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TOWARDS A LARGE-SCALE ASSESSMENT OF THE RELATIONSHIP BETWEEN BIOLOGICAL AND CHRONOLOGICAL AGING: THE INSPIRE MOUSE COHORT

 

Y. Santin1, S. Lopez2, I. Ader5, S. Andrieu3,4, N. Blanchard6, A. Carrière5, L. Casteilla5, B. Cousin5, N. Davezac2, P. De Souto Barreto3,4, C. Dray1, N. Fazilleau6, D. Gonzalez-Dunia6, P. Gourdy1, S. Guyonnet3,4, N. Jabrane-Ferrat6, O. Kunduzova1, F. Lezoualc’h1, R. Liblau6, L.O. Martinez1, C. Moro1, P. Payoux7, L. Pénicaud5, V. Planat-Bénard5, C. Rampon2, Y. Rolland3,4, J.-P. Schanstra1, F. Sierra9, P. Valet1, A. Varin5, N. Vergnolle8, B. Vellas3,4, J. Viña10, B.P. Guiard2, A. Parini1

 

1. Institut des Maladies Métaboliques et Cardiovasculaires, Inserm, Université Paul Sabatier, UMR 1048 – I2MC, Toulouse, France; 2. Centre de Recherches sur la Cognition Animale (CRCA), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, Toulouse, France; 3. Gerontopole of Toulouse, Institute of Ageing, Toulouse University Hospital (CHU Toulouse), Toulouse, France; 4. UPS/Inserm UMR1027, University of Toulouse III, Toulouse, France; 5. STROMALab, CNRS ERL 5311, Etablissement Français du Sang-Occitanie (EFS), National Veterinary School of Toulouse (ENVT), Inserm U1031, University Toulouse III Paul Sabatier, Toulouse, France; 6. Centre de Physiopathologie Toulouse Purpan, INSERM/CNRS/UPS UMR 1043, University of Toulouse III, Toulouse, France; 7. ToNIC, Toulouse NeuroImaging Center, Université de Toulouse, Inserm, UPS, France
8. IRSD, Université de Toulouse, INSERM, INRA, ENVT, UPS, U1220, CHU Purpan, CS60039, 31024, Toulouse, France; 9. Division of Aging Biology, National Institute on Aging (NIA), National Institutes of Health (NIH), Bethesda, Maryland, USA; 10. Freshage Research Group-Dept. Physiology-University of Valencia, CIBERFES, INCLIVA, Valencia, Spain.
Corresponding author: Professor Angelo Parini, Institut des Maladies Métaboliques et Cardiovasculaires, Inserm/Université Paul Sabatier UMR 1048 – I2MC, 1 avenue Jean Poulhès BP 84225 31432 Toulouse Cedex 4 – France, Phone: (+33)561325601, e-mail: angelo.parini@inserm.fr

J Frailty Aging 2020;in press
Published online August 7, 2020, http://dx.doi.org/10.14283/jfa.2020.43

 


Abstract

Aging is the major risk factor for the development of chronic diseases. After decades of research focused on extending lifespan, current efforts seek primarily to promote healthy aging. Recent advances suggest that biological processes linked to aging are more reliable than chronological age to account for an individual’s functional status, i.e. frail or robust. It is becoming increasingly apparent that biological aging may be detectable as a progressive loss of resilience much earlier than the appearance of clinical signs of frailty. In this context, the INSPIRE program was built to identify the mechanisms of accelerated aging and the early biological signs predicting frailty and pathological aging. To address this issue, we designed a cohort of outbred Swiss mice (1576 male and female mice) in which we will continuously monitor spontaneous and voluntary physical activity from 6 to 24 months of age under either normal or high fat/high sucrose diet. At different age points (6, 12, 18, 24 months), multiorgan functional phenotyping will be carried out to identify early signs of organ dysfunction and generate a large biological fluids/feces/organs biobank (100,000 samples). A comprehensive correlation between functional and biological phenotypes will be assessed to determine: 1) the early signs of biological aging and their relationship with chronological age; 2) the role of dietary and exercise interventions on accelerating or decelerating the rate of biological aging; and 3) novel targets for the promotion of healthy aging. All the functional and omics data, as well as the biobank generated in the framework of the INSPIRE cohort will be available to the aging scientific community. The present article describes the scientific background and the strategies employed for the design of the INSPIRE Mouse cohort.

Key words: INSPIRE program, biological aging, mouse cohort, frailty, biomarkers.


 

Introduction

The improvement of medical care and living conditions has increased life expectancy. Although being a progress per se, the extension of life expectancy is associated with an elevated risk of all types of chronic diseases as well as the decline in intrinsic capacities (1). Research on the basic “biology of aging” aims to increase life expectancy and to improve the quality of life. In this context, geroscience has emerged as a new interdisciplinary field seeking to define the biological underpinnings of aging that lie at the crossroads of age-dependent biology, chronic disease and health (2, 3). The geroscience hypothesis postulates that, since aging plays a major role in most chronic diseases, addressing aging physiology will reduce or delay the onset of multiple age-associated defects.
Frailty is a clinical state of increased vulnerability resulting from aging-related decline in function and reserve across multiple physiological systems, that carries an increased risk for poor health outcomes including falls, incident disability, hospitalization, and mortality (4). Even though frailty is an age-associated syndrome, the idea that it is not a normal and inevitable part of aging is growing. Hence, frailty can be conceptualized as a result of accelerated biological aging (5), and elucidating its etiology is thus critical for its prevention and/or treatment. Therefore, there is a pressing need to discover markers to differentiate biological age from chronological age and to identify individuals at higher risk of developing chronic diseases, ultimately with the goal to propose pharmacological and non-pharmacological approaches targeting biological processes underlying aging.
According to this integrated view, the INSPIRE research program has been created to foster research in the field of geroscience and healthy aging. INSPIRE aims at promoting healthy aging and preventing dependency through, among other strategies, the constitution of a bio-resource platform going from animals to humans in order to provide clinical, biological and technological resources for research and development on aging (for a detailed review on the INSPIRE program, see [6]). Besides the implementation of digital medicine (ICOPE program from the WHO) and the constitution of an INSPIRE Human Translational Cohort (6, 7), the INSPIRE program will create a unique Mouse Cohort dedicated to basic research, whose setup and design will be described in the present article.

 

Overview of the INSPIRE Mouse cohort

The primary goal of the INSPIRE Mouse cohort is to foster an understanding of the close relationship between the molecular mechanisms of biological aging and the onset of clinical frailty. This approach will importantly lead to the identification of frailty biomarkers. Complementarily, the INSPIRE Mouse cohort will enable to better characterize frailty in mice by implementing already existing tools such as the “Valencia Score”, a frailty score mainly based on neuromuscular alterations (8), or the “Howlett and Rockwood frailty index” relying on a list of deficits that accumulate during aging (9). This will eventually lead to the creation of an “INSPIRE Frailty Score” suitable for mice and as close as possible to the clinical scenario in humans. Since multiple molecular pathways are involved in the aging process and can contribute to various aspects of frailty, a panel of valid biomarkers in combination with functional measures of frailty would allow both diagnosis and follow up in preclinical and clinical settings (10).
A major asset of the INSPIRE Mouse cohort is its “mirroring” of the INSPIRE Human cohort in order to facilitate the translation of results from basic science to humans (Figure 1). To further improve the extrapolation of the results to the clinic, “humanized” living conditions, i.e. high fat/high sucrose diet and sedentary lifestyle, will be studied as common risk factors of accelerated aging. A particular attention has also been paid to the selection of a mouse strain that congruently mimics human heterogeneity. Besides, as main studies on frailty have been done on either male or female mice, comparison of frailty between genders will also be a major advantage of the INSPIRE Mouse cohort. These important considerations should facilitate the crosstalk between humans and experimental models, therefore speeding up the discovery process (Figure 1).
Here, we provide detailed information on the INSPIRE Mouse cohort setup, by putting forward an innovative methodology ranging from the study design to comprehensive phenotyping. This will be done by integrating measures evaluating different dimensions of frailty including cognitive/motor capacities, cardiac function assessment, body composition, metabolic parameters, urinary incontinency and immune function as defined in humans (syndrome diagnosis). Importantly, tissue biobanking for frailty biomarkers identification is implemented.

 

Study design

The INSPIRE Mouse cohort was designed to be as close as possible to human lifestyle. As a major issue in aging studies in mice is that most are carried out in inbred strains, the INSPIRE Mouse cohort will gather a genetically heterogeneous mouse stock to better mimic human diversity. In addition, besides normal aging, physical activity/exercise we will be studied as a human-relevant paradigm of delayed aging, while obesity/overweight will be evaluated as a risk factor for accelerated aging in mice. These are well-known risk factors for frailty in humans (11–14) and, as compared to other experimental approaches, they are particularly suitable to promote cognitive (15, 16), cardiometabolic (17, 18) and immune dysfunctions (19), that are involved in progressive/long-term frailty (20, 21). These aspects are described below.

Selection of mouse strain

Animal models have been critical tools in biomedical research, and among them, the laboratory mouse is undoubtedly the most commonly used experimental non-human model. The prevalence of mouse models in biomedical research, in particular in the field of aging, is unsurprisingly considerable given that mice require relatively inexpensive care, reproduce quickly, and have a high genetic similarity to humans (22). Especially, inbred strains (like C57Bl/6J mice or BALB/c mice), transgenic and congenic mice with inbred backgrounds are most used. An inbred strain is defined as a strain that has been through at least 20 generations of sib-mating, making animals from the same inbred strain effectively genetically identical (i.e. isogenic) (23). However, such strains do not reflect genetically diverse human populations, and therefore constitute only a small part of the picture. Hence, outbred stocks (as contrary to inbred mice that are referred as strains, outbred mice are referred to as stocks) represent new research options that parallel or even exceed human genetic diversity, offering more generalizability of responses across populations. Unfortunately, unfamiliarity with outbred mice and concerns about difficulty, genetic variability and lack of reproducibility have impeded their widespread use by the research community. Nevertheless, while there is a common belief suggesting that inbred strains should present less variability of outcomes (24), presenting practical and ethical advantages, a recent review of the literature shows this to be erroneous. Indeed, several studies have shown that for a majority of readouts, inbred and outbred mice showed comparable phenotypic variations (25–27). In addition, Tuttle et al. performed a systematic review of the primary literature, and found that strain type (i.e. inbred or outbred) did not have any effect on within-strain variability regardless of trait category including anatomy, behavior, immune function, molecules and organ function (26). Therefore, except in cases where precise genotypic regulation or standardization is required, it appears that outbred stocks from heterogeneous backgrounds are more appropriate models in many biomedical research applications.
Among the available outbred stocks, the SWISS mice are commonly used. The initial stock was bred at the Centre Anti-Cancéreux Romand in Lausanne, Switzerland, in the 1920s and consisted of two male and seven female albino mice derived from a non-inbred stock. These mice have many advantages for long-term studies, as they are inexpensive, robust and commercially available. They have been used for mouse transgenesis experiments, principally due to efficient breeding and large litter sizes. Importantly, they have a large genetic diversity, which is similar to that found within and between human populations (28). In addition, SWISS mice are sensitive to high fat diets (29–33) and have been used in aging studies (34, 35), the latter being of primary importance for the INSPIRE project. Indeed, Antoch and collaborators reported that mean life expectancy of these mice was 121.1 ± 9.2 weeks for males and 109.6 ± 6.9 weeks for females, with maximum lifespan being of 150 weeks and 164 weeks respectively (35).
For the reasons stated above, the INSPIRE Mouse cohort will gather SWISS mice as a model mimicking the genetic heterogeneity of human populations. It is important to note that females are often underrepresented in animal studies, leading to a compromised understanding of female biology and resulting in poorer treatment outcomes for women. By looking primarily in males, important biological effects can be missed or misinterpreted, partly due to hormonal and genetic intrinsic differences. In addition, contrary to a common belief, recent analyses have found that variability in female performance without regard for the estrous phase is not higher than performance variability in males (36). We thus decided to include both male and female SWISS mice in the INSPIRE cohort in order to further improve the reliability and representativeness of our findings (Table 1). When planning a study that includes an advanced age group, it is important to provide extra animals to ensure sufficient statistical power as a result of early mortality. Therefore, the number of males and females in each group was statistically adjusted considering both spontaneous and high fat diet-induced mortality (35, 37) (Table 1). Finally, as the tracking of each mouse is critical to carry out an individual follow-up, microchips will be implanted in mice so they will be easily identified using a microchip reader.

