Expression of aquaporins 1 and 4 in the brain of spontaneously hypertensive rats

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Research Report

Expression of aquaporins 1 and 4 in the brain of spontaneouslyhypertensive rats

Daniele Tomassoni, Vincenzo Bramanti, Francesco Amenta⁎

Anatomia Umana, Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università di Camerino, 62032 Camerino, Italy

A R T I C L E I N F O A B S T R A C T

Article history:Accepted 6 February 2010Available online 12 February 2010

Aquaporins (AQP) 1 and 4 arewater channel proteins localized respectively at the level of theblood–cerebrospinal fluids (CSF) and blood brain (BBB) barriers. These barriers represent thesites of exchange between blood and nervous tissue and between blood, choroid plexus andCSF in brain ventricles respectively. Damage of these barriers may alter transfer ofsubstances between blood and nervous tissue. In spontaneously hypertensive rats (SHR)chronic hypertension may induce BBB dysfunction and pronounced defects in the integrityof the blood–CSF barrier. AQP1 is expressed in the apical membrane of choroid plexusepithelium. AQP4 is expressed by astrocyte foot processes near blood vessels. The presentstudy has assessed the expression of AQP1 and AQP4 in the brain of SHR in pre-hypertensive(2 months of age), developing hypertension (4 months of age) and established hypertension(6 months of age) stages. Age-matchedWistar–Kyoto (WKY) rats were used as normotensivereference group. AQP1 expression is increased in choroid plexus epithelium of 6-month-oldSHR. An increased expression of AQP4 was found in frontal cortex, striatum, andhippocampus of 4- and 6-month-old SHR compared to younger cohorts and age-matchedWKY rats. These findings suggest that the increase in AQP expression may alter fluidexchange in BBB and/or in blood–CSF barrier. This situation in case of an acute or excessivelyelevated rise of blood pressure can promote BBB changes causing the brain damageoccurring in this animal model of hypertension.

© 2010 Elsevier Inc. All rights reserved.

Keywords:HypertensionBlood brain barrierAquaporinNeurodegenerationSpontaneously hypertensive rats

1. Introduction

Blood brain barrier (BBB) and blood–cerebrospinal fluid (CSF)barrier represent respectively the site of exchange betweenblood and the nervous tissue in brain capillaries and the site ofexchange between blood, choroid plexus and CSF. Damage ofthese barriers may alter physiological balance between bloodand nervous tissue. It has been documented that chronichypertension may induce BBB dysfunction in cerebral cortex,deep gray matter and hippocampus of adult spontaneously

hypertensive rats (SHR) (Knox et al., 1980; Fredriksson et al.,1987; Ueno et al., 2004). In spontaneously hypertensive rats(SHR) chronic hypertension is accompanied by ventricularenlargement and potential defects in the integrity of theblood–CSF barrier ( Al-Sarraf and Philip, 2003).

Aquaporins (AQPs) are hydrophobic intrinsic membraneproteins working as “water channels” in cells involved in fluidtransport (Rash et al., 1998; Amiry-Moghaddam and Ottersen,2003; Badaut et al., 2007; Papadopoulos and Verkman, 2007).AQP1 is the member of this family first identified in red blood

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⁎ Corresponding author. Anatomia Umana, Scuola di Scienze del Farmaco e dei Prodotti della Salute, Via Madonna delle Carceri, 9, 62032Camerino, Italy. Fax: +39 0737 403325.

E-mail address: [email protected] (F. Amenta).

0006-8993/$ – see front matter © 2010 Elsevier Inc. All rights reserved.doi:10.1016/j.brainres.2010.02.023

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cells and renal proximal tubular epithelium. In brain it isexpressed by apical membrane of choroid plexus epitheliumand has a role in the production of CSF (Wu et al., 1998; Badautet al., 2002; Speake et al. 2003; Longatti et al., 2004). It is alsoexpressed by capillary endothelium, except in brain (Speakeet al., 2003). AQP4 is the predominant brain AQP and isdistributedmainly in plasmamembrane of end-feet processesof astrocytes close to brain vessels (Amiry-Moghaddam andOttersen, 2003; Badaut et al., 2007).Water flux throughAQP4, orother AQPs, is bidirectional and driven by osmotic gradients.Hence, perivascular AQP4 might have negative effect inconditions of water accumulation. AQP4 probably is involvedin oedema formation and CSF absorption (Papadopoulos et al.,2004; Papadopoulos and Verkman, 2007) and null mice for thisprotein are protected from cellular brain oedema induced byvarious causes (Manley et al., 2000). AQP4 is also up-regulatedafter brain ischemia or traumatic injury (Taniguchi et al.,2000) and its deletion aggravates vasogenic (fluid leak)cerebral oedema (Papadopoulos et al., 2004; Papadopoulosand Verkman, 2007). A possible role of AQP4 in processesunrelated to brain oedema such as astrocyte migration(Saadoun et al., 2006; Hu and Verkamn, 2006) and neuronalexcitability (Binder et al., 2004, 2006) was suggested as well.

