Omega‐3 fatty acids are associated with blood–brain barrier integrity in a healthy aging population

Abstract In aging populations, omega‐3 polyunsaturated fatty acids (PUFAs) have been associated with better cognitive function, slower rates of cognitive decline, and lower risk of developing dementia. Animal studies have shown that diets rich in omega‐3 PUFAs reduce blood–brain barrier (BBB) disruption associated with aging, but this has yet to be observed in humans. Forty‐five healthy subjects (mean age, 76 years) were recruited and underwent cognitive assessment (verbal learning and memory, language, processing speed, executive function, and motor control) and measurement of PUFAs. Forty of the same subjects also underwent magnetic resonance imaging (MRI) to measure BBB integrity (K trans using dynamic contrast‐enhanced MRI). The long chain omega‐3 score (DHA+EPA) was negatively correlated with K trans values in the internal capsule, indicating higher omega‐3 levels were associated with greater BBB integrity in this region (r = –0.525, p = .004). Trends were observed for a positive correlation between the long chain omega‐3 score and both memory and language scores, but not with executive function, speed, or motor control. The omega‐6 score was not significantly correlated with any cognitive scores or K trans values. The significant correlations between long chain omega‐3 levels and BBB integrity provide a possible mechanism by which omega‐3 PUFAs are associated with brain health.


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, and an overall lower risk of developing dementia (Barberger-Gateau et al., 2007;Morris et al., 2003). Neuroanatomically, higher omega-3 PUFA intake has been associated cross-sectionally with larger gray matter volume in healthy older adults (Conklin et al., 2007;Tan et al., 2012;Titova et al., 2013) and longitudinally with lower rates of atrophy in the hippocampus and amygdala in communitydwelling adults over the age of 65 (Samieri et al., 2012). However, the basis for these relationships is not completely understood in humans.
Research on animals has been highly suggestive that diet impacts cognition through maintenance of the blood-brain barrier (BBB) (Davidson et al., 2012;Kanoski et al., 2010;Sparks et al., 2000;Takechi et al., 2013). Animal models have shown that diets rich in omega-3 PUFAs reduce BBB disruption associated with aging (Kuo et al., 2010) and poor diet (Takechi et al., 2013). The BBB has long been implicated in neurodegeneration and AD Zlokovic, 2011 ), and omega-3 PUFA supplementation in murine models decreased the amount of Aβ and neuronal loss in the brain (Hooijmans et al., 2012).
Existing studies of the BBB in humans  have almost entirely relied on either examinations of the cerebral spinal fluid (CSF), which gives no spatial information about where leakage occurs and requires an invasive lumbar puncture, or positron emission tomography (PET) and computed tomography (CT) exams, which utilize harmful radiation. Previous magnetic resonance imaging (MRI) studies of the BBB have had reduced sensitivity due to limited data acquisition and postprocessing techniques (Starr et al., 2009;Su et al., 1998;Wang et al., 2006). More sophisticated dynamic contrast enhanced (DCE) MR techniques have recently been developed, which allow accurate detection of subtle changes in BBB integrity Cramer et al., 2015;Montagne et al., 2015;Taheri et al., 2011). These new advances allow cognitive decline and BBB integrity to be carefully and noninvasively examined in humans. They have also provided evidence to suggest that reduced BBB integrity is a key mechanism early in cognitive decline and AD (Huisa et al., 2015;Montagne et al., 2015;Nation et al., 2019). Specifically, studies have shown a reduction of BBB integrity in the hippocampus to be associated with aging and additional loss of BBB integrity in individuals with mild cognitive impairment, a stage preceding dementia, compared to cognitively intact age-matched controls . However, whether specific dietary factors, including omega-3 PUFAs, are associated with preserving BBB integrity has not yet been investigated in humans.
This study explores the relationship among omega-3 PUFAs, cognitive function, and BBB integrity in a healthy elderly adult population, thereby secondarily considering the utility of measuring BBB integrity with MRI as an early, noninvasive, and sensitive biomarker of cognitive impairment risk. We hypothesize that higher levels of omega-3 PUFAs will be inversely associated with K trans levels measured using DCE MRI, an indicator of BBB integrity, which may help to preserve cognitive function in aging. We preliminarily investigate whether associations are specific to brain regions for which BBB changes could affect cognition.

