mTOR drives cerebral blood flow and memory deficits in LDLR −/− mice modeling atherosclerosis and vascular cognitive impairment

Animal, diets and experimental design

All studies were performed under approval of the UTHSCSA Institutional Animal Care and Use Committee (Animal Welfare Assurance Number: A3345-01) – which adheres to the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, the Public Health Service Policy on Humane Care and Use of Laboratory Animals, and the US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training – and also in compliance with the ARRIVE guidelines (Animal Research: Reporting In Vivo Experiments) for reporting animal experiments. Group size calculations for in vivo experiments were based on MWM because it has the highest variability. We estimated group sizes of n = 12 as sufficient to provide .85 power to detect an effect f = 0.4 with overall SD = 15% and alpha = 0.05 in RM-ANOVA). SAA and IL-6 were measured in serum and brain of the same animals that were tested in the MWM, respectively. CBF studies utilized animals that were not included in MWM and had been generated to be matched for age with those in MWM studies. From our prior studies, ²⁰ we determined that five mice per group would provide .8 power to detect an effect f = 0.9 with overall SD = 7% and alpha = 0.05 in ANOVA. We verified that our MWM and CBF studies were adequately powered (n = 4 to have .85 power to detect an effect f = 1.15 with overall SD = 10.4% and alpha = 0.05 in ANOVA, and n = 9 to have 0.84 power to detect an effect f = 1.36 with overall SD = 9.5% and alpha = 0.05 in RM-ANOVA for MWM and CBF studies respectively). Studies of brain vascular density (BVD) using n = 4 provided .85 power to detect an effect f = 1.14 with overall SD = 6.33% at alpha = 0.5 in ANOVA. HFD-fed LDLR^−/− mice showed accelerated weight loss and high mortality as a result of intraperitoneal injection of rapamycin; thus, this group was removed from ANOVA of CBF and VD data.

Tissues from animals that were used in CBF experiments were not utilized for any other determinations. For all studies, treatment groups were randomized by parentage. We minimized potential misidentification of animals in behavioral and functional imaging studies by using unique animal identifiers that were permanently tattooed on the tail.

Aortic arch lesion area studies used estimated group sizes of n = 6 as sufficient to provide .95 power to detect an effect f = 0.5 with overall SD = 5% and alpha = 0.05 in 2W-ANOVA. For comparisons of two independent means group sizes using t tests, we estimated group sizes of n = 6 as sufficient to provide 0.8 power to detect an effect d = 1.8 with overall SD = 5% and alpha = 0.05 in two-tailed Student’s t test. For ventricle mass studies, we estimated group sizes of n = 8 as sufficient to provide 0.8 power to detect an effect f = 0.38 with overall SD = 8% and alpha = 0.05 in two-way ANOVA.

Personnel performing experiments identified animals only by the animal identifier and were blinded from genotype and treatment assignment. Male and female LDLR^−/− (B6.129S7-Ldlr^tmlHer/J, Jackson Laboratories, Bar Harbor, ME) or C57Bl/6 (hereafter referred to as ‘WT’, Jackson Laboratories, Bar Harbor, ME) mice were housed ≤5/cage and maintained on a 12-h light/12-h dark cycle. WT and LDLR^−/− mice were fed maintenance (AIN-76A, BioServ) diet, while additional LDLR^−/− mice were fed high fat diet (HFD; 21% saturated milk fat, 0.2% cholesterol, supplemented into AIN-76A, BioServ). Encapsulated rapamycin (14 ppm) prepared as described ²¹ was supplemented into HFD. Control HF diet was also prepared which contained eudragit only (HF Eud); Eudragit is the encapsulation material for rapamycin that aids in enteric drug absorption. Animals were euthanized by isoflurane overdose followed by cervical dislocation or by CO₂ overdose followed by cervical dislocation. Brain was dissected within ∼1.5–2 min of death. Blood was collected post-mortem from heart.

