
      ]); D, adapted from Ehrlich et al. [
      • Daniela Ehrlich
      • Michael Pirchl
      • Humpel Christian
      Effects of long-term moderate ethanol and cholesterol on cognition, cholinergic neurons, inflammation and vascular impairment in rats.
      ].

      1.3.3 Cholinergic neurons

      The cholinergic neurons located in the septum and nucleus basalis of Meynert (nbM; Fig. 1B) play an important role in cognition and memory and are severely impaired in AD [
      • Mufson E.J.
      • Counts S.E.
      • Perez S.E.
      • Ginsberg S.D.
      Cholinergic system during the progression of Alzheimer's disease: therapeutic implications.
      ]. For immunohistochemistry brains were removed and fixed in 4% paraformaldehyde. Then brains were frozen under CO2 snow and sectioned into 60-μm slices with a cryostate (Leica Jung CM3000). Slices were immunohistochemically stained for the enzyme choline-acetyltransferase (ChAT) that serves as markers for cholinergic neurons. ChAT-positive neurons of the nucleus basalis of Meynert (nbM) between Bregma −0.7 to −3.1 were counted at a 40× magnification under the microscope.

      1.3.4 Inflammation

      Inflammation plays a role in both AD as well as in vaD [
      • Helmy A.A.
      • Abdel Naseer M.M.
      • El Shafie S.
      • Nada M.A.
      Role of interleukin 6 and alpha-globulins in differentiating Alzheimer and vascular dementias.
      ]. In our studies levels of inflammatory markers (e.g. interleukine-1β, tumor-necrosis-factor-α, monocytochemotactic-protein-1, macrophage-inflammatory-protein-2) in the cortex were explored by multiplex ELISA (Fig. 1C) (Searchlight, Aushon Biosystems), as described recently by us [
      • Pirchl M.
      • Ullrich C.
      • Humpel C.
      Differential effects of short- and long-term hyperhomocysteinemia on cholinergic neurons, spatial memory and microbleedings in vivo in rats.
      ]. Briefly, the brains were removed, the frontal cortex dissected and immediately frozen. Cortex tissue was homogenized in ice-cold sodium phosphate buffer with a protease inhibitor, centrifuged and subsequently the supernatant was added to the prespotted plates and incubated for 3 h. After washing the biotinylated antibody was added and incubated for 30 min. After being washed streptavidin-horseradish peroxidise reagent was added and the luminescent signal was detected by the Searchlight CCD imaging and analysis system. Sample values were calculated from the standard curve in a liner range.

      1.3.5 Vascular disruptions

      Vascular risk factors may induce leakage of the blood–brain barrier allowing certain substances to migrate into the brain. For immunohistochemistry brains were removed and were frozen under CO2 snow and sectioned into 20-μm sections with a cryostate (Leica Jung CM3000). Blood–brain barrier integrity was assessed by immunohistochemical staining against rat immunoglobulin G (IgG), as described in detail by us [
      • Pirchl M.
      • Ullrich C.
      • Humpel C.
      Differential effects of short- and long-term hyperhomocysteinemia on cholinergic neurons, spatial memory and microbleedings in vivo in rats.
      ]. IgG-positive spots in the cortex were counted under the microscope at a 40× magnification.

      1.4 Effects of long-term moderate vascular risk factors in vivo in rats

      Treatment with the vascular risk factors cholesterol, homocysteine and ethanol over months resulted in memory impairment, dysfunction of the cholinergic system, blood–brain barrier leakage and inflammation in adult Sprague Dawley rats.