Table 1
Mouse cohort organization

For the cross-sectional study, end-point analysis will be performed at 6, 12, 18 and 24 months which roughly correspond to 30, 42, 56 and 70 years in humans. Two conditions that affect human and mouse health will be studied: high fat high sucrose (HFHS) obesity and exercise. For the longitudinal study, mice will be allowed to live their natural lifespan, and mean and maximal lifespans will be then calculated. Both male (M) and female (F) SWISS mice will be included in the cohort.

 

High fat high sucrose (HFHS) diet-induced obesity as a model of accelerated aging

There is strong evidence that excessive adiposity contributes to the impairment of several parameters of frailty, notably reducing the ability of older adults to perform physical activities, impairing different forms of memory and increasing metabolic instability (38). Many obesity-related conditions including low-grade inflammation, insulin resistance, type 2 diabetes and low physical activity are risk factors for frailty. In order to study the biological and molecular changes that occur during aging, and to depict the differences between accelerated and normal aging, a model of diet-induced obesity will be used to induce accelerated aging.
At present, there is a range of commercial high-fat diets that have been demonstrated to make small rodents obese. However, some of these diets contain levels of dietary fat that are much higher than the levels that humans routinely consume. The typical American or European diet contains about 35–40% fat by energy, and a tolerable high-fat human diet might contain 50–60% of energy as fat. However, the 60% fat rodent diet often used in experimental paradigms presents a much greater distortion of the fat content of a normal rodent chow. Thus, rodent studies with a 60% fat content might not be as relevant to human physiology as those which use a 40-45% fat diet (39). Moreover, mice fed with 60% fat diet become more obese, and do so faster than the ones fed with 40-45% fat diet. Thus, while many researchers use the 60% rodent diet as a matter of economics and convenience, it is not the best option for long-term follow-up studies. It is noteworthy that fatty acid (FA) composition of the diet should also be considered besides the percentage of fat in the diet. Moreover, it has been suggested that HFD with high sugar content better mimic the human western diet (40).
For all the aforementioned reasons, we decided to use a customized high fat high sucrose (HFHS) diet containing 40 % energy from animal and vegetal fat (among which 41% saturated fatty acids, 45% monounsaturated fatty acids and 14% polyunsaturated fatty acids) and 25% by weight sucrose. This diet or its corresponding customized control diet will be given to the mice from 6 to 24 months (Table 1). Interestingly, comparable HFHS diets have been shown to promote sarcopenia, bone loss and impaired neurological function in mice (41, 42). These findings represent some of the major features observed in aging humans, suggesting that HFHS diet-fed mice represent a useful model for studying accelerated aging.

Voluntary activity through running wheel access as a model of decelerated aging

Behavioral paradigms that are commonly used to model human exercise training in mice include forced treadmill running, forced wheel running and voluntary wheel running. Mice running behavior in voluntary wheels is closer to the natural running pattern than forced exercise, as it is performed under non-stress conditions, does not require a negative stimulus, and does not interfere in the normal nocturnal-diurnal rhythmicity of the animal (43). Remarkably, laboratory mice run spontaneously when they have access to running wheels, and this behavior is also observed in feral mice when running wheels are placed in nature (44). Voluntary wheel running thus consists of a rewarding behavior and not a stereotypic behavior that can result from environmental restriction and devoid of any goal or function (45). Another advantage of voluntary wheel running is that, since no direct intervention from the experimenter is required, it can be easily used in long-term studies. Hence, voluntary activity will be assessed in the INSPIRE Mouse cohort by giving mice access to upright running wheels (Table 1). To obtain continuous recording throughout the lifespan, we will use a sophisticated method connected to an analysis software that will record detailed activity parameters, including the number and duration of each running period, as well as the number of revolutions, speed, total distance and time, and dark/light cycle activity patterns on running wheels. As mice will be identified by microchips, parameters will be obtained for each single mouse, and at the time of this writing, we are developing a “toll like” detection system to measure individual mouse voluntary activity. Of note, to avoid enrichment/steric hindrance-linked bias, wheels will be placed in all cages. Nevertheless, in the control groups for which the effect of «no physical exercise” will be assessed, running wheels will be blocked (Table 1).

Spontaneous mobility

Mobility is among the most studied and most relevant parameters affecting quality of life with strong prognostic value for disability and survival. Indeed, locomotor impairments in older adults represent a pre-clinical transitional stage towards disability (46). It is thus necessary to understand how aging-related changes in mobility in mice resemble changes in humans.
To this end, the INSPIRE program will provide a life-long measurement of aging-related locomotor activity in mice, through automated home cage monitoring. This technique enables to monitor animals over long periods of time without human intervention. The system we will use, known as Digital Ventilated Cages (DVC®), is designed to gather continuous animal activity data directly from the home cage while keeping cages into conventional Individual Ventilated Cages (IVC) racks (Supp material). It provides a reduction in animal distress thereby increasing welfare, minimization of biases and increased reproducibility of data (47, 48). Therefore, mice belonging to the INSPIRE cohort will be housed in DVC cages so locomotor activity of all mice will be continuously and automatically monitored throughout their life. This activity metric represents the overall in-cage activity generated by all mice in a cage from any electrode and is not tracking activity of individual group-housed animals. Therefore, this parameter will be complemented by the individual aforementioned measure, i.e. voluntary activity through running wheel, as well as neuromuscular function by Valencia Score and behavioral cognitive tests (see the following section).

 

Comprehensive phenotyping

In this section, we provide an overview of our methodology for the measurement of healthspan and frailty in naturally aging, diet-induced accelerated aging, and exercise-induced decelerated aging in mice. These methods cover a spectrum of highly relevant biological indicators of frailty including cognitive, neuromuscular, cardiac, metabolic and immune function as well as urinary incontinency (For precise timeline, see figure 2). The goal is to improve the currently available “Frailty Scores” with an extended “INSPIRE Frailty Score” suitable for mice, and taking into account accurate parameters to get closer to the human clinical settings (9, 49) (Figure 3).

Figure 1
Parallel between INSPIRE Mouse and Human cohorts

The animal cohort will mimic the human diversity in functional status by providing both healthy and frail animal models to investigations. Both cohorts will allow the normalization and optimization of clinical and biological parameters, and will provide common dataset with equivalent clinical (e.g., cognitive function, mobility) and biological tests. Running animal and human cohorts in parallel is expected to facilitate cross-talks between the experimental models and the clinic in order to 1) identify causal mechanisms of clinical frailty; 2) discover biomarkers associated with functional loss; and 3) develop new therapeutic strategies allowing healthy aging. Of note, the INSPIRE Research Initiative will also use Nothobranchius Furzeri (African Killifish) and pet dogs as additional cohorts to investigate aging process.

Figure 2
Representation of experimental timeline

For the cross-sectional study, the multiple tests at 6, 12, 18 and 24 months will be performed over a period of 3-4weeks. The nature of these tests is indicated below each end-point. One month before end-point analysis (5, 11, 17 and 23 months), bladder function will be assessed. At 9, 15 and 21 months, blood will be collected in a longitudinal way to mainly evaluate immune system modifications. Both mouse mobility and voluntary activity will be continuously recorded during the whole study. For the longitudinal study, mice will be allowed to live out their maximum natural lifespan. * indicates the start of the HFHS diet at 6 months. FBO: Feces, Blood, Organs.

 

Observational study: longitudinal vs cross-sectional

Both cross-sectional and longitudinal approaches are observational studies commonly used in aging research. In cross-sectional studies, data are collected as a whole to study a mouse population at a single point in time to examine the relationship between variables of interest. Conversely, in longitudinal studies, data are gathered from the same mouse repeatedly over an extended period of time.
In the case of age-related healthspan studies, data are collected at predetermined ages from multiple individuals within a population. The cross-sectional study design allows performing invasive or terminal procedures but precludes the evaluation of lifespan. In the case of the INSPIRE Mouse cohort, a major cross-sectional study will be conducted with endpoint analyses being performed in different groups of mice at the ages of 6, 12, 18 and 24 months-old (Table 1), which roughly correspond to ages from 30 to 70 years in humans. This will allow us to carry out a large number of tests to evaluate and characterize the onset of frailty in aging mice (Figure 2). Importantly, it will also enable to evaluate if some organs “age” prematurely compared to others, and to presume the role of different organ dysfunction in the onset and progression of frailty.
Conversely, longitudinal studies allow mice to live out their maximum natural lifespan, either dying naturally or being euthanized in case of major decline. Therefore, a longitudinal sub-cohort with 120 animals (60 males and 60 females) will be implemented to the INSPIRE cross-sectional study to evaluate the spontaneous mouse mortality, and to determine mean and maximal lifespans in our animal facilities (Table 1, figure 2).

Frailty evaluation by the “Valencia Score”

The development of frailty scores suitable for mice and which resemble those that are used in the clinical scenario has become an essential challenge in basic gerontological research. In pursuit of this goal, the “Valencia Score” has been recently developed to measure frailty in rodents (8). It is based on the human clinical parameters described by Linda Fried and co-workers [50], and thus facilitates the extrapolation to humans, as it relies on five robust clinical criteria including unintentional weight loss, weakness, poor endurance, slowness and low activity level, that can be easily measured in mice. According to this score, if a mouse fails three or more components out of five, it is considered as frail, if it fails one or two criteria, it is classified as prefrail, whereas if it does not fail any criteria it is considered as robust, which is equivalent to the clinical classification defined in the Fried Frailty Score. We decided to use the Valencia Score as a starting point to evaluate frailty, and the following parameters will be therefore primarily measured.

Body weight

Animals’ body weights will be recorded biweekly throughout their lifespan to have a precise follow-up of weight evolution. In order to have reliable and individual data, all the mice will be weighted. As suggested by Gomez-Cabrera and colleagues, a 5% weight loss over a one-month period will be considered positive for this frailty criterion (8), a parameter reflecting the unintentional weight loss commonly observed in frail people.
In order to avoid variability in locomotor activity and other parameters driven by differences in circadian rhythms, all testing will be done starting at the same time. Tests will be run in the order listed, from the least to the most stressful, thereby decreasing the chance that one test might affect the behavior evaluated in the subsequent paradigm.