Participants
The Adventist Health Study-2 (AHS-2) is a prospective cohort study  (Butler et al., 2008). The cohort is healthy: at baseline, high proportions reported being in excellent health. Forty-five percent of cohort members follow vegetarian diets (Rizzo et al., 2013) and nonvegetarians consume lower amounts of meat compared to the general US population (Rizzo et al., 2013;Tantamango-Bartley et al., 2013). Note that 1.1% are current smokers and 6.6% drink alcohol (Butler et al., 2008).
Since the cohort has aged, and the majority of the cohort is elderly, the AHS-2 presents an opportunity to study age-related chronic diseases.
In 2016, we identified 2685 members of the cohort for whom study records indicated were 60 years or older, community-dwelling and living within 75 miles of Loma Linda University (LLU). During [2016][2017][2018]199 were reached by telephone and invited to participate in the AHS-2 Cognitive and Neuroimaging (AHS2-CAN) substudy (Gatto et al., 2020). Of those, 168 (84%) agreed to participate and were screened for eligibility. Participants were excluded if they did not understand and speak English proficiently or had any acute medical conditions that could adversely impact cognitive function. One hundred and thirty-two otherwise healthy adults were enrolled in the AHS2-CAN study and completed the baseline study procedures, which included cognitive and physical assessments. Approximately 1 year later, participants were invited to return for a follow-up cognitive assessment and brain MRI scan. Additional exclusions were made for a diagnosed neurological condition, such as Parkinson's disease, epilepsy, or multiple sclerosis; a history of brain tumor, stroke, or other focal brain injury or head trauma; any acute medical condition, such as infections, nutritional deficiencies, or adverse drug reactions; a pacemaker or other implanted device; a history of kidney disease or diabetes; or claustrophobia. Forty-five AHS2-CAN study participants completed repeated cognitive assessment; 40 of those also completed the MRI scan. Of the five participants who did not complete the MRI, one had metal in his/her body, one was unable to lie flat due to back problems, and three declined due to symptoms of claustrophobia or concerns related to the contrast agent.

Neuroimaging
The MRI was collected on a 3T (Siemens Medical Systems, Erlangen, Germany) scanner using a 32-channel array head coil. Total imag-

Fatty acids
A drop of blood was collected on filter paper that was pre-treated with an antioxidant cocktail (Fatty Acid Preservative Solution, FAPS) and allowed to dry at room temperature for 15 min.

Image processing
FLAIR images were used to determine the volume of white matter lesions. Lesions were segmented by the lesion prediction algorithm (Schmidt, 2017) as implemented in the LST toolbox version 3.0.0 (www. statistical-modelling.de/lst.html) for SPM.
DCE data were processed with the software package ROCK-ETSHIP . The dynamic DCE series was motion corrected using AFNI 3dvolreg. First, brain extraction was performed using the automated brain extration tool HD-BET (Isensee et al., 2019). AFNI 3dvolreg was applied using a brain mask excluding the scalp which moves independently from the brain; heptic interpolation was utilized to minimize Gibbs ringing. An arterial input function (AIF) was selected from the jugular veins.

Statistics
Age-and sex-adjusted Pearson correlation coefficients were calculated between fatty acid scores and the composite cognitive scores.

RESULTS
The mean age of participants (n = 45) was 76.8 years (SD = 8.6, min age = 63 years, max = 97 years). Participants were predominantly white, followed vegetarian diets, and were well educated. Their esti-  (Table 1). There were no substantial differences in demographic factors between participants who participated in the MRI portion of the study (n = 40) and those who did not (Table 2).
Although we found no significant associations between omega-3 scores and cognition, the long chain omega-3 score was positively correlated with memory ( Figure 1, r = 0.292; p = .058) and language (r = 0.267, p = .083), but did not reach statistical significance. Processing speed, executive function, and motor control did not show an association with omega-3 (Table 3). The omega-6 index was not statistically significantly correlated with any cognitive measures (Table 3).
The long chain omega-3 score was negatively correlated with median K trans values in the internal capsule (Figure 2, r = −0.3, p = .004).
Correlations between the omega-3 and the omega-6 scores and other Abbreviation: WML, white matter lesion. ** bold p < 0.005 brain regions did not achieve statistical significance (Table 4). Post hoc analyses found that the correlation in the internal capsule was driven by a correlation with EPA (Table S1, r = −0.542, p = .003) and significantly correlated with DHA (r = −0.471, p = .01). K trans values in the superior corona radiata were also significantly correlated with EPA (r = −0.372, p = .036).
Median K trans values in the superior corona radiata were statistically significantly correlated with FTT scores (Figure 3, r = −0.43, p = .015).
Median K trans values in the hippocampus, white matter, corpus callosum, thalamus, caudate nucleus, and internal capsule were not statistically significantly correlated with scores of memory, language, executive function, and speed (Table 5).
Of the ROIs we attempted to analyze, 13% were excluded as the K trans values did not converge on a positive value. This may be due to excessive noise or motion artifacts in that area of the image.
The K trans values which did not converge were distributed across participants. However, since this is a substantial number of samples to exclude, we also performed all statistical analyses considering those regions as K trans = 0. While this approach decreased the strength of the correlations, it did not change any of the statistically