Animal cohorts

Cohort 1: mice of both sexes were treated at 8–10 weeks of age for 10, 20 or 30 weeks, corresponding to expected aortic lesion states I and II, I, II and III and I, II, III and IV. Cohort 2: males aged to 28 weeks were treated intraperitoneally (IP) with either rapamycin (10 mg/kg) or vehicle every other day for 16 weeks, at which time MRI studies to investigate CBF and BVD were performed. IP administration of rapamycin was utilized for these studies due to temporary unavailability of supplemented chow due to licensing of the formulation; IP delivery of rapamycin has been shown to be equivalent to enteric delivery via chow supplementation. ²² Cohort 3: a crossover design in which mice were fed a HFD +/− rapamycin for 15 weeks starting at 20 weeks of age, then switched to the opposite diet for 15 weeks. Cohort 4: an all-male group fed maintenance or HFD +/− rapamycin beginning at 28 weeks of age for 20 weeks. Only males were used to avoid potential confounds related to variability in female performance in the Morris water maze (MWM).^23,24 All behavioral testing was performed at 12 months of age. Prior studies reported cognitive deficits in LDLR^−/− mice at seven to eight months of age ²⁵ ; since we aimed to model treatment and not prevention of these deficits, Cohort 2 and 4 studies were designed accordingly.

Note: The LDLR^−/− model of atherosclerosis relies on the use of HFD. In an effort to remain consistent with prior knowledge in the field, we designed our experiments such that the efficacy of mTOR inhibition by rapamycin was tested in the best-established LDLR^−/− model of atherosclerosis, using HFD-fed LDL mice treated with Eudragit-supplemented or rapamycin-supplemented chow. Due to time limitations, a maximum of 60 animals can be tested in the Morris water maze (MWM) in a single study. Accordingly, maintenance chow-fed LDLR^−/−, rapamycin-fed groups were excluded for all experiments with the exception of Cohort 2 (MRI-based studies, Figure 4). The inclusion of maintenance chow-fed LDLR^−/− rapamycin-fed groups in MRI studies was possible in MRI studies because sample sizes adequate to identify differences in group means for those measures were smaller than those calculated for MWM studies.

Blood analysis

Mice were fasted overnight and sacrificed with blood collection via cardiac puncture. Rapamycin levels in whole blood were determined at 30 weeks by LC/MS. Remaining blood was centrifuged and plasma samples were collected and analyzed for total cholesterol (Total Cholesterol E Kit, Wako Chemicals, Richmond, VA) and trigylycerides (L-type Triglycerides M, Wako Chemicals, Richmond, VA).

Fasting glucose

Mice were fasted overnight for 16 h. Blood was collected from the tail vein and tested using a handheld glucose monitor (LifeScan One Touch Ultra).

Measurement of rapamycin (RAPA) using HPLC-tandem MS

The HPLC system consisted of a Shimadzu SCL-10A Controller, LC-10AD pump with a FCV-10AL mixing chamber, SIL-10AD autosampler, and an AB Sciex API 3200 tandem mass spectrometer with turbo ion spray. The analytical column was a Grace Alltima C18 (4.6 × 150 mm, 5 µ; Alltech (Deerfield, IL)) and was maintained at 60℃ during the chromatographic runs in a Shimadzu CTO-10A column oven. Additional details are available in the supplementary materials.

Measurement of markers of inflammation

Serum amyloid A (SAA) was measured in serum using an SAA ELISA Kit (Invitrogen). Briefly, samples were diluted in diluent buffer and added to microplate wells in duplicate. An equivalent volume of anti-SAA HRP was added and the plate was covered and incubated for 1 h at 37℃. Wells were then gently aspirated, washed 4× with wash buffer, and substrate was added to each well. The plate was covered and incubated for 15 min at RT. After addition of stop solution, absorbance at 450 nm was measured using 630 nm as a reference.