The present study has investigated in SHR at differentstages of hypertension the expression of AQP1 in choroidplexus and AQP4 in different brain areas known to be sen-sitive to hypertension (Knox et al., 1980; Nelson and Boulant,1981; Tajima et al., 1993; Sabbatini et al., 2001, 2002; Amentaand Tomassoni, 2004). This for clarifying the role of “waterchannel proteins” in arterial hypertension. Analysis wasperformed by immunochemical and immunohistochemicaltechniques.

2. Results

Data of body weight, brain weight and systolic pressure valuesin the three age groups of normotensive Wistar Kyoto (WKY)rats and SHR are summarized in Table 1. As shown, bodyweight values were similar in WKY rats and SHR of the sameage group, but showed a tendency to increase as a function ofage. Brain weight was slightly lower in SHR of 6 monthscompared with age-matched normotensive WKY rats(Table 1). Systolic pressure values were similar in WKY ratsof the 3 age groups examined. In SHR, systolic pressure values

were higher in comparison with age-matched WKY rats andincreased as a function of age (Table 1).

2.1. Western blot analysis

AQP1 antibody was bound to a band of approximately 28 kDain the choroid plexus of lateral ventricle (Fig. 1). Bands ofsimilar molecular weight were also observed in kidneyhomogenates used as a reference tissue (data not shown).Densitometric analysis revealed an increased expression ofAQP1 in 6-month-old SHR, compared to normotensive WKYrats (Fig. 1, lane A). Western blot analysis for AQP4 revealedthe development of a intense band of approximately 32 kDa indifferent brain areas, likely corresponding to the highlyabundant 32 kDa M23 isoform of AQP4 (Fig. 1, lane C, D, E).Densitometry showed an increased expression of AQP4 infrontal cortex, hippocampus and striatum of 4 and 6-month-old SHR compared to normotensive WKY rats of the same age(Fig. 1).

2.2. Light and confocal laser microscopyimmunohistochemistry

Light microscopy showed the localization of AQP1 immuno-reactivity in the choroid epithelium (Fig. 2). No immunereaction was observed in brain parenchyma or when choroidplexus sections were incubated with antibody previouslyabsorbed with AQP1, to verify the specificity of reaction (datanot shown). Positive immunostaining is located in the apicalmembrane but not in the basolateral membrane of choroidplexus epithelium (Fig. 2). A stronger immunostaining wasobserved in the choroid plexus of SHR. This increasewasmorepronounced in 6-month-old SHR compared to younger cohorts(Figs. 2 B, D, F).

Sections processed for AQP4 immunohistochemistry de-veloped a dark-brown staining around brain microvessels,confirming the localization of this water protein in astrocytefoot processes near blood vessels (Figs. 3 and 4). An increasedexpression of AQP4 was found in frontal cortex, matrix ofstriatum, and hippocampus of 6-month-old SHR compared tonormotensiveWKY rats of the same age (Table 2 and Fig. 3). Athigher magnification, optical and confocal laser microscopyconfirmed the increased expression of AQP4 in 6-month-oldSHR compared to age-matched normotensive WKY rats(Fig. 4).

Table 1 – Body weight, brain weight and systolic pressure values in normotensive Wistar Kyoto rats (WKY) andspontaneously hypertensive rats (SHR) of different ages.

WKY(N=8)

SHR(N=8)

WKY(N=8)

SHR(N=8)

WKY(N=8)

SHR(N=8)

2 months 4 months 6 months

Body weight (g) 215±12 204±10 265±15 258±15 298±13 279±9Brain weight (g) 2.1±0.1 2.0±0.2 2.1±0.2 2.0±0.1 2.3±0.2 2.0±0.1a

Systolic pressure (mmHg) 130±5 150±8a 138±8 170±7a 145±6 200±6a

Data are the mean±S.E.a p<0.05 vs. age-matched WKY rats.

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3. Discussion

SHR are an animal model of genetic hypertension are largelyused for investigating the pathophysiology of arterial hyper-tension aswell as the influence of pharmacological treatmentson the development and course of arterial hypertension(Folkow, 1982). Behavioural studies have reported the occur-rence of loss of memory, learning, and attention processes inSHR (Gattu et al., 1997a,b), with impairment of spatial learningand working memory (Terry et al., 2000; Wyss et al., 2000).Microanatomical changes occurring in the brain of SHRdevelop starting from the 4th month of age and are obvioussince the 6th month (Sabbatini et al., 2000).