      1.5 Effects of hypercholesterolemia

      Cholesterol may play a role in development of AD possibly by influencing APP processing which leads to enhanced levels of Aβ [
      • Ehehalt R.
      • Keller P.
      • Haass C.
      • Thiele C.
      • Simons K.
      Amyloidogenic processing of the Alzheimer beta-amyloid precursor protein depends on lipid rafts.
      ]. Indeed, inhibitors of cholesterol synthesis (statins) may have a protective effect against AD [
      • Tong X.K.
      • Nicolakakis N.
      • Fernandes P.
      • Ongali B.
      • Brouillette J.
      • Quirion R.
      • et al.
      Simvastatin improves cerebrovascular function and counters soluble amyloid-beta, inflammation and oxidativestress in aged APP mice.
      ] and the C-terminal transmembrane domain C99 of APP specifically binds cholesterol and favors the amyloidogenic pathway in cells by promoting localization of C99 in lipid rafts [
      • Beel A.J.
      • Sakakura M.
      • Barrett P.J.
      • Sanders C.R.
      Direct binding of cholesterol to the amyloid precursor protein: an important interaction in lipid–Alzheimer's disease relationships?.
      ,
      • Barrett P.J.
      • Song Y.
      • Van Horn W.D.
      • Hustedt E.J.
      • Schafer J.M.
      • Hadziselimovic A.
      • et al.
      The amyloid precursor protein has a flexible transmembrane domain and binds cholesterol.
      ]. We have shown that treatment of rats with a 5% cholesterol-rich diet for 5 months caused spatial memory deficits, a dysfunction of the cholinergic system, blood–brain barrier leakage and inflammation [
      • Ullrich C.
      • Pirchl M.
      • Humpel C.
      Hypercholesterolemia in rats impairs the cholinergic system and leads to memory deficits.
      ]. In the cortex of cholesterol-fed rats levels of APP, Aβ(1–42), as well as tau and phospho-tau 181 were significantly enhanced [
      • Mostofsky E.
      • Burger M.R.
      • Schlaug G.
      • Mukamal K.J.
      • Rosamond W.D.
      • Mittleman M.A.
      Alcohol and acute ischemic stroke onset: the stroke onset study.
      ]. Thus, hypercholesterolemia in rats resembled some AD-like pathology and has been suggested to provide a good model to study AD development as well as cerebrovascular dysfunction [
      • Mostofsky E.
      • Burger M.R.
      • Schlaug G.
      • Mukamal K.J.
      • Rosamond W.D.
      • Mittleman M.A.
      Alcohol and acute ischemic stroke onset: the stroke onset study.
      ].

      1.6 Effects of hyperhomocysteinaemia

      Elevated homocysteine plasma levels may enhance the risk for developing AD or vaD [
      • Ravaglia G.
      • Forti P.
      • Maioli F.
      • Martelli M.
      • Servadei L.
      • Brunetti N.
      • et al.
      Homocysteine and folate as risk factors for dementia and Alzheimer disease.
      ]. It is well established that hyperhomocysteinaemia induces memory and learning disabilities in several animal models [
      • Pirchl M.
      • Ullrich C.
      • Humpel C.
      Differential effects of short- and long-term hyperhomocysteinemia on cholinergic neurons, spatial memory and microbleedings in vivo in rats.
      ,
      • Stefanello F.M.
      • Monteiro S.C.
      • Matte' C.
      • Scherer E.B.
      • Netto C.A.
      • Wyse A.T.
      Hypermethioninemia increases cerebral acetylcholinesterase activity and impairs memory in rats.
      ]. We have shown that in adult Sprague Dawley rats hyperhomocysteinaemia for 5 months markedly affected spatial memory performance and the number of cholinergic neurons in the nbM [
      • Pirchl M.
      • Ullrich C.
      • Humpel C.
      Differential effects of short- and long-term hyperhomocysteinemia on cholinergic neurons, spatial memory and microbleedings in vivo in rats.
      ]. In the cortex blood–brain barrier leakage was enhanced after 12 months homocysteine treatment, but inflammation was not induced [
      • Pirchl M.
      • Ullrich C.
      • Humpel C.
      Differential effects of short- and long-term hyperhomocysteinemia on cholinergic neurons, spatial memory and microbleedings in vivo in rats.
      ]. Interestingly, hyperhomocysteinaemia in rats did not show an AD-like pathology, such as Aβ-plaques, tau-pathology or inflammation, but resulted in spatial memory impairment and dysfunction of the cholinergic system which might be initiated by blood–brain barrier impairment [
      • Pirchl M.
      • Ullrich C.
      • Humpel C.
      Differential effects of short- and long-term hyperhomocysteinemia on cholinergic neurons, spatial memory and microbleedings in vivo in rats.
      ].

