High-Sodium Diet Has Opposing Effects on Mean Arterial Blood Pressure and Cerebral Perfusion in a Transgenic Mouse Model of Alzheimer’s Disease

Abstract

Background: Cerebral ionic homeostasis impairment, especially Ca²⁺, has been observed in Alzheimer’s disease (AD) and also with hypertension. Hypertension and AD both have been implicated in impaired cerebral autoregulation. However, the relationship between the ionic homeostasis impairment in AD and hypertension and cerebral blood flow (CBF) autoregulation is not clear.

Objective: To test the hypothesis that a high-salt diet regimen influences the accumulation of amyloid-β (Aβand CBF) and CBF, exacerbates cognitive decline, and increases the propensity to AD.

Methods: Double transgenic mice harboring the amyloid-β protein precursor (APPswe), and presenilin-1 (PSEN1) along with control littermates, 2 months of age at initiation of special diet, were divided into 4 groups: Group A, APP/PS1 and Group B, controls fed a high-sodium (4.00%) chow diet for 3 months; Group C, APP/PS1 and Group D, controls fed a low-sodium (0.08%) regular chow diet for 3 months. Mean arterial blood pressure (MAP) and CBF were measured noninvasively using the tail MAP measurement device and magnetic resonance imaging, respectively. Aβ plaques numbers in the cortex and hippocampus of APP/PS1 were quantified.

Results: In contrary to controls, APP/PS1 mice fed a high-salt diet did not show markedly elevated mean systolic and diastolic blood pressure (134±4.8 compared with 162±2.8 mmHg, and 114±5.0 compared with 137±20 mmHg, p< 0.0001). However, a high-salt diet increased CBF in both APP/PS1 and controls and did not alter the cerebral tissue integrity. Aβ plaques were significantly reduced in the cortex and hippocampus of mice fed a high-salt diet.

Conclusion: These data suggest that a high-salt diet differently affects MAP and CBF in APP/PS1 mice and controls.

Keywords

Alzheimer’s disease amyloid-β blood pressure cerebral blood flow cerebral blood volume magnetic resonance imaging

INTRODUCTION

Hypertension, a chronic elevation of mean arterial blood pressure (MAP), has been implicated in several age-related cognitive impairment and dementia such as, presymptomatic stage of cognitive impairment [1], vascular cognitive impairment [2, 3], and Alzheimer’s disease (AD) [4]. AD is the most common cause of cognitive impairment in elderly [5] that suspected to have a rather complex link to hypertension. Epidemiological studies have revealed that midlife hypertension could increase the risk of AD, whereas the later life hypertension could play a protective role [6]. Recent experimental studies support the synergistic effects of hypertension and aging on expression of genes involved in amyloid-β (Aβ) generation in AD [7].

Hypertension causes structural and functional alterations of cerebral small vessels [8]. Although, non-vascular mechanisms have been recognized that link hypertension to AD neurodegeneration in hippocampus [9 –11], the vascular alteration is the most likely link between hypertension and the AD pathology [2, 12]. Chronic hypertension affects cerebral blood flow (CBF) and the lower limit of CBF autoregulation mechanism [13 –16]. In AD, however, apart from hypertension, the autoregulation mechanism is impaired [17]. This happens most likely because of elevated Aβ in AD. Accumulated Aβ impedes the relaxation effect of extracellular Ca²⁺ on arteries [18]. But, it is not clear if hypertension and AD pathogenesis have synergistic effect on CBF and CBF autoregulation.

Many factors may contribute to the pathogenesis of hypertension [19]. Excess dietary salt intake is the single most important controllable factor responsible for the rise in MAP with advancing age [20 –22]. The mechanisms by which the increase in salt intake leads to the development of salt-dependent hypertension are not completely understood. A high-salt diet regimen in salt-sensitive rats elevates plasma and cerebrospinal fluid (CSF) sodium concentration ([Na⁺]) [23]. Increase in central nervous system (CNS) [Na⁺] is primary detected by sodium exchanger (Na_x) channels in glial cells located in the circumventricular regions [24]. Chronic elevation of brain [Na⁺] promotes sustained hypertension mediated by central endogenous ouabain and the Na⁺ pump [25]. However, other ionic receptors may be in the junction of hypertension and AD pathogenesis.