This study has assessed the expression of the two waterchannel proteins AQP1 and AQP4 in the brain of SHR. Ourfindings confirm the presence of AQP1 in the apicalmembrane(CSF-facing) of the rat choroid epithelium (Nielsen et al., 1993;Wu et al., 1998; Speake et al., 2003; Kimelberg, 2004), whichexhibits the highest expression of AQP1, than any other tissue(Mobasheri and Marples, 2004). AQP-1 has probably a majorrole in mediating water transport across the apical membraneduring CSF secretion (Oshio et al., 2003, Kimelberg, 2004). AQP1expression increases in SHR compared to WKY rats. Choroidplexus of SHR contains a higher percentage of water comparedto WKY rats (Al-Sarraf and Philip, 2003) and high bloodpressure in SHR might enhance filtration of water andelectrolytes from the choroid capillaries into intra- andextracellular space. Our findings suggest that this excess ofwater can be eliminated via CSF through an AQP1-mediatedmechanism (Oshio et al., 2003). The route of entrance of waterat the cell basolateral (blood-facing) side, remains to bedetermined. Since water flux through AQP1 is bidirectional,this protein can be involved in greater drainage of CSF into thevenous system of choroid plexus. Deletion of AQP-1 reducesosmotically driven water permeability across choroid epithe-lium determining a reduction of CSF production and ofintracranial pressure (Oshio et al., 2005). In SHR the increasedexpression of AQP1 is likely related to an enhanced increase ofCSF secretion and to a faster turnover of CSF compared tonormotensive rats (Al-Sarraf and Philip, 2003).

AQP4 is the main water channel protein in the centralnervous system (Frigeri et al., 1995; Rash et al., 1998; Satohet al., 2007). Probably is involved in brain oedema formationand resolution, although its exact role in oedema pathophys-iology is unclear (Papadopoulos and Verkman, 2005; Tomás-Camardiel et al., 2005; Fu et al., 2007). AQP4 may increaseoedema and its deletion protects mice brain from oedema(Manley et al., 2000; Vajda et al., 2002). AQP4 over expressionhas also been found to decrease cytotoxic oedema in ahypoxia–ischemia rat model (Meng et al., 2004). An increasedexpression of AQP4 protein was also found in the cerebralcortex of stroke-prone SHR of 20 weeks and in SHR of 32 weekscompared to age-matched WKY rats (Ishida et al., 2006;Tayebati et al., 2009). It has been suggested that AQP4 playsan important role in BBB function and in the pathogenesis ofhypertensive cerebral injury (Ishida et al., 2006; Tayebati et al.,2009). In spite of the above data, the role of AQP4 in keepingBBB integrity has not been clarified yet. AQP4 deletion altersBBB integrity and glial fibrillary acidic protein (GFAP) immu-

noreactivity in AQP4 null mice (Zhou et al., 2008), whereasother studies did not findmajor brain structural abnormalitiesin a murine model of AQP-4 deletion (Saadoun et al., 2009).These inconsistencies indicate that further studies are neces-sary to elucidate regulatory mechanisms and role(s) of AQP4expression in brain tissue.

The present study has confirmed the increased expressionof AQP4 in different brain areas of SHR. Since the rise in AQP4expression occurred between 4 and 6 months of age, the sameelapse of timewhenbrain injury develops in this animalmodel(Ritter and Dinh, 1986; Tajima et al., 1993; Sabbatini et al., 2000,2002), it cannot be excluded that AQP4 is involved in thepathophysiology of brain damage in SHR. The remarkableincrease of AQP4 is SHR is not related to the hypertrophy ofastrocytes, as in 6-month-old SHR no changes in perivascularastrocytes, those expressing selectively AQP4 were observed(Tomassoni et al., 2004). It cannot be excluded that an up-regulation of AQP4may induce changes in brainwater contentand this can alter autoregulation of cerebral blood flow in SHR(Wei et al., 1992; Al-Sarraf and Philip, 2003). Additionally or asan alternative hypothesis, changes in the expression of AQP4could be associated with the development of cytotoxicoedema, which may represent a first step in the pathophys-iology of brain lesions documented in this rat strain.

In summary, an increased expression of AQP1 in the choroidepithelium and of AQP4 in astrocyte foot processes of frontalcortex, striatum, and hippocampus of 6-month-old SHR wasfound. It is probable that this increase alters autoregulation ofcerebral blood flowand/or predisposes brain to a latent status ofoedema. This situation in case of an acute or excessivelyelevated rise of blood pressure can promote BBB injury (Quickand Cipolla, 2005) with subsequent brain damage extensivelydocumented in SHR (Nelson and Boulant, 1981; Sabbatini et al.,2000, 2002; Amenta and Tomassoni, 2004).