Total RNA extraction

RNA extracts were prepared from microvessel-depleted brain samples using the RNAqueous-4PCR Kit (Ambion) under RNase-free conditions following the manufacturer’s protocol. Additional details of this method can be found in the supplementary materials.

cDNA synthesis and real-time quantitative PCR

cDNA synthesis and real-time quantitative PCR were performed as described previously. ²⁶ Details of this method can be found in the supplementary materials.

Preparation of brain vascular lysates

Brain vascular lysates were prepared as described previously. ²⁰ Details of this method can be found in the supplementary material.

Immunoblotting

Samples were resolved by SDS-PAGE (Bio-Rad, 10% Mini-PROTEAN TGX) and electroblotted onto nitrocellulose membrane (GE Healthcare). Additional details are provided in the supplementary material.

Antibodies

The following primary antibodies were used for immunoblotting: phospho-rpS6 (Cell Signaling, 1:1000); rpS6 (Cell Signaling, 1:1000); Actin (UBP Bio, 1:5000). The following secondary antibodies were used where appropriate: Goat anti-Mouse IgG Poly HRP (Pierce, 1:20,000); Goat-anti-Rabbit IgG Poly HRP (Pierce, 1:20,000).

Analysis of atherosclerosis

Atherosclerosis was analyzed as previously described.^27,28 Further detail is available in the supplementary material.

Non-invasive measurement of cardiopulmonary parameters

Mice were temporally anesthetized (1–4% inhaled isoflurane), and left ventricle hypertrophy was measured using the VEVO 2100 digital ultrasound microimaging system (VisualSonics) as previously described. ²⁹

Morris water maze

The Morris water maze ³⁰ was used to test spatial memory as described. ²⁰ Further detail is available in the supplementary material.

Spatial novelty

Mice were trained with three visually distinct but comparably sized objects. During testing, one object was moved to a new location within the chamber and exploration was monitored. Additional details of this task are available in the supplementary material.

Cerebral blood flow measurements

Quantitative CBF (mL/g/minute) was measured using MRI-based continuous arterial spin labeling techniques ³¹ as previously described ²⁰ Briefly, measurements utilized a horizontal 7T/30 cm magnet and a 40 G/cm BGA12S gradient insert (Bruker, Billerica, MA, USA). A small circular surface coil (ID = 1.1 cm) was placed on top of the head and a circular labeling coil (ID = 0.8 cm), built into the cradle, was placed at the heart position for continuous arterial spin labeling. The two coils were positioned parallel to each other, separated by 2 cm from center to center and were actively decoupled. Paired images were acquired in an interleaved fashion with field of view = 12.8 ×12.8 mm², matrix = 128 × 128, slice thickness = 1 mm, 9 slices, labeling duration = 2100 ms, TR = 3000 ms, and TE = 20 ms. Continuous arterial spin labeling image analysis employed codes written in Matlab (Natick, MA, USA) ³¹ and STIMULATE software (University of Minnesota, Minneapolis, MN, USA) to obtain CBF.

Vascular density measurement

Magnetic resonance angiography was acquired as previously described. ²⁰ Briefly, three-dimensional gradient-echo MR imaging with flow compensation was used with following parameters: repetition time/echo time = 26/4.3 milliseconds; field of view = 12.8 × 12.8mm²; acquisition matrix = 128 × 128 × 192; repetition n = 4. The magnetic resonance angiography was taken before and after monocrystalline iron oxide nanoparticle (MION) injection through the tail vein (30 mg/kg) and blood vessel intensity was calculated as the change in transverse relaxivity between images. Image analysis was performed using custom-written programs (Matlab; MathWorks, Natick, MA, USA). “S-1/3” indicates the overall MRI signal as calculated by the equation ΔR2/(ΔR2*)^2/3 as described. ³²

Statistical analyses

Statistical analyses were performed using GraphPad Prism (GraphPad, San Diego, CA, USA) and included two-way ANOVA, one-way ANOVA, or Student’s t test as appropriate, as denoted in the figure legends. All data are means ± SEM with p < 0.05 considered significant.

N.B. Additional methods and details of those listed above can be found in the supplementary material.