Evidences support a role for calcium sensing receptors (CaSR) in high MAP modulation [26]. The impairment in Ca²⁺ homeostasis is well connected to high MAP [26]. In support of this, the dietary Ca²⁺ intake is shown to prevent the development of hypertension [27]. The Na⁺ gradient may also regulate the cellular Ca²⁺ through Na⁺/Ca²⁺ antiporter exchange. The Na⁺/Ca²⁺ exchanger’s family of membrane transporters is widely distributed in cells and tissues and constitutes one of the most important mechanisms for extruding Ca²⁺ from the cell [28]. In non-pathological conditions, the elevation of Na⁺ gradient through a high-salt diet regimen is considered as the reason for imbalance of Na⁺/Ca²⁺ and one of the causes of hypertension [29]. Though the Na⁺ gradient may influence Ca²⁺ homeostasis and CaSRs, the effect of Na⁺ gradient on competing with excess Aβ to affect the Ca²⁺ homeostasis and CaSRs in AD is unclear.

In summary, the mechanism that relates salt-induced hypertension to AD pathogenesis is not completely understood. Hypertension is suspected to causes structural and functional alteration of cerebral small vessels which may contribute to the AD pathology more likely as small vessel pathology [30]. The impaired Ca²⁺ homeostasis in elevated MAP may also exacerbate vascular inflammation in AD. Despite the growing recognition of the importance of vascular risk factors in pathogenesis of AD and dementia-like symptoms, it is still not clear if a high-salt diet regimen exacerbates cerebrovascular complications of AD. The purpose of this study was to test the hypothesis that a human lifestyle with a salty diet regimen influences the accumulation of Aβ, exacerbate cognitive decline, and increase the propensity to AD.

MATERIALS AND METHODS

Subjects

Double transgenic mice (2XTg-AD) harboring amyloid-β protein precursor (APPswe), and a mutant human presenilin-1 (PS1) were originally purchased from the Jackson Laboratory (Bar Harbor, ME) weighted between 25 and 30 g and bred in the animal care facility at the Medical University of South Carolina. Wild type littermates were used as controls (10–20 w, 25–30 g). Mice were group-housed (5 per cage) in a temperature- and humidity-controlled vivarium on a reversed 12 : 12-h light-dark cycle. Mice received ad libitum food and water (Harlan, Indianapolis, IN, USA). All animal procedures were conducted in accordance with the “Guide for the Care and Use of Laboratory Rats” (Institute of Laboratory Animal Resources on Life Sciences, National Research Council, 1996) and approved by the IACUC of the Medical University of South Carolina (protocol 3182).

Genotyping

Standard genotyping protocols were followed to confirm the presence of APP^swe/PSEN1^dE9 transgene in in-house breed mice (see http://jaxmice.jax.org/strain/005864).

Experimental groups

APP/PS1 mice along with control littermates, 2 months of age at initiation of special diet, were divided into 4 groups: Group A, APP/PS1, n= 15, and Group B, controls, n= 15 fed a high-sodium (4.00%) chow diet for 3 months; Group C, APP/PS1, n= 15 and Group D, controls, n= 15 fed a low-sodium (0.08%) regular chow diet for 3 months.