4. Experimental procedures

4.1. Animals and tissue treatment

Male SHR of 2-, 4-, 6-months of age (n=8 per age group) and age-matchedmalenormotensiveWKY rats (n=8 per age group)wereused. These ageswere chosen as they represent respectively thepre-hypertensive, developing hypertension and establishedhypertension phases (Sabbatini et al., 2000). Animals werehandled according to internationally accepted principles forcare of laboratory animals (European Community CouncilDirective 86/609, O.J. n° L358, Dec. 18, 1986). Rats were received4 weeks before experiments and kept under a constant light–dark cycle (7:00 a.m. to 7:00 p.m. light period), at an ambienttemperature of 22±1 °C,with free access towater and laboratorychow. Animals had blood pressure measured before sacrifice.They were then anesthetized with an i.p. injection of 50 mg/kgsodiumpentobarbital and perfused through the ascending aortawith a 0.9% NaCl solution containing 20 international units ofheparin. At the end of perfusion animal were killed bydecapitation. Brains were removed, washed in ice-cold 0.9%saline solution and divided into two halves by a sagittalcut through interhemispheric scissure. From the right hemi-sphere of each brain, frontal cortex, hippocampus, striatum and

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lateral ventricle choroid plexuswere dissected out, weighed andhomogenizedasdetailed below. Left hemisphere of fouranimalswas fixed in a Histochoice® (Amresco INC, Solon U.S.A.) solutionand embedded in paraffin. The brain of remaining rats wasembedded in a cryoprotectantmediumand frozen in isopentanecooled with liquid nitrogen. Samples were cut serially using amicrotome or a microtome cryostat for microanatomicalanalysis and immunohistochemistry respectively.

4.2. Western blotting

Samples (0.1±0.02 g) of frontal cortex, striatum, hippocampusand choroids plexus were homogenized in a Mixer Mill MM300(Qiagen, Hilden, Germany) with 800 μl of 0.1 M phosphate buffersaline (PBS) pH 7.4, 0.1% IGEPAL CA-630, 1 mM CaCl2, 1 mMMgCl2, 0.1% NaN3, 1 mM phenyl-methyl-sulphonil-fluoride(PMSF), aprotinin and 1mM sodium orthovanadate. After two

Fig. 2 – AQP-1 immunohistochemistry in sections of choroid plexus exposed to AQP1 antibody. A, C, E: 6-month-old WKY rats;B, D, F: 6-month-old SHR. A–D Conventional light microscope micrographs; E and F Confocal laser microscopy micrographs.Both conventional light and confocal laser microscopy demonstrated a positive immunostaining in the apical membrane butnot in the basolateral membrane of choroid plexus epithelium. Note the increased AQP1 immune reaction in the apicalmembrane of choroid plexus epithelium of SHR. Calibration bars: A–B: 25 μm; C–F: 10 μm.

Fig. 1 –Western blots of AQP1 (A) andβ-actin (B) in rat choroid plexus and of AQP4 in rat frontal cortex (C), hippocampus (D) andstriatum (E). F is β-actin band used as a reference protein. 1: 2-month-old WKY rat; 2: 2-month-old SHR; 3: 4-month-old WKYrat; 4: 4-month-old SHR; 5: 6-month-old WKY rat; 6: 6-month-old SHR.

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centrifugations at 13,000 rpm (10 min at 4 °C) aliquots of thesupernatant were used for protein assay against a standardof bovine serum albumin (BSA) using a BIO-RAD proteinassay (BIO-RAD, Munich, Germany). Equal amounts ofprotein (40 μg) were separated by 12% sodium dodecylsulphate polyacrylamide gel electrophoresis and transferredto nitrocellulose membrane by electroblotting in Towbinbuffer. Transblotted membranes were incubated with amonoclonal anti-AQP1 antibody (Calbiochem Cat. No178611) diluted 1:2000 in PBS 0.1 M, BSA (1%) and Tween-20(0.05%), or anti-AQP4 (Calbiochem, Cat. No 178614 diluted1:2000, in PBS 0.1 M, BSA (1%) and Tween-20 (0.05%). AQP1and AQP4 immunochemistry product was visualized using as

HRP-biotinylated antibody (donkey anti-rabbit IgG-HRP SantaCruz Biotecnology U.S.A., Cat. No. Sc-2313) followed by achemiluminescence detection system (SuperSignal West PicoChemiluminescent substrate, Pierce) with computer-drivendensitometry.