Reduced phosphorylation of mTOR targets in rapamycin-treated LDLR-/- mice

To determine cardiovascular and cerebrovascular consequences of mTOR attenuation in LDLR^−/−mice, we generated four experimental cohorts (Table 1 and Methods).

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Table 1.

Animal cohorts.

	Age at start	Route and dose	Duration	Sex	Rapamycin blood levels	Measures
Cohort 1	2–2.5 months	Enteric (chow) 14 ppm	10, 20 or 30 weeks	Male and female	1.62 ng/ml (F) 0.78 ng/ml (M)	Plaque area (IHC) Ventricle mass
Cohort 2	7 months	Intraperitoneal (10 mg/kg)	16 weeks	Male	Not measured (liver rapamycin levels of 14.1 pg/mg equivalent to 3–4 ng/ml in blood)	CBF and BVD (MRI)
Cohort 3	5 months	Enteric (chow) 14 ppm	15 weeks crossover	Male and female	Not measured	Plaque area (IHC)
Cohort 4	7 months	Enteric (chow) 14 ppm	16 weeks	Male	3–4 ng/ml	Memory (NOR, MWM)

Analysis of Cohort 1 samples demonstrated no significant differences in uncoagulated blood rapamycin levels in females and males (1.62 ± 0.48 ng/ml for females and 0.78 ± 0.23 ng/ml for males at 30 weeks of treatment, p = 0.137, Figure 1(a)). To determine whether rapamycin treatment attenuated mTOR activity in our studies, we measured rpS6 phosphorylation, a downstream target of mTOR signaling whose phosphorylation is reduced by rapamycin treatment in mouse visceral adipose tissue, heart, and brain.^21,33–35 In agreement with previous studies,^21,33,36 phosphorylation of rpS6 was significantly decreased in aortas of Cohort 1 LDLR^−/− mice following 20 weeks of rapamycin treatment as compared to control-treated animals (Figure 1(b)). Similarly, phosphorylation of rpS6 was significantly decreased in brain vasculature of rapamycin-treated HFD-fed LDLR^−/− animals in Cohort 4 (Figure 1(c)). No differences in mTORC2 signaling measured as phosphorylation of Akt at Ser473 were observed between rapamycin- and control-treated animals (Supplementary Figure 1). OPEN IN VIEWER

It has been shown that rapamycin does not accumulate in liver, and that liver levels of rapamycin represent an effective proximal measure of blood levels of the drug. ³⁷ Indeed, chronic rapamycin-supplemented HFD-feeding of Cohort 4 animals resulted in average liver rapamycin levels of 14.1 pg/mg (Figure 1(d)), which were comparable to rapamycin levels in livers of male C57BL/6 mice treated with the same chow as reported,^33,38 and correspond to 3–4 ng/ml in blood. ³⁸ Cohorts 2 and 3 mice were not tested as these groups underwent comparable treatment to Cohort 4 mice in regards to treatment onset and duration.

Chronic mTOR attenuation decreases body weight and fat mass in aged HFD-fed LDLR−/− mice

Female, but not male HFD-fed LDLR^−/− mice of Cohort 1 exhibited significantly lower body weight following chronic mTOR attenuation with rapamycin (Figure 2(a)) when compared to LDLR^−/− mice fed HFD containing only eudragit, the encapsulation material for rapamycin. Both sexes showed a significant elevation in plasma triglycerides (Figure 2(a)), despite no differences in blood glucose (Figure 2(f)) nor cholesterol levels (Figure 2(g)). In spite of the observed elevation in plasma triglyceride levels, percent fat mass decreased in rapamycin-treated mice in this cohort (Figure 2(b)). Similarly, chronic mTOR attenuation in Cohort 4 mice lowered both overall body weight (Figure 2(c)) and percent fat mass (Figure 2(d)). As expected, weight gain was significantly increased in HFD-fed LDLR^−/− mice of Cohort 4 as compared to animals on maintenance diet, with the most dramatic effects seen in the first eight weeks before reaching a stable range of body weight. Interestingly, this HFD-mediated weight gain was significantly attenuated in rapamycin-treated LDLR^−/− mice (Figure 2(c)), suggesting that systemic attenuation of mTOR activity may inhibit fat accumulation in LDLR^−/− mice either by changes in fat metabolism or storage. OPEN IN VIEWER