Experiments time line

The time line for the experiment and imaging sessions are depicted in Fig. 1. The mice were weaned at 22 days after birth, fed a regular diet for one month, then fed either regular chow diet or high salt diet for three months. At 70 days on the diet, blood pressure was measured three times each a week apart. Seven days after that, mice underwent magnetic resonance imaging (MRI). The MRI session included anatomical, arterial spin labeling (ASL), and diffusion MRI scans. At the end of the experiment, mice were euthanized and brains were harvested for histological analysis (control + APP; high-salt diet; n= 20; 21±0.6 weeks; range 4 to 5 weeks, and control + APP; regular diet n= 20; 21±1.2 weeks; range 4 to 5 weeks)

Diet

Mice were fed a low-salt (0.08% NaCl) or a high-salt (4% NaCl) diet (Harlan, Indianapolis, IN, USA) for about 3 months. A diet with 4% NaCl was implemented to induce experimental hypertension [31]. This diet was also recommended by nutrition experts to induce high blood pressure in APP/PS1 mice while reducing unwanted side effects and mortality. This special diet was purchased from Harlan (Harlan Laboratories Inc. Indianapolis, IN “TD09078”). To comply with IACUC regulation, the diet was irradiated. As described before, mice were divided into two experimental groups and fed a standard chow diet (control group) or a high-salt (4% NaCl) diet (Harlan, Indianapolis, IN, USA).

Quantification of Aβ deposition load

Brain tissue preparations

The brain tissues of mice were prepared soon after euthanasia in a two-day-long process. Specifically, mice were euthanized by exposure to a saturated atmosphere of isoflurane. Immediately after death, the brain of each animal was isolated. The brain was removed and placed in 4% paraformaldehyde overnight. The brains fixed in 4% paraformaldehyde were processed and embedded in paraffin. Ten serial 30μm-thick sections through the brain were obtained using a microtome. Cryosections of the brain hemispheres were washed three times (5 min/wash) with Tris-buffered saline (TBS) (pH 7.4) buffer, followed by washing once with 0.1% Triton X-100-TBS buffer for 5 min. Sections were then incubated in 3% H₂O₂ and TBS buffer for 30 min at room temperature to eliminate endogenous peroxidase activity. After 1 h of blocking with 5.0% serum (horse or goat), the sections were be incubated overnight with the following primary antibodies: GFAP (1 : 200 dilution, 2E1; BD Biosciences, San Jose, CA) to detect astrocytes and Aβ (1 : 500 dilution, Aβ peptide antibody, 4G8, Covance, Princeton, NJ) to detect amyloid deposits. The following day, sections were washed three times (5 min/wash) with 0.1% Triton X-100 and TBS buffer to remove excess primary antibody. Thereafter, primary antibodies were detected using horseradish peroxidase-conjugated IgG Vectastain ABC kit and DAB/substrate reagents (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions. The reactions were stopped in the water and coverslips were mounted after treatment with xylene. Amyloid load was assecced in brain sections by immunostaining for Abeta using Abeta antibody 10D5 (Elan Pharmaceutics) as described previously [32]. Slides with tissue samples were analyzed soon after they are ready for microscopic examination and the number of plaques was counted in each section.

Plaque counts and amyloid load determination

The total number of amyloid plaques was obtained by counting the number of plaques in each section and adding the count values to determine the number of plaques per hemisphere in each brain. The amyloid area in each section was determined by a computer-assisted image analysis system, consisting of a Power Macintosh computer equipped with a Quick Capture frame grabber card, Hitachi CCD camera mounted on an Olympus microscope and a camera stand. NIH Image Analysis Software (ImageJ, v. 1.49) was used. The images were captured and the total area of amyloid determined over the ten sections. A single operator blinded to treatment status was performed all measurements.

Blood pressure measurements

Conscious heart rate and systolic arterial blood pressure (SAP) and diastolic arterial blood pressure (DAP) were measured noninvasively at 20-22 weeks of age using tail blood pressure volume measurement device (CODA system, Kent Scientific Corporation, Torrington, CT, USA) [33] before the end of a three-month controlled diet (Fig. 1).