Grip strength

The grip strength test is a simple non-invasive method designed to assess neuromuscular function through animal’s limb strength. It takes advantage of the animal’s tendency to grasp a horizontal metal bar or grid while suspended by its tail. It allows to determine the maximum force, or peak of force, developed by a mouse when the operator tries to move it away from the bar or grid. The measurement is carried out using a high-precision sensor and an electronic device, guaranteeing a perfect capture and display of the maximal force. As suggested by the “Valencia Score”, a cut-off point below which 20% of the observations may be found has to be calculated, and all the animals ranking below this 20th percentile will be considered to fulfill the frailty criterion of weakness, which is frequently measured in the clinical setting.

Motor coordination

The tightrope test is a method for evaluating neuromuscular coordination and vigor. It is positively correlated to lifespan in rodents and has been extensively validated as a behavioral marker of aging since it was first described in the seventies (51, 52). When animals are placed on a tightrope, they are able to grasp the string with the four legs and tail and move to reach a side pole. Mice are scored positive if they are unable to reach the side pole before a 60 sec time-limit or if they fall from the rope. Usually, obese and aged mice cannot lift their hind legs and, after hanging for a few seconds from the forepaws, fall on the cage bedding. In this case, mice are scored as “positive” for this frailty criterion.

Incremental treadmill test

Poor endurance and slowness are key components of the diagnosis of frailty in humans. These parameters can be evaluated in mice by measuring the running time and speed values when performing an incremental intensity test in a treadmill. For endurance, the running time values will be measured. Then, similar to the grip test, a 20th percentile will be calculated as a cut-off point. The animals that will report a running time under this “threshold” will fulfill this frailty criterion. Besides endurance, running speed will be measured as an index of “slowness”. The same aforementioned calculation will be performed to define a threshold under which mice will be considered as positive for the “slowness criterion”. Of note, very old animals are usually unable to keep even the lowest running intensities. In our study this is likely to be exacerbated in older mice fed the HFHS diet. As in clinical practice, subjects that are unable to perform any one test are categorized as positive for that criterion.
In the case of the INSPIRE Mouse cohort, the Valencia Score will be used as a primary indicator to evaluate frailty in mice. However, as this score is mainly based on neuromuscular alterations that are commonly observed in frail people, implementation of additional parameters would be of great value to better characterize frailty onset and progression. Therefore, in order to detect early signs of frailty that might not be detected by the Valencia Score, complementary measurements will be carried out on the INSPIRE Mouse cohort in order to propose an extended “INSPIRE Frailty Score”, including cognitive, cardiac, metabolic as well as other biological functions (Figure 2). These measurements are described in the following sections.

Behavioral cognitive tests

Behavioral indicators of healthspan in mice include gait/ataxia, motivated activity, cognition, and affective function (53). In the context of aging, we will primarily use the spontaneous alternation Y-maze, which assesses prefrontal cortex- and hippocampus-dependent spatial working and reference memory, reflecting changes in cognitive performance (54).
The Y-maze spontaneous alternation test is based on rodent’s innate curiosity to explore previously unvisited areas and is used to assess spatial working memory. When placed in a Y-shaped maze, a mouse will show a tendency to enter previously unexplored arms, thus showing alternation in the arm visits. The number of arm entries and the successive entry sequences in the 3 arms are recorded in order to calculate the percentage of alternation. An entry occurs when the four legs are in the arm.

Cardiac function

Cardiac dysfunction is a main issue in elderly people, and its assessment could be of great interest in the diagnosis and the better characterization of frailty. Despite the absence of underlying pathologies like hypertension or myocardial infarction which lead to heart failure with reduced ejection fraction (HFrEF), the ‘normal’ aged heart usually exhibits changes like arterial stiffening, increased myocardial stiffness, decreased diastolic myocardial relaxation, increased left ventricular (LV) mass and decreased peak contractility (55). In addition, aging and related comorbidities (obesity, hypertension, diabetes, chronic obstructive disease, anemia and chronic kidney disease) may initiate or aggravate chronic systemic inflammation that may further affect cardiac remodeling and dysfunction (56). Therefore, the majority of elderly patients exhibit heart failure but have a preserved systolic LV function, a syndrome known as heart failure with preserved ejection fraction (HFpEF). Patients with this syndrome have severe symptoms of exercise intolerance, frequent hospitalizations and increased mortality. Despite the importance of HFpEF, optimal treatments remain largely insufficient. The INSPIRE Mouse cohort thus represents a model to better understand HFpEF pathophysiology within a ‘systemic’ perspective. Of note, approximately 85% of elderly HFpEF patients are overweight or obese, and the HFpEF epidemic has largely paralleled the obesity epidemic (57). Therefore, HFHS diet-induced obesity also represents a congruent mouse model of HFpEF.
For the evaluation of cardiac function, we have selected echocardiography. In addition to traditional parameters reflecting systolic function (ejection fraction and ventricular wall thickness), particular attention will be given to the measurement of diastolic (dys)function. In particular, the evaluation of mitral inflow will be assessed, as it is very informative and plays an important role in grading diastolic dysfunction (Supp material). Of much interest, these parameters will be complemented with strain imaging to measure the regional and global deformation of the myocardium, which allows for early detection of subclinical LV dysfunction.
The combination of the aforementioned cardiac parameters will allow to better highlight HFpEF in mice and to upgrade the Valencia Frailty Score with the degree of diastolic dysfunction.

Metabolic function

During aging, there are changes in body composition, including a loss of lean body mass, bone mass, body water, and a relative increase of fat mass. The bone deteriorates in composition, structure and function, which predisposes to osteoporosis. Furthermore, the increase in fat mass is distributed more specifically in the abdominal region, which is associated with cardiovascular disease and diabetes (58). Changes in body composition often occur in the absence of weight fluctuations, being due to alterations in energy balance, with a positive balance leading to weight gain and a negative balance resulting in weight loss. These key parameters will thus be assessed in the INSPIRE Mouse cohort.

Body composition and bone analysis

Magnetic Resonance Imaging
Body composition analysis will be performed by Magnetic Resonance Imaging (MRI) which provides an accurate estimate of whole-body fat, lean, free water, and total water masses in live mice. This technology combines simplicity of use, short scan times, and the comfort of animals which do not need to be anesthetized.

X-ray micro computed tomography
Bone analysis will be done by micro-computed tomography (micro-CT), which can provide ultrahigh-resolution images with resolution of less than 10 µm. This analysis will be performed after bone collection following terminal anesthesia. This technique will evaluate key parameters of bone microarchitecture like cortical thinning, cortical porosity, thinning of the trabeculae and loss of trabecular connectivity.

Plasmatic metabolic profiling

In addition to the aforementioned parameters, key plasmatic markers will be measured in plasma collected 2h after fasting, at the time of euthanasia. The combination of biochemical and multiplex immunoassay analysis will allow us to determine a broad range of metabolic markers in mice. These markers include, but are not limited to, hepatic enzymes, lipids and lipoproteins, incretins, glycated proteins, glucose, lactic acid, glucagon, insulin, leptin, PYY, amylin, peptide C, ghrelin and others.
The consideration of metabolic function in the INSPIRE frailty score will be of great importance to correlate body and bone compositions, and plasmatic metabolic profiling with neuromuscular and cardiac alterations, which will allow a better characterization of the sequential progression of frailty.

Bladder function

Urinary incontinence is a major problem in the elderly population, especially among women (59). Affected individuals often make great efforts to deny or hide urinary incontinence, which can lead to psychosocial hindrance. Its consideration is thus important in the characterization of frailty, but unfortunately its measurement is often undervalued in aging research, in particular in animal cohorts.
We thus decided to measure urinary incontinence in the INSPIRE Mouse cohort in order to study lower urinary tract function during aging. To this end, a spontaneous void spot assay (VSA) will be performed (Supp material), so urinary spotting patterns will be used as an indirect way of measuring bladder function and outlet control (60). As urinary incontinence is usually considered as a feature of frailty in humans, its measurement in mice will improve the scoring of frailty to be closer to the clinical evaluation.

Immune function

A crucial component of aging is a set of alterations in the immune system that can manifest as a decreased ability to fight infection, diminished response to vaccination, increased incidence of cancer and constitutive low-grade inflammation (61). The latter, which has been called “inflammaging”, has drawn particular attention in the field of aging, as recent studies have provided evidence that a pool of molecules can be secreted by senescent cells, a process known as senescence-associated secretory phenotype (SASP). This SASP includes cytokines, chemokines, proteases and growth factors that can affect neighboring cells via autocrine/paracrine pathways.
Immunological markers will be assessed in the INSPIRE Mouse cohort at different time points, i.e. 9, 15 and 21 months through submandibular blood collection and 6, 12, 18 and 24 months through terminal blood collection in the posterior vena cava (Figure 2). These markers will be measured in plasma by multiplex immunoassays and include, but are not limited to, IL6, IL-1 beta, TNF alpha, IL-12, IFN gamma, IL-2, IL-10, TGF beta, IL-4, IL-13, IL-17, CCL2, CXCL9, CXCL10, CCL22, CCL17, CRP. In addition, end-point blood collection will also serve at determining the white blood cell count of mice.
Adding some key markers reflecting immune system modifications in the characterization of frailty would be of great interest, as this feature is not considered in the current evaluation of frailty in mice.

Organ collection, biobanking and multi-omics analysis

After phenotyping, mice will be sacrificed and urine, feces, blood and tissues will be collected for biobanking as appropriate. Mice will be fasted 2h before euthanasia. Urine will be collected after placing mice in metabolic cages for 12 hours, the day before euthanasia. Feces will be collected during mouse handling, just before euthanasia and directly frozen. Blood will be collected just after euthanasia from the posterior vena cava, which is recommended for terminal stage studies in order to collect a maximal volume of blood. The fluids will be prepared as appropriate (e.g. for plasma collection), aliquoted and stored at -80°C before further investigations. Concerning the tissues, as many tissues as possible will be collected. Each tissue will be then subdivided into two pieces: the first one will be included in Optimal Cutting Temperature (OCT) compound, paraformaldehyde (PFA) or glutaraldehyde as appropriate, and cut into ultrathin slices for complete anatomopathological analysis. The other piece will be flash frozen in liquid nitrogen and, shortly before analysis, tissues will be fragmented with a biopulverizer into tiny pieces the size of grains of sand or course powder. This technique was selected for different reasons: 1) it reduces the number of collector tubes; 2) it limits sampling bias during organ collection; and 3) it optimizes subsequent rapid and complete lysis using lytic solutions or mechanical homogenizers. All the samples will be stored in a Biological Resource Center dedicated to the conservation of biological resources according to strict criteria of ethics and quality.
Multi-Omics analysis will be performed on biological fluids, feces and tissues. To facilitate the transfer of the results to the Human cohort, priority will be given to the analyses in plasma, urine (in particular proteomics and metabolomics profiling) and feces (microbiota analysis). The goal of this approach is to define a set of robust and accurate biomarkers for normal, accelerated, and decelerated aging. The tissues will be then dedicated to the multi-Omics-designed identification of novel tissue-specific candidate biomarkers for frailty and accelerated/decelerated aging, and to the validation of the novel candidate targets for prevention and treatment of accelerated aging (Figure 3).

Figure 3
Graphical abstract of the proposed INSPIRE Frailty Score

The goal of the INSPIRE Mouse cohort is to propose a clinically relevant “INSPIRE Frailty Score”, combining both functional and biological parameters, which will bring important knowledge on frailty characterization, assessment and target identification.