To determine whether chronic mTOR attenuation may affect body mass composition, we performed quantitative magnetic resonance on Cohort 4 mice at 12 months of age. Whereas percent fat mass in maintenance diet-fed LDLR^−/− mice (∼25–30%) did not differ from WT, percent body fat was increased to ∼45% in HFD-fed LDLR^−/− mice (Figure 2(d)). Notably, percent body fat was significantly lower in LDLR^−/− mice that were fed HFD supplemented with rapamycin (Figure 2(d)). With the exception of a transient initial spike in the HFD-fed groups in Cohort 4, food consumption was not significantly different amongst experimental animals regardless of diet or treatment (Figure 2(e)). Despite their significant weight gain, however, HFD-fed mice of Cohort 4, similar to those in Cohort 1 (Figure 2(a)), did not exhibit any difference in fasting glucose levels compared to either maintenance diet-fed or WT mice (Figure 2(f)) and were therefore not insulin deficient.

Chronic attenuation of mTOR does not affect hypercholesterolemia in LDLR−/− mice

LDLR^−/− animals fed a HFD develop hypercholesterolemia. ³⁹ Given the established effects of mTOR attenuation with rapamycin on glucose and lipid metabolism^40,41 and prior studies suggesting that rapamycin administration results in hypercholesterolemia, ⁴² we investigated the effects of rapamycin treatment on circulating cholesterol levels in HFD-fed LDLR^−/− mice. Since LDLR is responsible for clearing LDL cholesterol from the periphery, it was not surprising that LDL cholesterol levels were significantly higher in LDLR^−/− mice compared to WT. Notably, serum LDL cholesterol levels were comparable between all LDLR^−/− mice regardless of diet or treatment (Figure 2(g)), suggesting that HFD does not confer any further cholesterol burden at this age. Similarly, rapamycin treatment had no effect on cholesterol levels in WT C57BL/6 J.

Chronic mTOR attenuation reduces atherosclerotic lesion area in the aortic arch

Previous studies^18,43 have suggested that mTOR has a role in atherogenesis. To determine whether chronic mTOR attenuation affected the development of atherosclerotic lesions in HFD-fed LDLR^−/− groups, we measured aortic arch lesion area using both a time-course (Cohort 1) and a crossover design (Cohort 3, Methods and Table 1). Aortic arch lesion area in HFD-fed LDLR^−/− mice was significantly reduced 20 and 30 weeks after initiation of rapamycin treatment in both male and female mice of Cohort 1 (Figure 3(a)). OPEN IN VIEWER

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Figure 4.

Restored cerebral blood flow and preserved brain vascular density by mTOR attenuation in LDLR^−/− mice. (a) Profound CBF deficits in 12 month old LDLR^−/− mice were restored to levels indistinguishable from those of WT mice as a result of chronic rapamycin treatment (F(2,7) = 21.22, p = 0.001, Tukey’s test, one-way ANOVA); (b–c) Reduced BVD (calculated as S = ΔR2/(ΔR2*)^2/3) in control-treated but not in rapamycin-treated LDLR^−/− mice (F(2,7) = 41.48, p = 0.0001, Tukey’s test, one-way ANOVA; (b) Representative MRI angiograms; (c) Quantitative analyses of angiogram data. n = 3–4. Data are means ± SEM. N.B. Data for the single surviving HFD-fed rapamycin-treated LDLR^−/− animal are provided (a,c) but were not included in the ANOVA. Values for WT CBF (a) and BVD (c) are included as a reference and correspond to measures performed on separate groups of WT C57Bl/6 J mice of comparable age; these values were not included in the ANOVA.