      1.7 Effects of ethanol

      Ethanol consumption might cause cerebrovascular diseases, such as stroke and vaD, and may induce cognitive decline and cholinergic dysfunction [
      • Mostofsky E.
      • Burger M.R.
      • Schlaug G.
      • Mukamal K.J.
      • Rosamond W.D.
      • Mittleman M.A.
      Alcohol and acute ischemic stroke onset: the stroke onset study.
      ] and AD [
      • Floyd E.A.
      • Young-Seigler A.C.
      • Ford B.D.
      • Reasor J.D.
      • Moore E.L.
      • Townsel J.G.
      • et al.
      Chronic ethanol ingestion produces cholinergic hypofunction in rat brain.
      ,
      • Arendt T.
      Impairment in memory function and neurodegenerative changes in the cholinergic basal forebrain system induced by chronic intake of ethanol.
      ,
      • Arendt T.
      • Brückner M.K.
      • Krell T.
      • Pagliusi S.
      • Kruska L.
      • Heumann R.
      Degeneration of rat cholinergic basal forebrain neurons and reactive changes in nerve growth factor expression after chronic neurotoxic injury—II. Reactive expression of the nerve growth factor gene in astrocytes.
      ]. However, there is increasing evidence that moderate chronic ethanol has protective effects on AD development [
      • Anstey K.J.
      • Mack H.A.
      • Cherubin N.
      Alcohol consumption as a risk factor for dementia and cognitive decline: meta-analysis of prospective studies.
      ]. We have shown that long-term treatment (12 months) of adult Sprague Dawley rats with 20% ethanol in drinking water ad libitum resulted in cognitive decline, cholinergic dysfunction and blood–brain barrier leakage, but did not dramatically induce cortical inflammation [
      • Daniela Ehrlich
      • Michael Pirchl
      • Humpel Christian
      Effects of long-term moderate ethanol and cholesterol on cognition, cholinergic neurons, inflammation and vascular impairment in rats.
      ]. In the cortex of ethanol treated rats only monocyte chemotactic-protein-1 (MCP-1) was enhanced suggesting that MCP-1 may take part in a signaling cascade deviating from its role as a pro-inflammatory cytokine [
      • Daniela Ehrlich
      • Michael Pirchl
      • Humpel Christian
      Effects of long-term moderate ethanol and cholesterol on cognition, cholinergic neurons, inflammation and vascular impairment in rats.
      ]. MCP-1 is also secreted by activated microglia cells [
      • Kaul M.
      • Garden G.A.
      • Lipoton S.A.
      Pathways to neuronal injury and apoptosis in HIV-associated dementia.
      ] and indeed, after moderate long-term ethanol treatment cortical microglia reactivity was enhanced in rats [
      • Daniela Ehrlich
      • Michael Pirchl
      • Humpel Christian
      Effects of long-term moderate ethanol and cholesterol on cognition, cholinergic neurons, inflammation and vascular impairment in rats.
      ]. Interestingly, ethanol did not resemble an AD-like pathology, but rather suggested a protective effect against AD development [
      • Daniela Ehrlich
      • Michael Pirchl
      • Humpel Christian
      Effects of long-term moderate ethanol and cholesterol on cognition, cholinergic neurons, inflammation and vascular impairment in rats.
      ]. In summary, the cerebrovascular risk factor ethanol may markedly contribute to some pathology of vaD, but did not resemble an AD-like neuropathology [
      • Daniela Ehrlich
      • Michael Pirchl
      • Humpel Christian
      Effects of long-term moderate ethanol and cholesterol on cognition, cholinergic neurons, inflammation and vascular impairment in rats.
      ].