DISCUSSION
Omega-3 PUFAs are essential components of cell membrane phospholipid bilayers and are also concentrated in synaptic membranes in the brain. They play an important role in brain physiology and biological activities, such as by increasing fluidity of neuronal membranes, modifying endothelial function and cerebral blood flow, synthesizing anti-inflammatory mediators, and slowing degradation of nerve tissues (O' Donovan et al., 2019). High levels of omega-3 PUFAs in the blood have also been associated with reduced cognitive decline in normal aging (Beydoun et al., 2007). Studies have shown a beneficial effect of omega-3 PUFAs in neurological disorders due to anti-inflammatory, anti-apoptotic, and neuroprotective properties (Arab, 2003;Brookmeyer et al., 2007;Campbell et al., 2019;Harris & Thomas, 2010;Lin et al., 2012;Tan et al., 2012;Tu et al., 2013).
In our study, the long chain omega-3 score was marginally associated with cognitive performance in the domains of memory and lan- . Furthermore, higher omega-3 levels have also been shown to reduce volume loss in the hippocampus, a region of the brain critical for learning and memory (Pottala et al., 2014). The relationship between omega-3 PUFAs and other cognitive domains, including speed and executive function, is less clear, with some studies reporting a positive association (Milte et al., 2011;Tan et al., 2012), while others report no association (Ammann et al., 2013;Sydenham et al., 2012 ) or a negative association (Danthiir et al., 2014). Consistent with this literature, we did not observe associations with cognitive performance in the areas of processing speed or executive function.
Studies on animals have shown that omega-3 PUFAs are important for preserving BBB integrity (Andreone et al., 2017). Our observation of an inverse correlation between long chain omega-3 PUFAs and This was a small exploratory study and thus did not find statistically significant correlations between fatty acids and neuropsychological measures or between K trans values, and some of the neuropsychological measures may have been a result of our being underpowered. Our cohort was exceptionally healthy with a relatively low rate of overall cognitive impairment and had a narrow range of variability in fatty acid values. In the general population, omega-3 scores of ≤4% are considered high vascular risk, 4%-8% intermediate risk, and >8% is considered low risk (Harris & Von Schacky, 2004).
Around 65% of the study cohort had omega-3 scores putting them in an intermediate vascular risk group and only 7% was in the high-risk group. Thus, a more heterogeneous sample with a greater number of participants with low omega-3 scores would be useful for future studies. With cross-sectional data, we cannot infer the directionality or temporality of the associations that we detected. It could be that declines in cognitive function precede BBB disruption, but we expect that reduction of BBB integrity as an underlying mechanism would be manifested through observable cognitive impairments (Huisa et al., 2015;Montagne et al., 2015). We adjusted correlations for age, sex, and white matter lesions, which does not rule out the potential that other factors such as education or comorbidities could confound the associations (Dias et al., 2014;Montagne et al., 2020). We also did not adjust correlations with omega-3 and omega-6 scores for other fatty acids to examine independent correlations. However, measurements of the individual fatty acids which contributed to these scores were expressed as a percentage of total fatty acids. Many of the variables we examined are not truly independent, so a Bonferroni correction for multiple testing was not done as this would be overly conservative, particularly given our limited sample size. Instead, we minimized the number of comparisons by reducing the number of variables examined in analyses using composite cognitive scores, omega-3 and 6 scores, and a limited number of brain regions for K trans values. A larger study which would allow more thorough analysis of individual cognitive tests, fatty acids, and a more diverse set of BBB measurements is merited.
In conclusion, we found significant correlations between long chain omega-3 levels and BBB integrity and cognition, providing evidence of a possible mechanism by which omega-3 may contribute to brain health.

ACKNOWLEDGMENTS
This work was supported by a GRASP award from Loma Linda University.