 

Conclusions and perspectives

Belonging to the global INSPIRE platform on geroscience, the INSPIRE Mouse cohort represents a unique way to model and better characterize frailty in mice. Although excellent institutions like the Buck Institute and the National Institute on Aging also carry out comparable studies in mice dedicated to investigate biological aging, the main originality of the INSPIRE Mouse cohort relies on the focus on getting closer to the human lifestyle to define the time course and the mechanisms of frailty/accelerated aging onset. Within this line, the selection of outbred mice that better parallel human genetic diversity, is a determining parameter offering more generalizability of responses across populations. In addition, including both males and females, and mimicking “humanized” lifestyles through voluntary physical activity and HFHS-diet induced obesity further approach real human living conditions.
Through a large functional and biological phenotyping of mice, a first objective of the INSPIRE project is to define the age at which early signs of frailty arise. Indeed, frailty is considered as a clinical syndrome appearing in advanced ages (62), but this is because the definition of frailty is mainly based on clinical criteria becoming discriminating in old patients. However, it is likely that the biological mechanisms leading to frailty and accelerated aging may be induced and detectable much earlier than the actual clinical signs of frailty. The goal here is to define the early signs of premature aging and to correlate them to the normal/altered functional phenotype to 1/ define the age of frailty onset and 2/ identify the organ/system(s) primarily altered in the frailty process. To this aim, the development of a clinically relevant score for frailty in mice is essential. Within this line, the “Howlett and Rockwood frailty index” is a simple and noninvasive index, based on 31 health-related variables like alopecia, distended abdomen, hearing loss and breathing rate (9). Although this 31-item check list is based on deficit accumulation during aging, we believe that investigator bias may play a critical role in diagnosis of frailty, which may affect the comparison of results across studies. More recently, the Valencia Score has been developed to determine frailty in naturally aging mice, based on five clinical components previously reported for humans by Fried and co-workers (8, 50). Despite its undeniable interest, this approach is primarily focused on the in-depth study of aging-related neuromuscular alterations and does not evaluate other key aspects of frailty such as cognitive, cardiac or metabolic impairments. Therefore, for the INSPIRE Mouse cohort, mice will be initially labeled as ‘frail/pre-frail/robust’ based solely on the Valencia test. Then, functional phenotyping will allow us to know if other aspects of frailty that are currently undervalued (e.g. cardiac or metabolic alterations) are detectable earlier than neuromuscular defects, which could greatly refine frailty detection. Then, a cut-off will be empirically determined for each parameter in order to set a more accurate frailty score. This method will bring key information on frailty by 1/ evaluating the effect of HFHS-induced overweight and sedentarism on frailty onset and 2/ including clinically relevant criteria like cognitive, cardiac, metabolic, bladder and immune parameters in addition to the currently measured neuromuscular deficits (Figure 3). Importantly, all these parameters, which will be supplemented by the longitudinal follow up of mouse mobility and voluntary activity, closely reflect changes observed in humans and therefore better approach the human frailty criteria.
Besides phenotypic measures, molecular biomarkers will be highly valuable and complementary in the prediction of healthy/unhealthy aging. Through a better understanding of the close relationship between the molecular mechanisms of cell premature aging and the onset of frailty/accelerated aging, the INSPIRE Mouse cohort will foster the identification of a panel of robust and sensitive frailty biomarkers that have not been extensively studied so far. Multi-Omics analysis of blood, urine and feces will allow to rapidly identify such biomarkers’ profiles (that can be conceptualized as a “frailty ID”), which might inform timely pharmacological and non-pharmacological preventive strategies acting directly on aging and contributing to a healthy state even in late ages. Then, these multi-Omics approaches will be extended to tissues to eventually discover novel tissue-specific putative biomarkers and therapeutic targets of frailty/accelerated aging (Figure 3).
An important notion, tightly linked to frailty is resilience, which is defined as the capacity to respond to or recover from clinically relevant stresses (63). Therefore, resilience must be evaluated in aging studies and necessitates the development of new animal models, which would be of particular great value for testing the benefits of geroprotectors. However, modelling resilience in mice is challenging, as there is no consensus on its precise definition or on how best to measure it (64). Although some models are currently available, there is very little data related to the characterization of the multiple deficits caused, especially in aged animals. As of this writing, INSPIRE investigators (gathering physicians, pharmacists, epidemiologists, geriatricians, clinicians, molecular biologists and others interested in the process of aging) are working on the tremendous question of “resilience modelling”, aiming at reaching a consensus on the suitability of such models.
To sum up, the INSPIRE Mouse cohort will importantly lead to the precise functional characterization of frailty together with the identification of robust molecular biomarkers to predict healthy/unhealthy aging. The resulting INSPIRE Frailty Score, combining both functional and biological parameters, will thus allow to refine frailty characterization and detection in animal models (figure 3). Therefore, by belonging to the global INSPIRE platform on geroscience (6, 65) and through its interaction with the INSPIRE Human Translational cohort and the INSPIRE Icope Care Cohort (6, 7, 66), the INSPIRE Mouse cohort should speed up the discovery process in the field of aging, with the final goal to increase access to healthy aging for the current and next generations.

 

Acknowledgments: We thank Massimiliano Bardotti, Rémy Burcelin and Sarah Gandarillas for their help in the design of the cohort. The Inspire Program was supported by grants from the Region Occitanie/Pyrénées-Méditerranée (Reference number: 1901175), the European Regional Development Fund (ERDF) (Project number: MP0022856), and the Inspire Chairs of Excellence funded by: Alzheimer Prevention in Occitania and Catalonia (APOC), EDENIS, KORIAN, Pfizer, Pierre-Fabre.
Conflict of interest: All authors of the paper “Towards a large-scale assessment of relationship between biological and chronological aging: The INSPIRE Mouse cohort” declare no conflict of interest related to this manuscript.
Permissions: This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, duplication, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.
Open Access: This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, duplication, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

 

SUPPLEMENTARY MATERIAL

 

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THE INSPIRE BIO-RESOURCE RESEARCH PLATFORM FOR HEALTHY AGING AND GEROSCIENCE: FOCUS ON THE HUMAN TRANSLATIONAL RESEARCH COHORT (THE INSPIRE-T COHORT)

 

S. Guyonnet1,*, Y. Rolland1,*, C. Takeda2, P.-J. Ousset2, I. Ader3, N. Davezac4, C. Dray5, N. Fazilleau6, P. Gourdy5, R. Liblau6, A. Parini5, P. Payoux7, L. Pénicaud3, C. Rampon4, P. Valet5, N. Vergnolle8, S. Andrieu9, P. de Souto Barreto10, L. Casteilla3, B. Vellas1 for the INSPIRE Platform group

 

1. Inserm UMR 1027, Toulouse, France; University of Toulouse III, Toulouse, France; Gérontopôle, Department of Geriatrics, CHU Toulouse, Toulouse, France; 2. Gérontopôle, Department of Geriatrics, CHU Toulouse, Toulouse, France; 3. STROMALab, Etablissement Français du Sang-Occitanie (EFS), Inserm 1031, University of Toulouse, National Veterinary School of Toulouse (ENVT), ERL5311 CNRS, Toulouse, France; 4. Centre de Recherches sur la Cognition Animale (CRCA), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, Toulouse, France; 5. Institut des Maladies Métaboliques et Cardiovasculaires, Inserm/Université Paul Sabatier UMR 1048 – I2MC 1 avenue Jean Poulhès BP 84225 31432 Toulouse Cedex 4 – France; 6. Centre de Physiopathologie Toulouse Purpan, INSERM/CNRS/UPS UMR 1043, University of Toulouse III, Toulouse, France; 7. ToNIC, Toulouse NeuroImaging Center, Université de Toulouse, Inserm, UPS, France; 8. IRSD, Université de Toulouse, INSERM, INRA, ENVT, UPS, U1220, CHU Purpan, CS60039, 31024, Toulouse, France; 9. Inserm UMR 1027, Toulouse, France; University of Toulouse III, Toulouse, France; Department of Epidemiology and Public Health, CHU Toulouse, Toulouse, France; 10. Inserm UMR 1027, Toulouse, France; University of Toulouse III, Toulouse, France; Gérontopôle, Institute of Aging, CHU Toulouse, Toulouse, France; *These authors contributed equally to this work.
Corresponding author: Sophie Guyonnet, Inserm UMR 1027, Toulouse, France; University of Toulouse III, Toulouse, France; Gérontopôle, Department of Geriatrics, CHU Toulouse, Toulouse, France, guyonnet.s@chu-toulouse.fr

J Frailty Aging 2020;in press
Published online July 10, 2020, http://dx.doi.org/10.14283/jfa.2020.38

 


Abstract

Background: The Geroscience field focuses on the core biological mechanisms of aging, which are involved in the onset of age-related diseases, as well as declines in intrinsic capacity (IC) (body functions) leading to dependency. A better understanding on how to measure the true age of an individual or biological aging is an essential step that may lead to the definition of putative markers capable of predicting healthy aging. Objectives: The main objective of the INStitute for Prevention healthy agIng and medicine Rejuvenative (INSPIRE) Platform initiative is to build a program for Geroscience and healthy aging research going from animal models to humans and the health care system. The specific aim of the INSPIRE human translational cohort (INSPIRE-T cohort) is to gather clinical, digital and imaging data, and perform relevant and extensive biobanking to allow basic and translational research on humans. Methods: The INSPIRE-T cohort consists in a population study comprising 1000 individuals in Toulouse and surrounding areas (France) of different ages (20 years or over – no upper limit for age) and functional capacity levels (from robustness to frailty, and even dependency) with follow-up over 10 years. Diversified data are collected annually in research facilities or at home according to standardized procedures. Between two annual visits, IC domains are monitored every 4-month by using the ICOPE Monitor app developed in collaboration with WHO. Once IC decline is confirmed, participants will have a clinical assessment and blood sampling to investigate markers of aging at the time IC declines are detected. Biospecimens include blood, urine, saliva, and dental plaque that are collected from all subjects at baseline and then, annually. Nasopharyngeal swabs and cutaneous surface samples are collected in a large subgroup of subjects every two years. Feces, hair bulb and skin biopsy are collected optionally at the baseline visit and will be performed again during the longitudinal follow up. Expected Results: Recruitment started on October 2019 and is expected to last for two years. Bio-resources collected and explored in the INSPIRE-T cohort will be available for academic and industry partners aiming to identify robust (set of) markers of aging, age-related diseases and IC evolution that could be pharmacologically or non-pharmacologically targetable. The INSPIRE-T will also aim to develop an integrative approach to explore the use of innovative technologies and a new, function and person-centered health care pathway that will promote a healthy aging.

Key words: Gerosciences, integrated care, biological aging, intrinsic capacity, biology of aging, translational research on aging.