4.3. AQP1 and AQP4 immunohistochemistry

The first of each group of five consecutive sections of lefthemisphere was stained with a 0.5% cresyl violet solution andused for assessing the volume of single brain areas investi-gated. This step was necessary since the brain of SHR issmaller than that of corresponding normotensive WKY rats

Fig. 3 – AQP4 immunohistochemistry in sections of rat frontal cortex (A, B), striatum (C, D) and hippocampus (E, F). A, C, E:6-months-old WKY rats; B, D, F: 6-months-old SHR. III: Zone III of frontal cortex; M: matrix; S: striosomes; O: stratum oriens;P: pyramidal layer; R: stratum radiatum; H: hilum. AQP4 immune reaction was located around brain microvessels (arrows) inthe different brain areas investigated. The immunoreactivity was increased in SHR compared to age-matched WKY rats.Calibration bar: 50 μm.

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(Nelson and Boulant, 1981; Tajima et al., 1993). The second ofeach group of three consecutive sections of the left hemi-sphere was used for the immunohistochemical detection ofAQP1 immunoreactivity (test Section 1). The third one wasused for immunohistochemical detection of AQP4 immuno-reactivity (test Section 2) whereas the fourth one was used toassess the specificity of immune reaction (control section).

Test sections were first pre-incubated for 1 h at 4 °C in a 3%normal goat serum dissolved in 0.1 M phosphate bufferedsaline (PBS)–Triton X-100. Sections were incubated overnightat 4 °C in the presence of the primary antibody anti-AQP1(Calbiochem, Cat. No 178611) diluted 1:500 with 0.3% PBS–Triton X-100 (second section) or in the presence of anti-AQP4(Calbiochem, Cat. No 178614) diluted 1:1000 with 0.3% PBS–

Fig. 4 – Sections of rat hippocampus processed for immunohistochemical detection of AQP4 localization in small arteries.A, C: 6-month-old WKY rats; B, D: 6-month-old SHR. A, B: Conventional light microscope micrographs; C, D: Confocal lasermicroscopy micrographs. Both conventional light microscope and confocal laser pictures revealed the localization ofimmunoreaction only along microvessels. An increased expression of AQP4 is noticeable in SHR compared to age-matchednormotensive WKY rats. Calibration bar: 10 μm.

Table 2 – Intensity of AQP4 immune reaction in normotensiveWistar Kyoto (WKY) rats and spontaneously hypertensive rats(SHR) of different ages.

WKY(N=8)

SHR(N=8)

WKY(N=8)

SHR(N=8)

WKY(N=8)

SHR(N=8)

2 months 4 months 6 months

Frontal cortex 10.9±0.9 13.1±1.1 11.6±0.5 14.5±1.7 10.3±1.1 17.7±1.5 ⁎

StriatumMatrix 12.1±0.5 14.9±0.6 17.4±0.2 19.3±0.7 ⁎ 17.3±1.2 25.6±2.1 ⁎

Striosome 7.5±0.2 9.8±0.3 10.1±0.4 10.6±0.5 10.3±1.1 15.7±1.4 ⁎

HippocampusCA1 Subfield 10.9±1.8 13.1±1.9 11.6±0.9 14.5±1.4 10.3±1.5 17.7±1.2 ⁎

CA3 Subfield 12.2±1.0 12.7±1.1 12.3±0.7 13.8±1.3 12.1±0.8 17.3±1.7 ⁎

Dentate gyrus 11.5±1.1 11.6±0.8 11.5±0.8 16.8±1.1 12.1±0.7 19.3±1.8 ⁎

Data are expressed in arbitrary units and microdensitometric analysis was performed as indicated in the Section 4.3.⁎ p<0.05 vs. age-matched normotensive WKY rats or younger SHR.

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Triton X-100 (third section). After rinsing several times with0.3% PBS–Triton X-100 at room temperature, sections wereincubated for 1 h at room temperature in a biotinylated (goatanti-rabbit Vectastain ABC Kit PK-6101) secondary antibodydiluted 1:200 with 0.3% PBS–Triton X-100. After another rinsefor removing excess of unbound antibody, sections wereexposed to the avidin–biotin complex to reveal the product ofthe immunohistochemical reaction using 3,3′-diaminobenzi-dine as a chromogen. After rinsing, sections were counter-stained in methyl green, air-dried, mounted in Micromount(Diapath S.r.l, Martinengo, Italy) and observed under a lightmicroscope. Series of sections obtained from the differentanimal groups were processed in the same incubation bath toavoid changes in the intensity of the immune reaction due todifferent incubation conditions. A group of cryostat sectionswere processed as above, but using as secondary antibody afluorescein–isothiocyanate (FITC) conjugated (Goat anti-rabbitIgG Fluor Chemicon AP 132F) at a dilution of 1:200. Incubationwith this secondary antibody was made at room temperaturefor 30 min. Control sectionswere processedas above, but usinga non-immune rabbit IgG instead of the primary antibody, orby omitting the primary antibody in the incubationmedium. Inthese conditions no specific immunostaining was observed.