The measurement protocol described below is in accordance with the manufacturer‘s recommendations [33, 34]. In brief, mice were immobilized in plastic holders and acclimated to the restrainer for 10 min per day for at least 3 days before the start of the BP study. Then, for data acquisition, unanesthetized mice were immobilized in plastic holders and allowed 10 min of acclimation before the tail cuff measurements of SAP and DAP, tail blood volume, and heart rate. Measurements were taken as the mean of 2 acclimation and 10 or 15 measurement cycles, with duration of approximately 20 min total. Any measurement sets with a standard deviation of >30 mmHg were discarded. The mouse tail temperature was monitored and maintained between 34°C and 36°C for the accuracy of BP measurements. To control for external stimuli that may affect blood pressure, conditions were kept as consistent as possible between days; this included taking blood pressures at the same time of day with the same room and acclimation conditions.

MR imaging

Imaging was performed on a 7T BioSpec research dedicated MR scanner (Bruker Biospin, Ettlingen, Germany), equipped with 500 mT/m (rise time 80–120μs) gradient set (for performing high resolution small animal imaging) and a small bore linear radio frequency (RF) coil (ID 119 mm) as the RF transmitter and a four channel surface array coil as the RF receiver. Mice were anesthetized using isoflurane gas (induction dosage 1–1.5%, maintenance dosage 0.5% –1%), at 1 L/min N₂O/O₂ (70/30) flow under spontaneous respiration, during which we collected data. Real time monitoring of physiological parameters (heart rate, respiratory rate, and body temperature) were done during the imaging session for signs of distress and to ensure normal physiological parameters by using SAII monitoring instrument (Small Animal Instruments Inc., Stony Brook, NY). Inside the magnet, core body temperature was constantly maintained with a controlled warm airflow.

MR protocol included anatomical, diffusion tensor imaging (DTI), and ASL for measuring regional CBF and CBV. T2-weighted (T2 w) images were acquired in the coronal plane centered 5 mm caudal from the posterior edge of the olfactory bulb using the RARE (Rapid Acquisition with Relaxation Enhancement) sequence (repetition time/echo time (TR/TE) 4000/65 ms, field of view (FOV) of 3.7 cm × 3.7 cm, slice thickness 2 mm, slice gap 0.1 mm, contiguous slices 12, matrix 256 × 128, and number of averages 5).

DTI-EPI sequence was used to acquire diffusion images with the same geometrical parameters as anatomical images but a smaller matrix size (128 × 128 and TR/TE 3800/44.7 ms, 30 diffusion per direction, number of averages 1, d/D = 5/10 ms, and b value of 0 and 1000 s mm^–2).

In vivo CBF and CBV measurement

Regional CBF was quantified with continuous arterial spin labeling (CASL) technique with single-shot, gradient-echo, flow sensitive alternating inversion recovery echo-planar image acquisition (FAIR-EPI) [35]. The labeling pulse was a 1.78-s, square radiofrequency pulse in the presence of 1.0 G/cm gradient along the flow direction (TR/TE 3300/29 ms, number of TIR 24, FOV 3.7 cm × 3.7 cm, slice thickness 1 mm, slice gap 0.1 mm, number of slices 3, matrix 128 × 128 and number of averages 1).

All the data were processed using custom software written in MATLAB (Mathworks, Natick, MA). The anatomical images were co-registered by rigid body alignment to a mice brain template. The transformation parameters of each animal were then applied to subsequent images. DTI data were processed to generate maps of mean diffusivity (MD) and fractional anisotropy (FA). CBF was calculated by using FAIR ASL fitting equation [36, 37]. A hemispheric average of CBF values of each slice was calculated by using manually drawn ROIs and then represented as mean±standard error of mean (SEM; ml/100 g/min).