 

Introduction

Aging is an important risk factor for several adverse health outcomes, particularly chronic and metabolic diseases and intrinsic capacity (IC) decline. Since chronological age differs from biological aging, operationally defining biological aging is an essential aspect to understand the interplay between aging and health outcomes. Individuals progress differently in the aging process (“normal” aging vs “accelerated” aging), which means, biological aging is a heterogeneous process. In this context, we need to develop researches to identify biomarkers of aging and healthy aging, and know how to measure biological aging. According to the Geroscience field, understanding aging and the links with age-related diseases would contribute to prevent and/or delay the onset of various diseases and the decline in IC domains, in particular in the six operational IC domains crucial for independent living defined by the World Health Organization (WHO) (mobility, cognition, psychological, vitality, hearing and vision capacities) (1-3).
The WHO has recently published the Integrated Care for Older People, ICOPE handbook Guidance, to support Healthy Aging and to propose to health-care providers appropriate approaches to detect and manage declines in IC. With this integrated and individualized approach, WHO aims to reduce the number of dependent people by 15 million Worldwide by 2025 (4-7).
Studying concomitantly biomarkers of aging and the natural history of IC evolution in people of different ages and functional status is to date very challenging to understand the relation between biological aging and health outcomes. In this context, the INSPIRE program was built to foster research in the field of Geroscience and healthy aging. INSPIRE is a research program dedicated to biological and healthy aging, aimed at constituting a bio-resource platform going from animals to humans, from cells to individuals, from research to clinical care. INSPIRE will provide clinical, biological and technological resources for research and development on aging. The resources will be open to both academic and industry worlds in order to promote healthy aging and prevent dependency. It is a public-private initiative that brings together internationally recognized experts from basic and translational science (in particular, in the fields of immunology, metabolism, neurosciences and mesenchymal stem/stroma cells), clinical gerontology (i.e., researchers, but also physicians and nurses involved in clinical care), primary care and public health (8).
One of the main challenges of INSPIRE is to identify markers capable of determining biological aging with the implementation of human and animal cohorts. The INSPIRE Human Translational Cohort (INSPIRE-T cohort) will recruit about 1000 individuals of several chronological ages (from 20 years to 100+) and functional capacity levels (from robust to frail, and even disabled) with baseline and follow-up biological, clinical, imaging and digital data over 10 years. These data should allow us to explore and identify a set of biomarkers of aging, age-related diseases and IC evolution. In addition, the INSPIRE-T cohort aims: i/ to test the feasibility and acceptability of a new app for smartphone and tablet developed to monitor the six IC domains (locomotion, cognition, vision, hearing, vitality/nutrition, psychological status) according to the WHO recommendations; and ii/ to explore the development of digital markers of aging. This paper describes the study design of the INSPIRE-T cohort.

 

Material and Methods

Study design

The INSPIRE-T cohort, started in October 2019, is a 10-year observational study. The study population will consist of 1000 subjects recruited in the city of Toulouse and surrounding areas, South-Western France, and covering the age range of 20 years and over (no upper limit for age). Several follow-up visits will be regularly scheduled during the 10-year period of this study. Additional visits will be conditioned by the remote monitoring of IC and the onset of other major clinical conditions.
At baseline, and then once a year, diversified data (clinical, digital, imaging) and biospecimens (blood, urine, saliva and dental plaques) are collected following standardized procedures. Data collection is performed in the Clinical Research Center (CRC) of the Gerontopole – CHU Toulouse. It can also be performed in participants’ home (for more frail and disabled volunteers), or in selected Gerontopole’s collaborating centers by a mobile research team trained by the CRC. Participants are assessed by appropriately trained clinical research members.
Between yearly waves of data collection, participants are asked to record major clinical information, including adverse events (e.g. new diagnosis, SARS COV-2 diagnosis, influenza, fracture…), medical consultations, hospitalizations, and changes in the drug prescription every 4-months. They also have the six IC domains monitored (with or without the help of a caregiver) (ICOPE program Step 1) (4,7) in either the application developed in collaboration with WHO (ICOPE Monitor app) or a web platform; or through a phone call by a Gerontopole’s trained research nurse. When declines are detected in the ICOPE Step 1, a phone call is organized by the research nurse within one week to confirm this decline and to investigate the causes in collaboration with the medical research team. Once an IC decline is confirmed, participants have a thorough clinical assessment following the recommendations of the ICOPE Step 2 (4,7) and blood sampling (data are collected by research nurses in a home visit or at the research facility). Such information will enable us to investigate the response of some markers of aging at the time declines are detected. The clinical assessments and biomarkers’ exploration also allow us to propose a personalized prevention care plan to maintain function according to the recommendations from the WHO ICOPE program for usual care (ICOPE Step 3) (4,7).
Participants are trained to monitor their IC during the baseline visit by the Gerontopole’ research team. The remote monitoring of IC will last the whole length of this research study, i.e., up to ten years. The figure 1 shows schematically the study procedures over one year. Table 1 describes the study flow chart with all data collected at each time point during the follow-up.
To ensure quality of data collected, standard operating procedures are implemented covering subject’s recruitment, biobanking, remote monitoring of IC, clinical assessments and digital data collection. All data are collected in the INSPIRE-T database. Preventive strategies to limit errors like miscoding, missing values, are applied before data entry to ensure the validity and quality of the performed data analysis. Tools will be implemented for data exploration and data sharing between INSPIRE consortium researches and later on with external scientists.

Table 1
The INSPIRE Human Translational Research cohort flow-chart

a. The cognitive composite score will be realized only in people lower than 70 years; b. Other examinations are proposed to a limited number of participants in a volunteer basis

Figure 1
INSPIRE-T study procedures over one year. The remote monitoring of intrinsic capacity will last the whole length of this research study, i.e., up to ten years

 

Objectives

The main objective of the INSPIRE-T cohort is the appropriate data collection of key variables and biospecimens for at least 1000 people at baseline and 800 people with at least four years of follow-up (i.e. four yearly post-baseline assessments) over the 10-year study. The key variables are clinical data on all six IC domains (locomotion, cognition, vision, hearing, vitality/nutrition, psychological status), and the collection of blood, urine, saliva and dental plaque samples. Secondary objectives include: i/the identification of (a set of) biomarkers of aging through the constitution of a comprehensive biobank; ii/the assessment of the feasibility and acceptability of the ICOPE Monitor app used to measure and monitor intrinsic capacity; iii/ the study of the evolution of IC domains over time and its association with health outcomes; and finally, iv/ the study of the correlation between digital biomarkers to biological/imaging biomarkers and IC domains (Figure 1, online consultation).
A mouse cohort in mirror of the human INSPIRE-T cohort is being built in order to cross the results of translational research found in humans on aging animal models, and vice versa (8). The main objective of the INSPIRE Animal cohort is to define the relationship between the molecular mechanisms of cell premature senescence and frailty/accelerated aging (8).

Study population

We will recruit about 1000 subjects, men and women, aged 20 years-old or over (no upper limit for age), and affiliated to a social security scheme. People having a severe disease compromising life expectancy at 5 years (or at 1 year for subjects living in nursing homes) and people deprived of their liberty by administrative or judicial decision, or under guardianship, are excluded. Recruitment is stratified per 10-year age groups, oversampling older people in order to be able to investigate major clinical events (e.g., declines on IC, onset of age-related diseases).
Due to the heterogeneity of biological aging, we opted for no too stringent eligibility criteria. By diversifying our recruitment sources and monitoring key risk factors for accelerated aging (e.g., age, obesity, frailty, and activities of daily living), we will be able to recruit participants with different trajectories of aging.
Sample size calculation was not relevant as many objectives of the INSPIRE-T cohort are exploratory. We therefore considered an approach based more on the potential of the INSPIRE-T cohort in terms of the ability to obtain parameter estimates with sufficient precision with a recruitment of 1000 subjects that corresponds to the maximum number of subjects that can be recruited and monitored with the funding provided. In case of evident underpowered population (for a particular subgroup of subjects), a reasoned additional recruitment of subjects may be considered in a second phase. To limit the attrition rate, subjects will be monitored by both active (visits, telephone calls) and passive ways (monitoring of several functions using new technologies via mobile phones or other connected devices).

Data collection

From all subjects enrolled, investigations include data collection at baseline and during follow-up visits (annual visits and additional visits planed in case of decline in IC). Upon written informed consent, the following set of information is obtained by using a standardized questionnaire:
• Demographic information: marital status, education, occupation and housing conditions, use of healthcare services;
• Physical examination comprising measurement of the following classical markers: medical history, medication, vaccination, current diseases, body mass index, waist and hip circumference, heart rate, blood pressure, self-reported visceral pain, skin elasticity (cutometer measurement), cutaneous itching/pruritus;
• Lifestyle information: physical activity, sedentarity time, smoking, alcohol consumption, solar exposure;
• Fried frailty phenotype (9);
• Functional status: Activities of Daily Living (ADL) (10) and Instrumental Activities of Daily Living (IADL)(11);
• Cognitive status: Mini Mental State Examination (MMSE) (12) and for people lower than 70 years, neuropsychological tests including free and total recall of the Free and Cued Selective Reminding Test (13), ten MMSE (12) orientation items, the Digit Symbol Substitution Test score from the Wechsler Adult Intelligence Scale—Revised (14), and the Category Naming Test (15) (2-minute category fluency in animals);
• Nutritional status: Mini Nutritional Assessment (MNA) (16), food frequency questionnaire (17);
• Oral status: Oral Health Assessment Tool (OHAT) (18);
• Depressive symptoms: Patient Health Questionnaire (PHQ-9) (19);
• Physical performance: Short Physical performance battery (20) and chair rise test (30 seconds)(21-22);
• Participant-reported outcome for cognition (CFI) (23) and mobility, fatigue, and social isolation (PROMIS) (24);
• Objective physical activity and sleep parameters (parameters are collected for one week using activPAL accelerometer);
• Vision: WHO simple eye chart, and the AMSLER Grid;
• IC domains (ICOPE Step 1) by using the ICOPE Monitor app. This app will be used throughout the study for the remote (at-distance) evaluation and monitoring (self-monitoring). All cut-offs operationalizing a deficit in IC comes from the WHO ICOPE program (4,7). At the first visit, the research team explains to the participants how to use the ICOPE Monitor app and monitor their IC domains over time. At each annual regular visit, the research team will confirm participants apply the correct evaluation procedures for assessing their IC. The 6 domains of IC evaluated by the ICOPE Monitor app are:
o Mobility measured by the time (in sec) spent to raise from a chair, at 5-repetition at a maximum speed. Declines will be considered when the time needed to complete the test is higher than 14 sec.,
o Cognitive measured by the 3-word remember test of the MMSE (12) and the following questions: Do you have problems with memory or orientation (such as not knowing where one is or what day it is)? Did you notice a worsening of these disorders in the last 4 months or since the last evaluation? What is the full date today? (day, month, year, day of the week). For the 3-word remember test, three different sets of words will be used to avoid memory bias between two close assessments. Declines are present if the individual is unable to remember at least one word or if he/she provide a wrong response to the orientation question,
o Psychological measured by the following two questions: Over the past two weeks, have you been bothered by: 1. Feeling down, depressed or hopeless? 2. Little interest or pleasure in doing things? One “YES” response determines a decline,
o Vitality/nutrition measured by the following two questions: Have you unintentionally lost more than 3 kg over the last 3 months? Have you experienced loss of appetite? One “YES” response determines a decline. One further question will be asked: what is your actual weight (in kilograms)?
o Sensorial-hearing measured by the Whisper test according to the following procedures: the evaluator must 1/stand about an arm’s length away behind and to one side of the person; 2/ ask the person or an assistant to close off the opposite ear by pressing on the tragus (the tragus is the projection in front of and partly covering the opening of the ear); 3/ Breathe out and then softly whisper a word with two syllables (a set of words will be selected by the Inspire research team), use a common word; 4/Ask the person to repeat the word; and 5/ Move to the other side of the person and test the other ear, use a different word. Not repeating the correct words determines a decline. If the Whisper test can’t be realized, two questions are asked: Did you notice a worsening of these disorders in the last 4 months or since the last evaluation? Does your family complain an acute recent hearing loss?
o Sensorial-vision measured by the following questions: Do you have any problems with your eyes: difficulties in seeing far, reading, eye diseases or currently under medical treatment (e.g. diabetes, high blood pressure)? Declines are considered present when a person responds «yes» to this question and if she did not recently consult an ophthalmologist.