Sections processed for immunohistochemistry were viewedunder a Leica DMR microscope at a final magnification of ×60connected, via a TV camera to an IAAS 2000 image analyzer(Delta Sistemi, Rome, Italy). Sections processed for immunoflu-orescence analysis were viewed under a confocal laser micro-scope (Bio-Rad MRC 600) with an objective lens of ×60. Theintensity of AQP4 immunostaining was assessedmicrodensito-metrically. The image analysis system was calibrated taking as“zero” the background developed in sections incubated withnon-immune serum and “100” as a conventional value ofmaximum intensity of staining.

4.4. Data analysis and chemicals

Values for individual animalswithin the groups investigated aremeans of measurements of the parameters considered. Groupmeans were then derived from individual means. Statisticalanalysis was performed by analysis of variance (ANOVA). Thesignificance of differences between mean values of differentparameters in the two animal groups was assessed by the two-tailed Student's t-test, taking a value of p<0.05 as theminimumlevel of significance.

Acknowledgments

Study supported by the grant from Italian Ministero Istru-zione Università e Ricerca Scientifica; MIUR-COFIN No.2006060985_003.

R E F E R E N C E S

Al-Sarraf, H., Philip, L., 2003. Effect of hypertension on the integrityof blood brain and blood CSF barriers, cerebral blood flow andCSF secretion in the rat. Brain Res. 975, 179–188.

Amenta, F., Tomassoni, D., 2004. Treatment with nicardipineprotects brain in an animal model of hypertension-induceddamage. Clin. Exp. Hypertens. 26, 351–361.

Amiry-Moghaddam, M., Ottersen, O.P., 2003. The molecular basisof water transport in the brain. Nat. Rev. Neurosci. 4, 991–1001.

Badaut, J., Lasbennes, F., Magistretti, P.J., Regli, L., 2002.Aquaporins in brain: distribution, physiology, andpathophysiology. J. Cereb. Blood Flow Metab. 22, 367–378.

Badaut, J., Brunet, J.F., Regli, L., 2007. Aquaporins in the brain: fromaqueduct to “multi-duct”. Metab. Brain Dis. 22, 251–263.

Binder, D.K., Oshio, K., Ma, T., Verkman, A.S., Manley, G.T., 2004.Increased seizure threshold in mice lacking aquaporin-4 waterchannels. Neuroreport 15, 259–262.

Binder, D.K., Yao, X., Sick, T.J., Verkman, A.S., Manley, G.T., 2006.Increased seizure duration and slowed potassium kinetics inmice lacking of aquaporin-4 water channels. Glia 53, 631–636.

Folkow, B., 1982. Physiological aspects of primary hypertension.Physiol. Rev. 62, 347–404.

Fredriksson, K., Kalimo, H., Westergren, I., Kåhrström, J.,Johansson, B.B., 1987. Blood–brain barrier leakage and brainedema in stroke-prone spontaneously hypertensive rats. Effectof chronic sympathectomy and low protein/high salt diet. ActaNeuropathol. 74, 259–268.

Frigeri, A., Gropper, M.A., Umenishi, F., Kawashima, M., Brown, D.,Verkman, A.S., 1995. Localization of MIWC and GLIP waterchannel homologues in neuromuscular, epithelial andglandular tissues. J. Cell Sci. 108, 2993–3002.

Fu, X., Li, Q., Feng, Z., Mu, D., 2007. The roles of aquaporin-4 inbrain oedema following neonatal hypoxia ischemia andreoxygenation in a cultured rat astrocyte model. Glia 55,935–941.

Gattu,M., Pauly, J.R., Boss, K.L., Summers, J.B., Buccafusco, J.J., 1997a.Cognitive impairment in spontaneously hypertensive rats: roleof central nicotinic receptors. Part I. Brain Res. 771, 89–103.

Gattu, M., Terry, A.V., Pauly, J.R., Buccafusco, J.J., 1997b. Cognitiveimpairment in spontaneously hypertensive rats: role of centralnicotinic receptors. Part II. Brain Res. 771, 104–114.

Hu, J., Verkamn, A.S., 2006. Increased migration and metastaticpotential of tumors cells expressing aquaporin water channel.FASEB J. 20, 1892–1894.

