The worldwide expansion of aging populations has become a major public health challenge (Song and Chen, 2015). Increased lifespan has resulted in a steady rise of debilitating diseases related to aging, including neurodegenerative disorders, such as Alzheimer’s disease (AD), the most common type of dementia (Alzheimer’s Association, 2015), and age-related macular degeneration (AMD), the leading cause of visual loss and blindness worldwide (Lim et al., 2012). Unfortunately, to date there is no effective treatment for AD (Iqbal et al., 2014) or even mild cognitive impairment (MCI), its precursor stage (Cooper et al., 2013). Likewise, no effective preventive strategies exist for AMD, although nutritional and behavioral modifications can delay disease progression once initiated (Lim et al., 2012). In the continuum from normal cognition to MCI and dementia there is age-related cognitive decline, the onset and progression of which depends on a number of factors, including genetic variation, the level of education, environmental factors, particularly dietary habits, and the presence of cardiovascular risk factors or disease (Baumgart et al., 2015). Similar risk factors, albeit with a different genetic background, play a role in the pathophysiology of AMD (Lim et al., 2012). Optimal cognitive and visual functions are essential for quality of life and developing effective primary prevention strategies to reduce the economic and societal burden of such common age-related diseases would be of utmost public health importance.

Oxidative stress and inflammation are thought to play a pivotal role in precipitating neurodegenerative diseases, including AD (Schrag et al., 2013; Heneka et al., 2015) and AMD (Khandhadia and Lotery, 2010). Consequently, as derived from epidemiologic studies, a promising hypothesis in recent years has been that antioxidant-rich foods, such as fruit, vegetables, and particularly seeds and berries (Nooyens et al., 2011; Valls-Pedret et al., 2012; Ros and Hu, 2013; Barbour et al., 2014; O’Brien et al., 2014), and plant-based dietary patterns, especially the Mediterranean diet and the DASH (Dietary Approaches to Stop Hypertension) dietary pattern (Otaegui-Arrazola et al., 2014; Tangney et al., 2014) may protect from cognitive decline and AD. Nutrients such as n-3 polyunsaturated fatty acids (n-3 PUFA) and antioxidants, namely polyphenols, carotenoids and vitamins C and E, may have a role in preventing both cognitive impairment (Macready et al., 2009; Dangour et al., 2012; Valls-Pedret et al., 2012; Devore et al., 2013; Barnes et al., 2014; Otaegui-Arrazola et al., 2014) and macular degeneration (Age-Related Eye Disease Study Research Group, 2001; Parisi et al., 2008; Tan et al., 2009; Majumdar and Srirangam, 2010; Merle et al., 2013). Nuts in general and walnuts in particular have a rich matrix of these bioactive components and through additive effects have the potential to beneficially impact neuronal function in the brain and the retina (Poulose et al., 2014). Prospective studies have provided support for the association between nut consumption and improved cognitive performance (Valls-Pedret et al., 2012; O’Brien et al., 2014) and lower incidence of AMD (Tan et al., 2009; Amirul Islam et al., 2014). Among the different tree nuts, walnuts contain n-3 PUFA, specifically α-linolenic acid (ALA; C18:3n-3). ALA has long been believed to improve brain health indirectly via modest conversion to docosahexaenoic acid (DHA; C22:6n-3) (Domenichiello et al., 2015), which has been found to modulate brain plasticity and counteract neuroinflammation in experimental studies (Dyall, 2015). However, studies in rodents uncovered brain benefits of ALA by itself, including increased brain plasticity (Blondeau et al., 2009), reduced cell death and calcium dysregulation (Carey et al., 2013), and reduced amyloid-beta deposition (Gao et al., 2016). Other bioactive compounds in walnuts, such as arginine, tocopherols, folate, melatonin, and polyphenols also support neurological health and cognitive wellness by modulating blood pressure, HDL function, glucoregulation, endothelial vasodilator function, arterial compliance, oxidative status, and vascular inflammation (Carey et al., 2013; Del Rio et al., 2013; O’Brien et al., 2014). In support, experimental feeding studies in aged rodents have shown that walnuts improve age-related motor and cognitive deficits as assessed by rod walk, plank walk, and Morris water maze tests (Willis et al., 2009).