      1.8 Combination of vascular risk factors

      The combination of ethanol and cholesterol did not dramatically affect the changes in spatial memory, cholinergic neurons, blood–brain barrier impairment and inflammation [
      • Daniela Ehrlich
      • Michael Pirchl
      • Humpel Christian
      Effects of long-term moderate ethanol and cholesterol on cognition, cholinergic neurons, inflammation and vascular impairment in rats.
      ]. However, ethanol counteracted some of the cholesterol-induced effects: it reduced weight, plasma cholesterol levels and cortical Aβ(1–40) and Aβ(1–42) content, suggesting a protective role of ethanol in the development of AD [
      • Daniela Ehrlich
      • Michael Pirchl
      • Humpel Christian
      Effects of long-term moderate ethanol and cholesterol on cognition, cholinergic neurons, inflammation and vascular impairment in rats.
      ]. In contrast, cholesterol did not markedly affect the ethanol-induced changes showing only a prominent reduction of plasma ethanol levels [
      • Daniela Ehrlich
      • Michael Pirchl
      • Humpel Christian
      Effects of long-term moderate ethanol and cholesterol on cognition, cholinergic neurons, inflammation and vascular impairment in rats.
      ]. Cholesterol may modulate the absorption of ethanol into the blood, possibly due to a prolonged retention of ethanol in the stomach after cholesterol-rich nutrition [
      • Gentry T.R.
      Effect of food on the pharmacokinetics of alcohol absorption.
      ]. Taken together, a combined treatment of ethanol and cholesterol did not potentiate the effects of a single treatment but rather counteracted some of the ethanol- or cholesterol-induced effects [
      • Daniela Ehrlich
      • Michael Pirchl
      • Humpel Christian
      Effects of long-term moderate ethanol and cholesterol on cognition, cholinergic neurons, inflammation and vascular impairment in rats.
      ]. A combined cholesterol and homocysteine diet for 5 months resulted in spatial impairment and reduced cholinergic neurons similar to a single treatment, but homocysteine counteracted the cholesterol-induced inflammation and reduced slightly cortical blood–brain barrier leakage.

      1.9 Long-term versus short-term effects: differences

      Short-term (5 months) 5% cholesterol diet markedly reduced spatial memory performance, the number of cholinergic neurons in the nbM, induced inflammation as well as blood–brain barrier leakage in the cortex [
      • Ullrich C.
      • Pirchl M.
      • Humpel C.
      Hypercholesterolemia in rats impairs the cholinergic system and leads to memory deficits.
      ]. Interestingly, chronic ethanol or cholesterol treatment for 12 months had similar effects in vivo, although they were not as pronounced as the 5-month cholesterol effects. Long-term treatment of cholesterol did not result in inflammation and the changes were not as pronounced, most likely due to adaptive and compensatory mechanisms. This is in line with Pirchl et al. [
      • Pirchl M.
      • Ullrich C.
      • Humpel C.
      Differential effects of short- and long-term hyperhomocysteinemia on cholinergic neurons, spatial memory and microbleedings in vivo in rats.
      ], who reported that short-term (5 months) treatment with the vascular risk factor homocysteine had more severe effects than long-term (15 months) homocysteine treatment. The cholinergic impairment after prolonged exposure to homocysteine was possibly counteracted by the upregulation of nerve growth factor [
      • Pirchl M.
      • Ullrich C.
      • Humpel C.
      Differential effects of short- and long-term hyperhomocysteinemia on cholinergic neurons, spatial memory and microbleedings in vivo in rats.
      ], which is the most potent trophic molecule to support survival of cholinergic neurons [
      • Levi-Montalcini R.
      • Skaper S.D.
      • Dal Toso R.
      • Petrelli L.
      • Leon A.
      Nerve growth factor: from neurotrophin to neurokine.
      ].

      2. Conclusion

      Moderate chronic vascular risk factors, such as cholesterol, homocysteine or ethanol impair spatial memory, decline cholinergic neurons and induce blood–brain barrier leakage in rats in vivo which may contribute to the development of vascular dementia or Alzheimer's disease (Table 1).
      Table 1Effects of vascular risk factors in Sprague Dawley rats in vivo.
      Risk factorSpatial memoryCholinergic neuronsInflammationBlood–brain barrier leakage
      Cholesterol
      Homocysteine
      Ethanol
      Ethanol+cholesterol
      Homocysteine+cholesterol
      Spatial memory was tested on the eight arm radial maze. The number of cholinergic neurons was measured by immunohistochemistry. Inflammatory markers were analyzed by multiplex Searchlight ELISA. Blood–brain barrier leakage was indirectly shown by anti-rat immunoglobulin G histochemistry. (↓ decreased, ↑ increased, – no change).

      Conflict of interest

      None.

      Acknowledgments

      This study was supported by the Austrian Science Funds (P191220-B05 and L429-B05). We thank Ursula Kirzenberger–Winkler for her excellent technical help.

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