Ishida, H., Takemori, K., Dote, K., Ito, H., 2006. Expression ofglucose transporter-1 and aquaporin-4 in the cerebral cortex ofstroke-prone spontaneously hypertensive rats in relation tothe blood–brain barrier function. Am. J. Hypertens. 19, 33–39.

Kimelberg, H.K., 2004. Water homeostasis in the brain: basicconcepts. Neuroscience 129, 851–860.

Knox, C.A., Yates, R.D., Chen, I., Klara, P.M., 1980. Effects of agingon the structural and permeability characteristics ofcerebrovasculature in normotensive and hypertensive strainsof rats. Acta Neuropathol. 51, 1–13.

Longatti, P.L., Basaldella, L., Orvieto, E., Fiorindi, A., Carteri, A.,2004. Choroid plexus and aquaporin-1: a novel explanation ofcerebrospinal fluid production. Pediatr. Neurosurg. 40, 277–283.

Manley, G.T., Fujimura, M., Ma, T., Noshita, N., Filiz, F., Bollen,A.W., Chan, P., Verkman, A.S., 2000. Aquaporin-4 deletion inmice reduces brain oedema after acute water intoxication andischemic stroke. Nat. Med. 6, 159–163.

Meng, S., Qiao, M., Lin, L., Del Bigio, M.R., Tomanek, B., Tuor, U.I.,2004. Correspondence of AQP4 expression andhypoxic–ischaemic brain oedemamonitored by magneticresonance imaging in the immature and juvenile rat. Eur. J.Neurosci. 19, 2261–2269.

Mobasheri, A., Marples, D., 2004. Expression of the AQP-1 waterchannel in normal human tissues: a semiquantitative studyusing tissuemicroarray technology. Am. J. Physiol. Cell Physiol.286, C529–C537.

Nelson, D.O., Boulant, J.A., 1981. Altered CNS neuroanatomicalorganization of spontaneously hypertensive (SHR) rats. BrainRes. 226, 119–130.

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Nielsen, S., Smith, B.L., Christensen, E.I., Agre, P., 1993.Distribution of the aquaporin CHIP in secretory andresorptive epithelia and capillary endothelia. Proc. Natl.Acad. Sci. U. S. A. 90, 7275–7279.

Oshio, K., Song, Y., Verkman, A.S., Manley, G.T., 2003. Aquaporin-1deletion reduces osmotic water permeability and cerebrospinalfluid production. Acta Neurochir. Suppl. 86, 525–528.

Oshio, K., Watanabe, H., Song, Y., Verkman, A.S., Manley, G.T.,2005. Reduced cerebrospinal fluid production and intracranialpressure in mice lacking choroid plexus water channelAquaporin-1. FASEB J. 19, 76–78.

Papadopoulos, M.C., Verkman, A.S., 2005. Aquaporin-4 genedisruption in mice reduces brain swelling and mortality inpneumococcal meningitis. J. Biol. Chem. 280, 13906–13912.

Papadopoulos, M.C., Verkman, A.S., 2007. Aquaporin-4 and brainedema. Pediatr. Nephrol. 22, 778–784.

Papadopoulos, M.C., Saadoun, S., Binder, D.K., Manley, G.T.,Krishna, S., Verkman, A.S., 2004. Molecular mechanisms ofbrain tumor edema. Neuroscience 129, 1011–1020.

Quick, A.M., Cipolla, M.J., 2005. Pregnancy-induced up-regulationof aquaporin-4 protein in brain and its role in eclampsia. FASEBJ. 19, 170–175.

Rash, J.E., Yasumura, T., Hudson, C.S., Agre, P., Nielsen, S., 1998.Direct immunogold labeling of aquaporin-4 in square arrays ofastrocyte and ependymocyte plasma membranes in rat brainand spinal cord. Proc. Natl. Acad. Sci. U. S. A. 95, 11981–11986.

Ritter, S., Dinh, T.T., 1986. Progressive postnatal dilation of brainventricles in spontaneously hypertensive rats. Brain Res. 370,327–332.

Saadoun, S., Papadopoulos, M.C., Watanabe, H., Yan, D., Manley,G.T., Verkam, A.S., 2006. Involvement of aquaporin-4 inastroglial cell migration and glial scar formation. J. Cell Sci. 118,5691–5698.

Saadoun, S., Tait, M.J., Reza, A., Davies, D.C., Bell, B.A., Verkman,A.S., Papadopoulos, M.C., 2009. AQP4 gene deletion in micedoes not alter blood–brain barrier integrity or brainmorphology. Neuroscience 161, 764–772.

Sabbatini, M., Strocchi, P., Vitaioli, L., Amenta, F., 2000. Thehippocampus in spontaneously hypertensive rats: aquantitative microanatomical study. Neuroscience 100,251–258.