Other examinations are proposed to a limited number of participants (all age ranges and functional status) in a volunteer basis: Dual energy X-ray absorptiometry (DXA) for body composition assessment; Whole body and brain magnetic resonance (MRI); cardiorespiratory fitness (maximum oxygen consumption (V02 max) with blood sampling before and after the effort, and maximal aerobic power), and isokinetic muscle strength. These examinations are proposed annually for the DXA and, every two years for the other tests (MRI, VO2 max, Isokinetic muscle strength). Participant-reported outcome for sarcopenia (SARQoL) (25) is completed for volunteers who perform cardiorespiratory fitness exploration.

Digital assessments

Innovative digital assessments are also planned to be tested, such as home sensors (e.g., for measuring walking speed and its variability in daily environment), automated video analysis of mobility, and 3D facial images for the detection of digital markers of aging. A subgroup of 100 patients monitored by ambient sensors at home or sensors worn on the wrist over the long term will allow to remote and continuous monitoring of the trajectories of the IC domains (especially mobility, sleep parameters and nutrition) (CART/SmartHome research ancillary study, legal authorizations in process). This sub-study, developed in partnership with the CART research project team in United States (ORCATECH Team, Oregon Health and Science University, OR, USA; PI, Jeffrey Kaye; www.ohsu.edu) will allow us to detect subtle changes that are not clinically perceptible, well before the appearance of signs and symptoms and therefore determine innovative digital biomarkers and decision thresholds. These digital biomarkers will be correlated with clinical data but also biological and imaging biomarkers.

Biobank

Biospecimens are collected during the INSPIRE-T cohort for the creation of a biobank.
Biospecimens, including blood, urine, saliva, dental biofilm, are collected from all subjects at baseline and then, annually (the genotyping sample will be collected only at the baseline visit). Nasopharyngeal swabs and cutaneous surface samples are collected from all subjects every two years. Feces, hair bulb, and skin biopsy, are collected optionally at baseline visit (see Table 2).
Aliquots of biological material are stored at -80°C (dental biofilm, saliva, serum, plasma and urine) or at -196° C liquid nitrogen (PBMC) at the central lab (CRB TBR, CHU Toulouse/IFB PURPAN, Toulouse, France). Analysis will be either performed in Toulouse by the local biological teams involved in the INSPIRE project or by any third party not yet determined. The modality of Laboratory Data Transfer from the central lab to other parties will be defined at a later stage. Samples from the biobank may be moved to other US and European countries if required.
The INSPIRE-T biobank is supervised by the CRB TBR where all measures are taken to ensure a quality service based on appropriate resources and adequate safety procedures: observance of Good Laboratory Practice guidelines (the CRB TBR is certified AFNOR since 2015), fully-equipped premises, appropriate, approved and safe equipment (17 freezers -80°C Eppendorf Cryocube, 2 liquid nitrogen tanks with manual and documented filling, Vigitemp probes provide metrological tracking), qualified personnel, safety test and system implementation, sample traceability (all of biological collections are tracked in a specific software (TD Biobank), and CHU servers are daily backed up). All freezers are equipped with an alarm system. Equipments are monitored three times per day. Every failure is reported in the Kalilab software as non-compliance statements.
All the participants will be tested for SARS COV-2 infection via serological tests from blood collection when these latter will be available.
All biological samples are processed within 110 min following a protocol elaborated for INSPIRE purpose and split into smaller aliquots at the INSPIRE-T biobank (Figures 2 &3, online consultation).

Table 2
Samplings proposed to the INSPIRE Human Translational Research Cohort participants for the creation of the biobank

 

Blood collection

For blood collection, all subjects are asked to donate blood (60 ml) by venipuncture after overnight fasting. The blood sample is processed to obtain whole blood, plasma, red blood cells, serum and peripheral blood mononuclear cells (PBMC).
For whole blood and serum, samples are immediately shipped after collection at room temperature to the CRB TBR for the preparation of whole-blood and serum aliquots for freezing at -80° in the INSPIRE-T biobank.
For PBMC, blood samples are immediately transported to the CRB TBR at room temperature and treated within 24 hours from time of collection. PBMC are collected after density gradient-based separation, counted and frozen at 8-14 millions/cells per vial. Frozen vials will be stored in liquid nitrogen.
For plasma and RBC (EDTA/Lithium Heparin/BDP100 Blood), aliquots are immediately prepared after collection in the CRC and stored at -80°C until their shipment to the INSPIRE-T biobank.
When visits are organized by the mobile research team, some blood samples are not performed to limit quality procedures deviations (it concerns the Lithium Heparin tube, the 2 whole blood EDTA tubes and the BD P100 blood tube).

Urine collection

Participants are asked to collect at least 20 ml urine in a sterile screw-top container. The obtained volume is transferred into two vacutainer tubes of 10 ml each and directly shipped at room temperature to the CRB TBR where urine aliquots of 1 ml are prepared and stored at -80°C in the INSPIRE-T biobank.

Saliva

Participants are asked to collect 10 ml saliva in 50 ml Falcon tube (at least 30 min after a meal). The Falcon tube is immediately shipped at room temperature to the CRB TBR where saliva aliquots of 1 ml are prepared and stored at -80°C in the INSPIRE-T.

Dental biofilm collection

Biofilm sampling consists in recovering the biofilm from the external surfaces of the teeth (from natural teeth in priority, from prosthetic teeth if not possible), at the juxta-gingival level by curettage at 4 sites distributed over the dental arches: a sample from the upper anterior teeth, a sample from the upper posterior teeth, a sample from the lower anterior teeth, a sample from the lower posterior teeth. The product of each curettage is individualized in a sterile 1 ml cryotube. The four cryotubes are frozen at -80° in the CRC after the collection and regularly shipped to the CRB TBR.

Nasopharyngeal swabs

Nasopharyngeal swab will be addressed to the Institute of Biology of the CHU Toulouse within 4 hours from time of collection for their analysis (detection and identification of multiple respiratory viral and bacterial nucleic acids). Residuals samples will be stored at -80° to the CRB TBR in the INSPIRE-T biobank.

Skin swab and stripping

Swab samples are done on a skin exposed area (posterior face of the forearm) and a non-exposed area (lower back). Specimens are stored immediately at -80°C in the CRC. Frozen tubes are regularly shipped to the CRB and stored at -80°C in the INSPIRE-T biobank.
In addition, two 14 mm diameter D-squames are applied successively on the exact same area of the posterior face of the forearm: the first one is discarded and the second one is stored in a 2 ml tube. The same procedure is performed on the lower back. Both tubes are regularly shipped to the CRB and stored at -80°C in the INSPIRE-T biobank.

Feces collection (optional)

Feces are collected at the first visit and immediately stored at -80°C. If it is not possible, the participant can return to the research facility within one week with its frozen sample in a coproculture pot, placed in a cool box. The frozen feces samples are regularly shipped to the CRB TBR and stored at -80°C in the INSPIRE-T biobank.

Hair bulb collection (optional)

Twenty hairs are taken with the bulb and immediately stored after the collection in a sterile 2 ml cryotube in the CRC until their shipment to the INSPIRE-T biobank.

Skin biopsy (optional)

A 4 mm skin biopsy is obtained by using a punch. Skin samples are prepared according two different procedures: half of the samples are immediately rinsed, dried and stored at -80°C until its shipment to the CRB TBR; the other are immediately placed in a cryotube with PBS for cells cultures to organize a biobank of skin fibroblasts.
A biobank scientific committee will be set up, in the aim of determining the scientific directions and research priorities, of evaluating ongoing projects and their state of progress, and of resolving any methodological and ethical concerns raised by the studies. It shall i/examine the relevance, feasibility and conditions of implementation of the propositions concerning any analyses; ii/ ensure that national and international partnerships are made formal; iii/ control use of data, especially sample use, and iv/ ensure that participants rights are protected. The data disclosed will be made anonymous (coded, traceable data).

Statistical methods

Since the primary outcome measure of the INSPIRE-T cohort is related to reaching prespecified numbers for recruitment and retention, we will use numbers and percentages. Hypothesis-testing statistics will be employed for some of the secondary outcome measures and the new hypothesis arising through the 10 year duration of the INSPIRE program. Specific statistical analysis plan (SAP) will be written to answer each research question. Big data methods of analysis will be considered when examining the large and diversified amount of data that will be gathered from clinical and para-clinical evaluations, biospecimens, and digital assessments.
Significance will be set at p ≤ 0.05. Analyses will be performed using Stata (v14, StataCorp), SAS (v9.4, Cary, NC, USA), and R (v3.5.2). Statistical analyses will be done by researchers of the Inspire program and professional statisticians. Analyses by gender will be conducted.

Ethical and regulatory considerations

The INSPIRE-T cohort is carried out in accordance with the declaration of Helsinki, which is the accepted basis for clinical study ethics, and must be fully followed and respected by all engaged in research on human beings. The INSPIRE-T cohort protocol has been approved by the French Ethical Committee located in Rennes (CPP Ouest V) in October 2019. This research has been registered on the site http://clinicaltrials.gov (ID NCT04224038).

 

Current progress of the INSPIRE -T cohort

Recruitment status

The first participant was recruited on October 16 2019. Our objective is to recruit at least, 1000 people at baseline (500 during the first year and 500 during the second year of the project) from 20 onwards, including robust, prefrail and frail older adults, as well as disabled people, to be able to better understand the biology of aging across age-ranges and functional status. All the recruiting work is currently carried out by the Toulouse Gerontopole research team on a single site; a mobile clinical research team is also currently active to recruit frailer population (e.g. people unable to come to research facilities) in Toulouse and surrounding areas. Current inclusion rates are 4 participants per day. This rate will allow us to reach our objective of 1000 inclusions in 2 years. Two hundred and forty participants have been included by March 13 2020 (137 women / 103 men; mean age: 74.6 years), and 400 new inclusions are planed until September 2020. Among the 240 enrolled participants, 168 are robust, 60 prefrail and 6 frail with Fried criteria (9). All participants gave their consent for the complete biobanking, 112 participants have accepted the skin biopsy, 231 hair bulb collection and 216 feces collection. All subjects have accepted the DXA, and 211 VO2 max and muscle strength assessment. The sub-study on MRI is planned to start on September 2020. However, recruitment has been temporarily suspended during the COVID-19 pandemic.

Recruitment strategies

Our first plans were to recruit a representative sample of users of primary care services, by inviting people to participate using patients’ list of several family physicians in different areas (with different deprivation levels) of Toulouse (all patients aged 20 years or over being invited to be screened for participation). However, this recruitment approach proved to be unfeasible, mainly because many physicians have been very busy taking care of several viral pathologies during winter 2019-2020 (including COVID-19 from February 2020) (26, 27). Consequently, we decided to diversify the sources of recruitment.
Current recruitment relies mainly on the following strategies: flyers, community outreach strategies, media coverage, newspaper advertising, posters, online promotion, mass mailing, presentations at public events, conferences, study website, dissemination through institution newsletters, identification of participants from previous studies or existing registries, onsite recruitment /medical records review (by investigators/clinical research assistants), dissemination through health care providers : coordination with primary care, memory centers, hospital outpatient clinics, medical centers, physicians (site investigators, primary care physicians), specialists, hospital inpatient lists, private clinics, and finally, dissemination through residential homes, and nursing homes. The recruitment channels of the participant included (and planed over the next 6 months) is detailed in Table 3. Applied strategies are constantly followed and adapted if necessary throughout the recruitment study period (weekly meeting with investigators and study staff).