Statistical analysis

For statistical analysis “R” (version 3.0.2, R Development Core Team 2013) was used. Parametric statistical comparisons between the data sets were made based on the representation of mean±SEM unless otherwise stated. Student’s t-test (parametric data) was used for statistical analyses. Statistical analyses between groups were performed using two-way repeated measurements, analysis of variance (ANOVA) with TukeyHSC post hoc tests for multiple comparisons. We have used Cox proportional hazards regression model [38], a semi parametric model, to analyze the effect of salt on survival after high-salt diet. APP/PS1 mice under regular chow diet (n= 16) and under a high-salt diet (n= 22) were monitored for 21 days for survival studies. p-value<0.05 was considered as a significant difference.

RESULTS

High-salt diet regimen did not increase MAP of APP/PS1 mice

We observed that a high-salt diet regimen did not increase MAP of APP/PS1 mice, an animal model of AD, though MAP was increased in control littermates under the same high-salt diet regimen. We investigated both systolic and diastolic arterial BP (SAP and DAP respectively) by using tail blood pressure and blood volume measurement device (CODA system, Kent Scientific Corporation, Torrington, CT, USA). High-salt diet (Na⁺ load) did not change in systolic blood pressure of APP/PS1 mice while it did increase systolic blood pressure of non-carrier mice in comparison to the low Na⁺ fed mice (p= 0.0002). In comparison to APP/PS1 mice, Na⁺ load increased systolic blood pressure of controls (p< 0.0001). Hemizygous (Hemi) + regular diet 120.0±7.6; Hemi + high-salt diet = 122.0±4.6; non-carrier (NCAR) + regular diet = 134.0±4.8; NCAR + high-salt diet = 162.1±2.8 mmHg (Fig. 2A). Na⁺ load did not change in diastolic blood pressure of APP/PS1 mice. However, the Na⁺ load did increase diastolic blood pressure of non-carrier mice in comparison to the low Na⁺ fed mice (p= 0.001). The Na⁺ load increased diastolic blood pressure of controls (p< 0.0001) but not of the APP/PS1 mice. Hemi + regular diet 93.67±5.6; Hemi + high-salt diet = 92.67±2.9; NCAR + regular diet = 114.75±5.02; NCAR + high-salt diet = 137.75±2.6 mmHg (Fig. 2B).

High-salt diet regimen increased CBF in both APP/PS1 and controls

Advanced quantitative MR imaging techniques were used to measure in vivo regional CBF and compare experimental groups for the effect of a high-salt diet on CBF. We observed that a high-salt diet regimen increased CBF of both APP/PS1 group and the control group (p= 0.022). Figure 3 shows both groups have a statistically significant increase in CBF (p= 0.022), though the APP/PS1 mice group have a slightly higher mean CBF value than the control group (non-significant).

We further investigated CBF in two different anatomical areas of the brain; cortical and subcortical area of the brain was analyzed separately for the effect of high-salt regimen on regional CBF. We observe that cortical area of APP/PS1 mice has lower CBF than the cortical area of controls (p= 0.036). Interestingly, our data show that a salty diet regimen increases CBF in the cortex of APP/PS1 mice more than in the cortex of controls. In both investigated anatomical areas we observed evidence of CBF improvement as a result of high-salt diet regimen (p= 0.037,Fig. 4).

High-salt diet reduces plaques count in the cortex and hippocampus of APP/PS1

Plaques were observed in the cortex and hippocampus of APP/PS1 transgenic mice (Figs. 5A and 5C, and 5B and 5D, respectively). Quantitative analysis was performed on cerebral tissues to count for Aβ plaques. We observed a statistically significant reduction in the plaques count in both the cortex and hippocampus (p < 0.05) in APP/PS1 mice under a high-salt diet regimen (Fig. 5). Moreover, reduced plaques size in mice on a salty-diet in comparison to mice fed a regular chow diet is noticeable (Fig. 5).

A high-salt diet regimen did not change water diffusion parameters in APP/PS1 mouse brain

We investigated the effect of a high-salt diet regimen on the integrity of cerebral tissues and white matter fibers in the brain of APP/PS1 mice and controls. Diffusion parameters FA and MD as a measure of white matter integrity and swelling edematic areas, respectively, were investigated between the groups. We did not observe any statistically significant difference between FA and MD in all four experimental groups (Fig. 6).