Sabbatini, M., Tomassoni, D., Amenta, F., 2001. Hypertensive braindamage: comparative evaluation of protective effect oftreatment with dihydropyridine derivatives in spontaneouslyhypertensive rats. Mech. Ageing Dev. 122, 2085–2105.

Sabbatini, M., Catalani, A., Consoli, C., Marletta, N., Tomassoni, D.,Avola, R., 2002. The hippocampus in spontaneouslyhypertensive rats: an animal model of vascular dementia?Mech. Ageing Dev. 123, 547–559.

Satoh, J., Tabunoki, H., Yamamura, T., Arima, K., Konno, H., 2007.Human astrocytes express aquaporin-1 and aquaporin-4 invitro and in vivo. Neuropathology 27, 245–256.

Speake, T., Freeman, L.J., Brown, P.D., 2003. Expression ofaquaporin 1 and aquaporin 4 water channels in rat choroidplexus. Biochim. Biophys. Acta 1609, 80–86.

Tajima, A., Hans, F.J., Livingstone, D., Wei, L., Finnegan, W.,DeMaro, J., Fenstermacher, J., 1993. Smaller local brain volumesand cerebral atrophy in spontaneously hypertensive rats.Hypertension 21, 105–111.

Taniguchi,M., Yamashita, T., Kumura, E., Tamatani,M., Kobayashi,A., Yokawa, T., Maruno, M., Kato, A., Ohnishi, T., Kohmura, E.,Tohyama, M., Yoshimine, T., 2000. Induction of aquaporin-4water channel mRNA after focal cerebral ischemia in rat. BrainRes. Mol. Brain Res. 78, 131–137.

Tayebati, S.K., Di Tullio, M.A., Tomassoni, D., Amenta, F., 2009.Neuroprotective effect of treatment with galantamine andcholine alphoscerate on brainmicroanatomy in spontaneouslyhypertensive rats. J. Neurol. Sci. 283, 180–188.

Terry, A.V.J., Hernandez, C.M., Buccafusco, J.J., Gattu, M., 2000.Deficits in spatial learning and nicotinic-acetylcholinereceptors in older, spontaneously hypertensive rats.Neuroscience 101, 357–368.

Tomás-Camardiel, M., Venero, J.L., Herrera, A.J., De Pablos, R.M.,Pintor-Toro, J.A., Machado, A., Cano, J., 2005. Blood–brainbarrier disruption highly induces aquaporin-4 mRNA andprotein in perivascular and parenchymal astrocytes: protectiveeffect by estradiol treatment in ovariectomized animals.J. Neurosci. Res. 80, 235–246.

Tomassoni, D., Avola, R., Di Tullio, M.A., Sabbatini, M., Vitaioli, L.,Amenta, F., 2004. Increased expression of glial fibrillary acidicprotein in the brain of spontaneously hypertensive rats. Clin.Exp. Hypertens. 26, 335–350.

Ueno, M., Sakamoto, H., Tomimoto, H., Akiguchi, I., Onodera, M.,Huang, C.L., Kanenishi, K., 2004. Blood–brain barrier is impairedin the hippocampus of young adult spontaneouslyhypertensive rats. Acta Neuropathol. 107, 532–538.

Vajda, Z., Pedersen, M., Füchtbauer, E.M., Wertz, K.,Stødkilde-Jørgensen, H., Sulyok, E., Dóczi, T., Neely, J.D., Agre,P., Frøkiaer, J., Nielsen, S., 2002. Delayed onset of brain edemaand mislocalization of aquaporin-4 in dystrophin-nulltransgenic mice. Proc. Natl. Acad. Sci. U. S. A. 99, 13131–13136.

Wei, L., Lin, S.Z., Tajima, A., Nakata, H., Acuff, V., Patlak, C.,Pettigrew, K., Fenstermacher, J., 1992. Cerebral glucoseutilization and blood flow in adult spontaneously hypertensiverats. Hypertension 20, 501–510.

Wu, Q., Delpire, E., Hebert, S.C., Strange, K., 1998. Functionaldemonstration of Na+-K+-2Cl- cotransporter activity in isolated,polarized choroid plexus cells. Am. J. Physiol. 275, C1565–C1572.

Wyss, J.M., Chambless, B.D., Kadish, I., van Groen, T., 2000.Age-related decline in water maze learning and memory inrats: strain differences. Neurobiol. Aging 21, 671–681.

Zhou, J., Kong, H., Hua, X., Xiao, M., Ding, J., Hu, G., 2008. Alteredblood–brain barrier integrity in adult aquaporin-4 knockoutmice. Neuroreport 19, 1–5.

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