Interestingly, lesion areas in the aortic root were reduced only in female mice of Cohort 3 that had been switched from control to rapamycin diet (Figure 3(b)), suggesting that mTOR has a role in development, but not in the initiation, of plaque deposition during atherogenesis in female mice. Notably, left ventricle hypertrophy was significantly reduced in females, but not in males (data not shown) after 30 weeks of rapamycin treatment (Figure 3(c)). Measured ejection fraction values were indistinguishable from WT for both male and female animals (data not shown), though the sample size of these studies was likely too small to detect differences in these in vivo measures of cardiac function.

Cerebrovascular rarefaction and cerebral hypoperfusion are reduced by mTOR attenuation

We used arterial spin labeling magnetic resonance imaging to measure CBF and MRI-based angiography to estimate BVD. CBF was significantly decreased in LDLR^−/− mice; this deficit was exacerbated by HFD (Figure 4(a)). Because relatively small groups provide sufficient statistical power in MRI studies (see Methods), we were able to include a maintenance diet-fed LDLR^−/− group that was treated with rapamycin in these experiments. Notably, chronic mTOR attenuation by intraperitoneal rapamycin administration restored CBF in maintenance diet-fed LDLR^−/− mice to levels comparable to WT mice, suggesting that mTOR has a critical mechanistic role in brain hypoperfusion in LDLR^−/− animals. The consequences of mTOR attenuation by rapamycin on CBF in HFD-fed LDLR^−/− mice could not be unequivocally determined due to accelerated weight loss and high mortality in this group, in agreement with the observed rapamycin-mediated reduction in body weight in HFD-fed LDLR^−/− (Figure 2(c)). Of note, and although this observation can only be considered anecdotal, a single surviving rapamycin-treated HFD-fed LDLR^−/− animal (n = 1) exhibited CBF levels indistinguishable from those of WT control mice (Figure 4(a)). To determine whether differences in CBF between control- and rapamycin-treated LDLR^−/− mice could be explained by changes in BVD as previously shown for hAPP(J20) mice modeling AD, ²⁰ we measured BVD using MRI-based angiography. Indeed, we found decreased CBF in LDLR^−/− was accompanied by proportionate differences in BVD (Figure 4(b) and (c)), suggesting that the preservation of BVD may underlie the restoration of CBF in rapamycin-treated LDLR^−/− mice.

Chronic mTOR attenuation improves memory in LDLR−/− mice

Decreased CBF is associated with cognitive dysfunction.^13,44,45 To determine whether decreased CBF resulted in impaired cognitive function in LDLR^−/− mice and ascertain the role of mTOR, we next assessed recognition memory in LDLR^−/− mice of Cohort 4 using the spatial novelty (SN) paradigm, a simple discrimination task which requires that the animals retain the prior locations of objects presented in the experimental arena and thus recognize novel object placement. All animals explored the three objects presented during training equally (Figure 5(a)), ruling out potential confounds arising from aversion/preference for a particular object or location. During testing, WT and maintenance diet-fed LDLR^−/− animals preferentially explored the object that had been moved (Figure 5(b)), indicating retention of the object’s prior location. HFD-fed LDLR^−/− mice, however, spent an equivalent percent of trial time examining each of the three objects (Figure 5(b)), indicating a deficit in retention of the prior location of the displaced object. Notably, rapamycin-treated HFD-fed LDLR^−/− mice performed indistinguishably from WT mice, suggesting that attenuation of mTOR activity restores hippocampus-dependent recognition memory in HFD-fed LDLR^−/− mice. OPEN IN VIEWER

To rule out potential task-specific effects of mTOR attenuation by rapamycin and define whether the observed improvement in SN recognition in rapamycin-treated HFD-fed LDLR^−/− mice was associated with improvements of other aspects of context-related learning and memory, we used the Morris water maze (MWM). MWM design limits the number of animals that can be included in an experiment to 60 due to time constraints inherent with keeping time-at-testing as constant as possible. For our studies, n = 60 corresponded to 4 groups of 15 animals each to have sufficient power to detect meaningful differences between group means (Methods). Because HFD-fed LDLR^−/− are the established model of atherosclerosis, we chose the four groups to be included in these studies to be (1) maintenance-fed WT controls; (2) maintenance-fed LDLR^−/− mice; (3) control-treated (eudragit) HFD-fed LDLR^−/− and (4) rapamycin-treated, HFD-fed LDLR^−/− mice in order to identify effects of mTOR attenuation on cognitive and brain vascular functional outcomes in HFD-fed LDLR^−/− mice.