DISCUSSION

Here we demonstrated that a high-salt diet regimen increases MAP of control mouse but not of the APP/PS1 mouse model of AD. Also we showed that a high-salt diet regimen increases CBF and decreases the Aβ burden of APP/PS1 mice. Two major advantages of the current study were 1) the induction of non-pharmacological hypertension in mouse by a high-salt diet regimen and 2) the employment of in vivo measurement methods to evaluate MAP and CBF. Furthermore, we characterized the differences between the diet regimen within the same group to delineate the effect of salty diet on CBF and MAP. Specifically, our data show that a high-salt diet regimen did not increase MAP but increased CBF of APP/PS1 mouse model of AD. In contrast, a high-salt diet regimen increased MAP of controls, which is in agreement with the findings from previous studies [39]. Histological comparisons corroborated the in vivo analysis, showing the reduced Aβ in mouse under a high-salt diet regimen. Taken together, these results confirm that a high-salt diet regimen can produce quite different effects on MAP and CBF of APP/PS1 and controls.

We observed a significant increase in regional CBF in the cortex and hippocampus of both controls and APP/PS1 mice after a high-salt diet regimen. At the same time a high-salt diet regimen failed to induce a significant change in MAP of APP/PS1 mice relative to controls. One would not expect to observe this in a healthy subject because in a healthy subject CBF autoregulation mechanism compensates CBF in response to changes in MAP. Cerebrovascular autoregulation mechanism responds to both transient hypoperfusion and hyperperfusion by controlling cerebrovascular reactivity [40]. However, a long term high MAP scenario is shown to negatively impact cerebrovascular reactivity [41]. This causes dysregulation of cerebral hypoperfusion or hyperperfusion. Previous studies also reported that a chronic high MAP causes alteration of the structure of cerebral blood vessels [42]. This is recognized as a leading cause of stroke and formation of other cerebral lesions. Formation of cerebral lesions is believed to correlate with cognitive decline and dementia [43]. However, contradicting reports question the existence of a causal association between high MAP and AD [44]. For example, in a large cohort of Czech patients with AD, it was observed that hypertension was diagnosed 14 years later in AD patients in comparison to controls [45]. Another study on arterial hypertension in elderly indicates that a decrease in MAP correlates with tau pathology and memory decline [46]. Our observations are in essential agreement with these previous studies that the mechanism that increases MAP in a healthy control can also reduce the AD pathology in an AD prone subject.

To the best of our knowledge, there is no report connecting diet-induced hypertension to cognitive decline and dementia. Previous reports on cognitive consequence of hypertension are based on epidemiological studies of dementia and the history of hypertension without any intervention [4 , 47–50]. Also, in studies with the experimental APP/PS1 mouse model of AD experimental hypertension was pharmacologically induced, (e.g., chronic infusion of angiotensin II, Ang II) [51, 52] or by transverse aortic coarctation [53, 54]. The results indicate that hypertension is associated with changes in hippocampal expression of APP binding proteins, Aβ and tau aggregation in the brain [51]. Although these experimental hypertensive models lead to interesting conclusions about the cognitive consequence of hypertension, they do not mimic factors, such as diet that may lead to hypertension in humans.

Results from models of experimental hypertension in mice that induced by Ang II and used to study Aβ concentration should be interpreted carefully. Because Ang II is not only involved in hypertension mechanisms but also involved in many other mechanisms that may impact the Aβ accumulation. For example, it is shown that Ang II impairs neurovascular coupling, and is of significant importance in neurodegenerative diseases [55, 56]. Thus, the Ang II model lacks specificity to be used as a model to investigate the effect of hypertension on cerebral hypoperfusion, and accumulation of Aβ in AD. Here a high-salt diet regimen implemented to induce experimental hypertension in mouse models of AD that mimics a human lifestyle with salty diet [31]. The implemented salty diet regimen successfully induced high MAP in control mice (Fig. 2). Therefore, this model has the potential to be used as an experimental model to investigate the effect of diets leading to hypertension.