Easy to implement lifestyle modifications that might help prevent chronic non-communicable disorders need to be uncovered, with the concept that even small changes in the right direction can lead to substantial benefit for global health. Thus, delaying the onset of AD by only 5 years would significantly reduce its prevalence by the next decade (OECD, 2014). Thus far randomized controlled trials (RCTs) testing various interventions in patients with established AD have failed to show any benefit, probably because at this stage neuropathology is far advanced and irreversible. This underlines the need to conduct RCTs in individuals at risk but cognitively healthy, when there are little or no underlying brain changes.

Although not primarily focused on neurodegenerative disorders, one such study, the PREvención con DIeta MEDiterránea (PREDIMED) study, is a landmark RCT conducted in older individuals at high cardiovascular risk that has shown beneficial effects of Mediterranean diets supplemented with extra-virgin olive oil or mixed nuts on several age-related disorders after intervention for ∼5 years (Martínez-González et al., 2015). In the PREDIMED trial, the Mediterranean diets reduced the incidence of cardiovascular diseases by 30%, compared with a low-fat control diet, while the risk of stroke was reduced by 34% by the olive oil diet and by 49% by the nuts diet (Estruch et al., 2013). In a PREDIMED sub-study, we showed better cognitive performance associated with consumption of polyphenol-rich Mediterranean foods at baseline (Valls-Pedret et al., 2012) and cognitive improvement in participants allocated the two Mediterranean diets compared with those in the control group after 4.1 years of follow-up (Valls-Pedret et al., 2015). Thus, data from PREDIMED suggest that nuts, including walnuts, are a good option for cardiovascular and brain health. Therefore, it is reasonable to surmise that usual walnut consumption may be an effective approach to preserve cognitive abilities and visual function. However, while there is sufficient rationale for the role of walnuts in neuroprotection, direct clinical evidence is lacking. The Walnuts and Healthy Aging (WAHA) study is the first RCT assessing cognitive function and macular health in an elderly cohort following daily ingestion of walnuts for 2 years.

As depicted in Figure 1, there are two main points of data collection, at baseline and at the end of the study after 2 years of intervention. However, there are intermediate points wherein relevant information is obtained. Thus, in mid-study (12 months) a physical activity questionnaire was administered and blood was collected for determination of safety biochemistry analyses, lipid profile, and some secondary outcome variables such as leukocyte telomere length. Nutritional information and office blood pressure was obtained every 6 months, and general health status, tolerance and side effects, medication changes, and anthropometric data were ascertained every 2 months.

The pre-intervention visit with the study’s clinician lasted 1 h and consisted of a face-to-face interview with the candidate wherein complete information on the trial was provided, medical information was collected, inclusion/exclusion criteria were recorded, office blood pressure was measured, an ECG was performed, and the MMSE were administered. If the candidate was suitable for the trial and willing to participate, the informed consent was signed and blood extraction and urine collection and the first neuropsychologist and ophthalmologic visits were scheduled for next week. In the Barcelona site, one part of the informed consent concerned brain MRI, and candidates were asked to fill it in if willing to undergo the procedure. Once the pre-established number of 30% of the cohort was reached, no further candidates were offered brain MRI.

The neuropsychologist visit included the comprehensive test battery described in the Section “Cognitive Testing,” which took 90 min on average to complete. Whenever a cognitive alteration was detected at this visit, the candidate was excluded from further studies and was referred to the local Neurology clinic with a report describing the test findings. In the Barcelona site, the neuropsychologist provided full details on the brain MRI examination to willing candidates and scheduled the procedure.

In the ophthalmologic visit, which lasted approximately 30 min, a general history of eye health was taken and the OCT was performed. Any candidate found to have advanced AMD or bilateral cataracts precluding retinal examination was excluded from the study and referred to the local Ophthalmology clinic.

Once candidates were considered fit to enter the study after assessment of the main outcomes, they were scheduled for the first visit with the dietitian wherein they were randomized to the walnut or control diets. Participants allocated to the walnut diet received their first 2-month allotment of walnuts plus extra packs for family needs. In the BCN site, after the first dietitian visit all participants were scheduled for the secondary outcome variables carotid ultrasound, DEXA for both body composition and bone mineral density, and ambulatory blood pressure monitoring. During the 2 years of the study, dietitians took care of all aspects of follow-up at the bimonthly visits. These included dietary compliance, tolerance and side-effects, anthropometric measurements, delivery of walnuts and recount of empty packages, and changes in clinical status and medication. Clinical and medication changes were always reported to the study’s clinician for confirmation and final recording in the online study’s management system.

At the end of the study, all examinations related to primary and secondary outcomes were repeated.