Table 3
Recruitment strategies implemented in the INSPIRE Human Translational Cohort

 

A mobile research team was implemented in January 2020 to recruit frailer population by collaborating with residential homes, long-term care facilities and post-acute and rehabilitation facilities. Next collaborations are considered with the CRCT in Oncopole-Toulouse (an institution dedicated to cancer research and care) and a private clinic focused on the management of obese people.
Retention strategies are implemented in parallel. It consists of participant-centered values and strategies including (but not limited to) identify proxy contacts, minimize waiting time during study visits, facilitate transportation from and to research facilities, adapt comfortable waiting room facilities, build relationships with study participants; remind nonresponsive participants (contact via phone or email, make phone calls during optimal hours; offering regular feed-back during the follow-up (mailing study updates); offering regular gadgets during the follow up and postcards.

Perspectives

The INSPIRE-T cohort will gather clinical, biological (including imaging), and digital data for subjects of several chronological ages and functional capacity status regularly followed over up to 10 years. One of the most innovative aspects of the INSPIRE-T cohort is that, through a close monitoring of participants with the ICOPE Monitor app, we will obtain clinical and biological data at the moment declines in IC come up. The cohort will provide us the needed resources to improve our understanding of the biological mechanisms of aging and the natural history of loss of IC leading to dependency during aging. By following and monitoring the IC of participants over time, this study will provide information about a new, function-centered healthcare pathway, which would agree with WHO recommendations for an integrated care for older people. At the medium term, this data may inform the development of a pragmatically interventional study testing the effects of this new healthcare pathway on clinical outcomes in older people; this healthcare pathway may be integrated in daily practice in healthcare systems, becoming thus the usual care. Innovative digital solutions (including sensors) proposed in the INSPIRE-T cohort are a promising way to remotely collect and analyze real-life and continuous health related data and thus longitudinal trajectories over time. It makes it possible to detect subtle variation in the IC before a clinical event.
The INSPIRE-T cohort will also perform relevant and extensive bio banking to allow basic and translational research in humans in the field of Geroscience and Healthy Aging. The INSPIRE-T biobank might lead to improving our understanding about molecular and physiological mechanisms involved in healthy aging, interacting with changes linked to specific chronic diseases. This may contribute to establish a set of biomarkers, that could be pharmacologically or non-pharmacologically targetable, and that would characterize biological aging and, then, permit to identify an accelerated aging phenotype. In their recent paper, Ahadi S et al (28) have defined different types of aging patterns in different individuals, termed “ageotypes”, on the basis of the types of molecular pathways that changed over time in a given individual. According to the authors, “ageotypes” may provide a molecular assessment of personal aging, reflective of personal lifestyle and medical history that may ultimately be useful in monitoring and intervening in the aging process”. One of the main objectives of the INSPIRE-T cohort is to identify biological markers that could detect the inter-individual variability of biological processes before it becomes clinically perceptible (29). The identification of biomarkers of aging may help us to identify individuals who are with a high risk of developing age-associated diseases, decline in IC or disability, and to propose personalized strategies, including innovative therapeutics, to prevent or restore impaired functions. Our clinical and biological data will give the opportunity to explore the interaction between changes with aging on inflammation, metabolism, gerosciences in general, and neurodegenerative process leading to Alzheimer’s disease (30, 31) or physical frailty (32), two major causes of loss of functions. The INSPIRE-T cohort will benefit from the availability of plasma neurodegenerative biomarkers (plasma amyloid beta 42/40, neurofilaments, plasma phospho tau) (31). The development of biological markers of frailty is also required to improve the treatment of frail individuals. The etiology of frailty is complex. Proposed biomarkers of frailty include markers of inflammation. As recently proposed by the ICSFR Task Force perspective on biomarkers for sarcopenia and frailty, «machine learning and information technology innovation could thus be used to develop risk scores that could be used in clinical and research settings. Other technologies, such as induced pluripotent stem cells (iPSCs) or skin fibroblasts, could be used to study markers of senescence and could also enable a move towards personalized medicine» (32). Interventions to promote healthy aging will be more effective in people with a risk of decline (29). Hallmarks of aging are under scrutiny in particular DNA alteration, epigenetics, unusual protein production, senescent cells secreting pro-inflammatory factors and others. New therapies aim to target senescent cells or their secretory proteins (the senolytic molecules) and therefore promote healthy ageing are presently under development (33-36).
Finally, the INSPIRE-T cohort gives us the opportunity to federate clinical and biological research teams in Toulouse and Occitania Region to build a research platform of gerosciences discovery to explore mechanisms of aging, and to implement comprehensive translational projects towards the goal of preventing the consequences of aging for a healthy, and long-lived society (37). The animal cohort, generated to “mirror” the human translational cohort, will facilitate the translation of results from basic research to humans and to the clinics. The identification of markers of aging will take advantage of three complimentary approaches to look for the best markers of aging: without a priori approaches (transcriptomics, proteomics, metabolomics); semi a priori approach (metabolism, inflammation, cell cycle, mitochondrial network…); and targeted approach (pre-identified targets such as (but not limited to) Growth Differentiating Factor 15 (GDF-15), apelin, senescent cells, amyloid protein in plasma) (38). From a biological viewpoint, the function-centred approach recommended by WHO represents a challenge due to the multidimensionality that characterizes IC’ trajectories during aging. We will develop an integrative view of biological aging (Figure 2). Three classes of parameters transversal to the whole organism, present in all organs, strongly interrelated and crucial in tissue homeostasis have been selected: i) inflammation and immunity that represents both a warning signals and the house keeping guard of tissue integrity, ii) mesenchymal stem/stroma cells (MSC) allowing support for all function and their adaptation and iii) metabolism that controls any cell decision and the fate of most of them. For all these transversal components, senescence mechanisms will be carefully investigated.

Figure 2
The INSPIRE approach: an integrative view of biological aging

 

Due to the Covid19, teleconsultation has been added for the pre-inclusion and some of the assessment, we will be able also to assess the” stay at home order” on the INSPIRE-T cohort subjects (39).
In conclusion, the INSPIRE-T cohort, nested in the INSPIRE Platform, will contribute to healthy aging and dependency prevention. The INSPIRE-T cohort will foster discoveries of human markers (i.e., biological, clinical, digital) of healthy aging capable of predicting functioning and resilience.

 

Acknowledgments: The Inspire Program is supported by grants from the Region Occitanie/Pyrénées-Méditerranée (Reference number: 1901175), the European Regional Development Fund (ERDF) (Project number: MP0022856), MSD Avenir and the Inspire Chairs of Excellence funded by: Alzheimer Prevention in Occitania and Catalonia (APOC), EDENIS, KORIAN, Pfizer, Pierre-Fabre.
Conflict of interest: All authors of the paper “The INSPIRE research initiative: a program for GeroScience and healthy aging research going from animal models to humans and the healthcare system” declare no Conflicts of Interest related to this manuscript.
Open Access: This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, duplication, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

 

SUPPLEMENTARY MATERIAL

 

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THE INSPIRE RESEARCH INITIATIVE: A PROGRAM FOR GEROSCIENCE AND HEALTHY AGING RESEARCH GOING FROM ANIMAL MODELS TO HUMANS AND THE HEALTHCARE SYSTEM

 

P. de Souto Barreto1,2,*, S. Guyonnet1,2,*, I. Ader3, S. Andrieu1,2, L. Casteilla3, N. Davezac4, C. Dray5, N. Fazilleau6, P. Gourdy5, R. Liblau6, A. Parini5, P. Payoux7, L. Pénicaud3, C. Rampon4, Y. Rolland1,2, P. Valet5, N. Vergnolle8, B. Vellas1,2
for the INSPIRE Program Group

 

1. Gerontopole of Toulouse, Institute of Ageing, Toulouse University Hospital (CHU Toulouse), Toulouse, France; 2. UPS/Inserm UMR1027, University of Toulouse III, Toulouse, France; 3. STROMALab, Etablissement Français du Sang-Occitanie (EFS), Inserm 1031, University of Toulouse, National Veterinary School of Toulouse (ENVT), ERL5311 CNRS, Toulouse, France; 4. Centre de Recherches sur la Cognition Animale (CRCA), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, Toulouse, France ; 5. Institut des Maladies Métaboliques et Cardiovasculaires, Inserm/Université Paul Sabatier UMR 1048 – I2MC 1 avenue Jean Poulhès BP 84225 31432 Toulouse Cedex 4 – France; 6. Centre de Physiopathologie Toulouse Purpan, INSERM/CNRS/UPS UMR 1043, University of Toulouse III, Toulouse, France; 7. ToNIC, Toulouse NeuroImaging Center, Université de Toulouse, Inserm, UPS, France; 8. IRSD, Université de Toulouse, INSERM, INRA, ENVT, UPS, U1220, CHU Purpan, CS60039, 31024, Toulouse, France.
*These authors contributed equally to this work
Corresponding author: Professor Philipe de Souto Barreto, Gérontopôle de Toulouse, Institut du Vieillissement, 37 Allées Jules Guesde, F-31000 Toulouse, France, Phone: (+33) 561 145 636, Fax: (+33) 561 145 640, e-mail: philipebarreto81@yahoo.com.br

J Frailty Aging 2020;in press
Published online April 16, 2020, http://dx.doi.org/10.14283/jfa.2020.18

 


Abstract

Aging is the most important risk factor for the onset of several chronic diseases and functional decline. Understanding the interplays between biological aging and the biology of diseases and functional loss as well as integrating a function-centered approach to the care pathway of older adults are crucial steps towards the elaboration of preventive strategies (both pharmacological and non-pharmacological) against the onset and severity of burdensome chronic conditions during aging. In order to tackle these two crucial challenges, ie, how both the manipulation of biological aging and the implementation of a function-centered care pathway (the Integrated Care for Older People (ICOPE) model of the World Health Organization) may contribute to the trajectories of healthy aging, a new initiative on Gerosciences was built: the INSPIRE research program. The present article describes the scientific background on which the foundations of the INSPIRE program have been constructed and provides the general lines of this initiative that involves researchers from basic and translational science, clinical gerontology, geriatrics and primary care, and public health.

Key words: Gerosciences, biological aging, healthy aging, intrinsic capacity.


 

Introduction

Aging is a major risk factor for the development of several burdensome chronic diseases. From this ancient observation, a new research field has been built with the objective of understanding the interplay between aging and chronic diseases: Gerosciences (1–3). Indeed, the Gerosciences’ area is born from the hypothesis that by manipulating biological aging processes, the onset of chronic diseases during aging might be prevented or postponed, and their severity decreased (4).
To date, the crucial role of aging on the onset of diseases and declines in functional outcomes has been observed mostly from an epidemiological perspective using a chronological approach, ie, chronological age. However, there is evidence supporting that aging (ie, biological and physiological processes) is a most reliable determinant of health than age (ie, the count of years lived) (5), being associated with health outcomes independently of individual’s chronological age (6–8). Although the field of biological aging has made outstanding progresses in the past decades, evidence linking this knowledge with human healthy aging are still limited. Healthy aging is strongly determined by the maintenance of optimal levels of functional capacities, such as mobility, cognition, psychological, nutritional, and sensorial capacities; therefore, linking biological aging not only to chronic diseases, but also to the evolution of functional capacities (9,10) is a research field that deserves to be explored.
Furthermore, the extent to which manipulating aging would modify health trajectories in the life course is almost completely unknown. In order to foster advances in the Gerosciences’ area, a better understanding about how to measure biological aging (ie, determining the aging phenotype) (11, 12) as well as the definition of a set of robust and sensitive biomarkers (including traditional markers from biospecimens, but also digital and other markers) capable of predicting healthy/unhealthy aging as defined by the World Health Organization (WHO) (13) is an indispensable step. Such advances in the measurement of biological aging and definition of biomarkers would ultimately permit individuals’ risk stratification early in the life-course. Moreover, such a set of biomarkers, allied to individuals’ risk stratification, might inform timely pharmacological and non-pharmacological preventive strategies acting directly on aging, contributing to a healthy aging even in late ages.
In this context, the creation of a research platform with baseline and follow-up biological, clinical and digital data for both humans and animals of several chronological ages and functional capacity status constitutes a crucial element to propel discoveries in this broad domain of healthful aging. The objective of the present article is to describe the INSPIRE research program, an initiative aimed at fostering research in the field of Gerosciences and healthy aging.