This behavioral task requires intact hippocampal function similarly to SN, but requires processing of more complex sets of spatial information to learn a specific location. As expected, spatial learning in the MWM progressively improved across four days of training for WT mice (Figure 5(c)). Performance of both HFD and maintenance diet-fed LDLR^−/− mice, however, was significantly impaired on training days 2 and 3 (Figure 5(c)). Increased latencies in LDLR^−/− mice fed a maintenance diet suggested delayed spatial learning in this group, a finding in contrast to their ability to retain simple contextual information (Figure 5(b)).

Distance swam can provide a better measure of performance when swimming speeds are significantly different amongst groups. We report no differences between experimental groups for distance swam (Figure 5(d)), but because reduced swimming speed of HFD-fed LDLR^−/− during training (Figure 5(e)) was a consequence of increased floating (Figure 5(f)), conclusions on spatial learning could not be unequivocally drawn for this group. Performance of rapamycin-treated HFD-fed LDLR^−/− mice, in contrast, was not confounded by decreases in swimming speed nor floating (Figure 5(e) and (f)) and was indistinguishable from that of WT control mice (Figure 5(c)), suggesting that attenuation of mTOR activity abrogates spatial learning defects in HFD-fed LDLR^−/− mice. All experimental groups performed comparably on day 4 of training, indicating that spatial learning is impaired but not abolished in LDLR^−/− mice. Furthermore, HFD-fed LDLR^−/− control (eudragit) mice exhibited a statistically significant deficit in percent time spent in the target quadrant during the probe trial (Figure 5(g)) compared to all other groups, whereas rapamycin-treated subjects were indistinguishable from the WT group. All experimental animals were confirmed to possess comparable motor and neurological competency prior to behavioral testing. Percent time engaged in thigmotaxis could not be used as a proximal measure of evoked anxiety because of increased floating, which confounds this measure.

mTOR attenuation suppresses markers of brain and systemic inflammation in LDLR-/- mice

Because a high systemic inflammatory state has been implicated in the pathogenesis of atherosclerosis in LDLR^−/− mice ⁴⁶ as well as in the etiology of cognitive impairment in vascular dementia, ⁴⁷ we investigated whether improved cardiovascular and neurovascular measures in LDLR^−/− mice as a result of mTOR attenuation with rapamycin were associated with reduced brain and systemic inflammation. To test this hypothesis, we measured levels of the pro-inflammatory cytokine IL-6 and of serum amyloid A (SAA) as markers of inflammation in brain and in serum, respectively, of Cohort 4 LDLR^−/− mice. Rapamycin treatment conferred a notable reduction in whole brain levels of IL-6 in HFD-fed LDLR^−/− mice, though this trend did not reach statistical significance due to high variability in the control-treated group (Figure 6(a)). Of note, variance was significantly lower in the rapamycin-treated group, suggesting that attenuation of mTOR may exert a regulatory effect over brain IL-6 levels that our studies were not sufficiently powered to detect. OPEN IN VIEWER

We next examined serum levels of SAA, a family of apolipoproteins secreted during acute inflammation, as a measure of systemic inflammatory state in all experimental groups. SAA was significantly elevated in sera of HFD-fed LDLR^−/− mice as compared to either maintenance diet-fed LDLR^−/− mice or WT control animals (Figure 6(b)). Rapamycin treatment, in contrast, conferred a significant reduction in SAA in HFD-fed LDLR^−/− mice.