APP/PS1 is a genetically modified trend designed to have Aβ accumulation in the brain. The genetic modifications may bear other defects such as impaired Ca²⁺ homeostasis. Excessive salt in the diet of this model may interfere with other genetically altered mechanisms that resulted in reduced Aβ load in comparison to controls. A high-salt diet regimen in this model may also interfere with salt-sensing mechanisms through different pathways. Therefore, we must be careful in interpreting the data on the effect of hypertension on cognitive decline. The best interpretation of the results of this study is that a high-salt diet regimen affects CBF and MAP in AD models different from controls.

The renin–aldosterone angiotensin system (RAAS) is a centrally active modifiable pathway that is involved in CBF autoregulation [57]. Ang II an element of RAAS is a key player in hypertension, impairs neurovascular coupling and has been found to be of significant importance in AD [55, 58]. Hypertension may alter RAAS system along other mechanisms that may influence the accumulation of Aβ [58]. Our finding is not incongruent with these studies on concluding that hypertension exacerbate AD pathology, rather our data could be used to conclude about the effect of salt, a comorbid of hypertension, on the case of excess Aβ without high blood pressure. Our experiment was designed to mimic a human lifestyle with a salty diet regimen and test if a high-salt diet regimen influences the accumulation of Aβ, and exacerbates cognitive decline. Regardless, the designed diet resulted in an overall increase in CBF and MAP in controls. A more interesting and striking difference occurred between peripheral blood pressure in APP/PS1 and controls. We did not find any difference between the heart rates of experimental groups.

Recent studies have shown that hypertension is associated with disrupted white matter integrity in humans [59 –61]. However, we did not find evidence of white matter integrity disruption in this study with mouse model of AD. Our acquired diffusion data by DTI do not support the hypothesis that AD or hypertension causes white matter integrity impairment. The most likely explanation for this discrepancy between our data and human data is the fact that those APP/PS1 models do not quietly represent all humans pathogenesis of AD. Also, those reported human data were mostly collected from the specific brain regions with high spatial resolution scanners. However, consistent with our findings, Holland et al. also showed that hypertension has minimal impact on white matter integrity in aged hypertensive rats fed a high-fat diet [62].

This study has several limitations. The lack of quantitative measurement of the food and water intake could be considered as a limitation to this study. The difference in the amount of food and water intake between controls and APP/PS1 group may cause the salt intake to be different between the groups. However, we monitored the body weight of all experimental groups weekly, especially during the special diet regimen. We did not find any significant differences between the groups’ body weights (data not shown). Here, we considered the body weight change as a surrogate measure of food intake. However, this assumption may not completely address the uncertainty in the exact amount of salt each mouse received. Another limitation is the lack of data on the effects of different concentrations of salt intake on Aβ load. In the present study, only one concentration of salt intake was investigated. This concentration was selected as a compromise between hypertension and mice survival. For future studies, we suggest investigating the effects of different concentrations of salt in the diet on the reduction of Aβ plaques in AD.

Despite these limitations, our data suggest that a salty diet regimen has different effects on APP/PS1 mouse models of AD compared to littermate controls. The effect of a salty diet regimen on CBF in relation to the pathophysiology of AD is important and merits further investigation with multiple interdisciplinary techniques. Also it would be valuable to investigate if a salty diet regimen delays the onset of AD. Moreover, studies that include behavioral pharmacology and dose-response curves on CBF changes will bring in details on CBF autoregulation mechanism in AD. Molecular, immediate early gene expression also will enhance our understanding of the neurobiological consequences of a high-salt diet on Aβ clearance from the brain.

Footnotes

ACKNOWLEDGMENTS

The study was supported by a grant from the Alzheimer’s Association (NIRG-12-242467) to S.T.

Authors’ disclosures available online ().

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