Data collection for the WAHA study was completed May 31, 2016. As shown in Figure 2, of the total 708 randomized subjects, 92% (n = 651) completed the first year of the study, while 89.8% (n = 636) completed the 2 years. At the end of the study, the dropout rate is 10.2%. Attrition rates were similar in the two groups, n = 38 in the walnut group and n = 34 in the control group.

The baseline characteristics of the study participants by group are provided in Table 2. The mean age was 69.1 years and 67.9% were women. The characteristics of participants in the two intervention groups were well balanced except for smoking status, as there were more current smokers in the walnut group than in the control group.

To objectively determine adherence to supplemental walnuts, we measured the RBC proportions of ALA, a fatty acid characteristic of walnuts, at baseline and after 1 year of intervention in a random sub-sample of participants. There were no significant differences in RBC ALA between intervention groups at baseline (Figure 3A). After adjusting for center, age, and sex, 1-year changes in RBC ALA were increases of 0.16% of total fatty acids (95% confidence interval, 0.144–0.183) and 0.02% (95% confidence interval, 0.001–0.039) for the walnuts and control group, respectively (P between groups < 0.001) (Figure 3B).

The WAHA study was designed to assess whether a nutritional intervention with walnuts for 2 years can prevent cognitive impairment and preserve retinal health in a healthy elderly population.

An unavoidable limitation of the study is not being able to blind participants to the intervention since it consists of a whole food. Another limitation is the difficulty in keeping older, free-living volunteers in the study during 2 years and ensuring compliance with the high-fat supplemental food in the walnut diet group. The bimonthly visits by dietitians and their availability to answer questions related to diet or body weight and solve any problems with walnut ingestion contributed to ensure participants’ loyalty. Thus, the final dropout rate was only 10% and there was good compliance with both eating the walnuts in the walnut group and not eating them in the control group, as attested by 1-year changes of RBC ALA, an objective biomarker of walnut consumption. Finally, by the design of the recruitment methodology, self-selected participants are in better health and have a higher educational level than the general population, which could lead to lower than expected age-related cognitive decline and a lower probability of improving cognitive function with the intervention. The mean baseline age of nearly 70 years, however, may partly overcome this healthy participant effect.

The WAHA study has several strengths. This is the first RCT that examines the effects of a single whole food, walnuts, on cognitive performance and retinal health. By being an RCT the results of the main outcomes should provide a high level of evidence, while those of secondary outcomes, such as brain MRI, carotid ultrasonography, 24-h ambulatory blood pressure, inflammatory markers, and telomere length, might shed light on the mechanisms of a putative beneficial effect of the walnut diet on age-related disorders. Having participants from two clinical centers located in different geographical regions with variations in background diet and lifestyle habits increases the external validity. We assessed the primary outcome cognitive decline with a battery of standardized neuropsychological tests designed to evaluate different cognitive domains, making it a comprehensive approach. Changes in the retina were also evaluated by a precise quantitative technique such as OCT. Finally the free-living aspect of the study makes it as close to real-life as possible since there were no major diet changes to be made except to either eat walnuts daily or abstain from eating walnuts. The results should thus be applicable to a wider population and might have a significant impact on global public health recommendations.

In summary, the WAHA study evaluates cognitive function and retinal health in an elderly cohort following daily ingestion of walnuts for 2 years. The results might provide high-level evidence of the benefit of regular walnut consumption on delaying the onset of age-related degenerative diseases. The findings should translate into public health policy and sound recommendations to the general population.

The study protocol was conducted in accordance with the guidelines of the Declaration of Helsinki and was approved by the institutional review boards of each center. All participants provided written informed consent.

ER and JS obtained funding and supervised the study. They also developed and drafted this study protocol in consultation with SR, CV-P, MC, MS-M, AP-H, RC-M, AS-V, and MD. SJ and MC were responsible for the recruitment process. CV-P and AA were in charge of cognitive assessment. Biochemical samples were managed and analyzed by SJ, MC, AS-V, and CC. MS-M, AP-H, IR, TF-S, EH, EB, NK, and LH were involved in dietary intervention and nutritional and anthropometric data collection. RC-M, SA, and JF were in charge of ophthalmologic assessment. MD and AL-I were responsible for clinical data, medical records, and blood pressure. All authors revised the manuscript critically for intellectual content and gave final approval of the version to be published.

The Principal Investigators of the two centers (JS and ER) have received grants for research through their institutions from the California Walnut Commission and are non-paid members of its Scientific Advisory Committee. All the other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.