 

The intrinsic capacity framework: creating the bridge between Gerosciences and healthy aging

Healthy aging was defined by the World Health Organization (WHO) as “the process of developing and maintaining the functional ability that enables well-being in older age” (13). In other words, healthy aging is not the absence of chronic diseases, but the ability to cope in daily life. Although the first foundations of the Gerosciences’ field relate to the interplays between the biology of aging and the biology of diseases (1, 4), recent literature recognizes the importance of functional capacity and frailty for Gerosciences’ investigations (14). Indeed, the Gerosciences’ field investigates the fundamental mechanisms of biological aging; these fundamental mechanisms are shared by several organs and, once their deterioration exceeds the individual’s resilience reserve, they will lead to clinical phenotypes (eg, frailty, disability, diseases, early mortality) (14) that may (or may not) be chronic diseases.
In recent years, experts from the field of biological aging converged into a list of nine major biological and physiological processes that determines aging, the so-called hallmarks of aging: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication (15). Elements of the hallmarks of aging are associated to both declines on functional capacity and the onset and severity of chronic diseases. As examples, we can cite: a) low-grade chronic inflammation, a marker of altered intercellular communication, is associated with declines on cognitive (16), mobility (17) and psychological functions (16), but also with the onset and worsening severity of frailty and diseases (18, 19), such as cardiovascular and metabolic diseases; b) reduction in the repair capacity of muscle fibers, a marker of stem cells exhaustion, may lead to sarcopenia (20) (which is a disease) and decreases in mobility function.
Several other examples might be provided, but the important message to retain is that chronic diseases and body functions share similar biological and physiological processes that partly determine individual’s health during aging. Therefore, it is plausible to accept that the Gerosciences field should embrace elements contributing to health (9, 21) other than chronic diseases, in particular the biological background affecting the levels of IC (body functions) and, thus, healthy aging.
The INSPIRE research program: an overview

INSPIRE is a research program dedicated to biological and healthy aging, aimed at constituting a bio-resource platform going from animals to humans, from cells to individuals, from research to clinical care. INSPIRE will provide clinical, biological and technological resources for research and development on aging open to both academic and business worlds in order to tackle one of the major societal challenges worldwide: promote healthy aging and prevent dependency.
INSPIRE is a public-private initiative that brings together internationally recognized experts from basic and translational science (in particular, in the fields of immunology, metabolism, and stroma), clinical gerontology (ie, researchers, but also physicians and nurses involved in clinical care), primary care and public health. Table 1 displays the main academic and care teams contributing to the INSPIRE initiative.

Table 1
Academic and care teams contributing to the INSPIRE Program

 

INSPIRE main objectives

1. Identifying biomarkers of aging. INSPIRE will provide clinical and digital data as well as biospecimens from both human and animals, to foster the identification and validation of (a set of) biomarkers of aging. Biomarkers are here understood in a broad meaning, going from traditional biomarkers from biofluids or other biomaterials and imaging, to alternative measurements, such as digital markers obtained from both innovative refined automated video and 3D photo analysis.
2. Implementing a function-centered healthcare pathway for aging. In the INSPIRE program, we will implement the Integrated Care for Older People (ICOPE) recommendations from the WHO; ICOPE recommends to focus the care of older individuals according to the five domains of intrinsic capacity (function-centered approach). INSPIRE will allow us to go beyond the simple implementation of ICOPE, by fostering investigations on both the biological changes related to healthy aging and the development of new technologies and metrics enabling the overtime monitoring of functions during aging.

 

INSPIRE main milestones

1. The creation of the INSPIRE Human Translational Cohort, with extensive biobanking. This human research cohort will foster discoveries of human markers (ie, biological, clinical, digital) of healthy aging capable of predicting functioning and resilience.
2. The creation of the INSPIRE Animal Cohort, with extensive biobanking. This animal cohort will be specifically dedicated to the discovery of biomarkers of aging, mechanisms of action, and target-specific investigations.
3. The implementation of the ICOPE program from the WHO in clinical practice, including the remote monitoring of IC domains. This implementation activity will give rise to the INSPIRE Clinical Care Cohort, which will provide information on the evolution of functioning in a real-life population and will inform about the feasibility (including in terms of using digital health tools) of implementing a function-centered model of care (instead of the traditional disease-centered model).

See Figure 1 for a schematic presentation of the INSPIRE program. We briefly describe below the most important characteristics of the three milestones of the INSPIRE Program.

Figure 1
The INSPIRE program for Gerosciences and healthy aging

 

The INSPIRE Human Translational Cohort

This is an observational study that will recruit about 1,000 subjects, men and women, aged 20 years-old or over (no upper limit for age), and affiliated to a social security scheme; will be excluded people having a severe disease with life expectancy lower than five years (lower than one year for the small fraction of INSPIRE participants who are disabled older adults), and those deprived of their liberty by administrative or judicial decision or under guardianship. The population of individuals 60 or over will be oversampled in order to capture major clinical events (eg, declines on functions, onset of disability, frailty, burdensome chronic diseases). Participants will be recruited mainly from the healthcare services (eg, ambulatory geriatrics services, medical records of primary care physicians located in different areas, with different social deprivation levels) in the city of Toulouse and surrounding regions, South-Western, France.
Participants will provide biospecimens: blood, urine, saliva, dental biofilm, nasopharyngeal/oropharyngeal swabbing, and skin swabbing and stripping. Several clinical measurements will be undertaken in all participants, including the five domains of IC (ie, locomotion, cognition, vitality/nutrition, psychological and sensorial capacities), frailty, functional ability, oral health, lifestyle (eg, physical activity (including objectively measured), diet, smoking, sun exposure), as well as participant-reported outcomes (eg, cognition, mobility, fatigue, social isolation). Subsamples of participants will have collected other biospecimens (eg, feces, hair bulb, skin biopsies) and/or will undertake more in-depth evaluations, such as Dual Energy X-ray Absorptiometry (DEXA), whole body and brain magnetic resonance imaging, cardiorespiratory fitness (maximum oxygen consumption (VO²max) and maximal aerobic power), and isokinetic muscle strength. Innovative digital assessments are also planned to be tested, such as home sensors (eg, for measuring walking speed and its variability in daily environment), automated video analysis of mobility, and 3D facial images for the detection of digital markers of aging.
Participants will be evaluated once a year for clinical assessments and for the collection of some biospecimens (eg, blood, saliva, urine). Between yearly waves of data collection, participants will record major clinical information, including adverse events (eg, new diagnosis, fracture), medical consultations, and changes in the prescribed drugs. Furthermore, participants will have their IC domains monitored (with or without the help of a caregiver) each 4-month through the use of either an app, developed in collaboration with WHO, a web platform, or through a phone call by a clinical/research nurse. Once IC declines are confirmed, participants will have a thorough clinical assessment and blood sampling; such information will allow us to investigate the response of some biomarkers of aging at the time declines on IC are detected. All participants will receive the usual care on the basis of the recommendations from the WHO ICOPE model.

 

The INSPIRE Animal Cohort

The animal cohort has been generated to “mirror” the human translational cohort and facilitate the translation of results from basic research to humans and to the clinics. The main objective of the INSPIRE Animal Cohort is to define the relationship between the molecular mechanisms of cell premature senescence and frailty/accelerated aging. With this scope, we first attempted to design a mouse model as close as possible to human frailty/accelerated aging.
To mimic the genetic heterogeneity of human populations, we decided to use outbred Swiss mice, which have a “controlled” genetic heterogeneity and are currently used in longevity studies. Sedentarity and overweight approaches will be investigated because: i) in humans, they are known risk factors of frailty(22–25); ii) as compared to other experimental approaches, sedentarity and overweight are particularly suitable to promote metabolic and immune/inflammation dysfunctions largely described to be involved in progressive/long-term frailty; and iii) the INSPIRE investigators have international reputation in the field of metabolic diseases as well as immune/inflammation dysfunctions.
The cohort will include between 1400 and 1500 (male and female) Swiss mice and four arms: control, high fat/high sucrose diet (HF/HSD), wheel (voluntary physical activity), HF/HSD+wheel. Mice will be tested at the following ages: 6, 12, 18 and 24 months corresponding to approximately 30, 42, 56 and 69 years in humans. The number of mice has been defined based on i) male/female spontaneous and HF/HSD-induced mortality and ii) variability of experimental data. Mice will arrive at the central (CREFRE) animal facility when they will be 3-4 weeks old (specific pathogen free status) and they will be housed in a rate of four per cage. HF/HSD will be administrated from month 6 to month 24. Spontaneous physical activity will be continuously recorded and analyzed using cage electromagnetic plates connected to computers (Techniplast Connected Digital Ventilated Cages – DVC™). Voluntary physical activity will be determined by monitoring the access and use of wheels into the cages.
The animal cohort will be comprehensively phenotyped. This phenotyping will allow: i) firstly, to determine the onset of frailty in relation to HF/HSD and physical activity. Frailty will be defined at the different ages by a basic Frailty Index score including functional cognitive/motor (eg, Y-T-maze test, motor coordination test) and blood (eg, cell count and formula, inflammatory cytokines) tests. Additional tests will be performed to define an extended Frailty Index; ii) secondly, to investigate the role of different organ dysfunctions in the onset/progression of frailty and accelerated aging using “field-specific phenotyping” (eg, metabolic, cardiovascular, immuno/inflammatory, motor/cognitive, stem/progenitor cells) performed at the different ages; thirdly, to identify biomarkers’ profiles predicting normal and accelerated aging using, as a first approach, omics “ID” for blood/urine (peptidome analysis) and feces (microbiota). The omics approaches will be then extended to tissues to identify novel tissue-specific putative biomarkers and therapeutic targets of frailty/accelerated aging.
INSPIRE will also use the accelerated aging model Nothobranchius Furzeri (African Killifish), the animal exhibiting one of the most reduced lifespan (4 to 6 months) among vertebrates; this model will be useful for isolating new hypothesis and validating identified mechanisms. Challenges, such as exercise, fasting or high-fat diet feeding, will be done in order to mimic the different conditions retrieved in human aging. Phenotyping of locomotion and cognition will be performed by camera video-tracking together with metabolic, immune and regenerative properties’ assessments. All the experiments will be performed in males and females aged from 1 to 6 months. A fish-dedicated transgenesis platform will be built to facilitate the validation of the isolated targets.
After phenotyping, mice and fish will be sacrificed and blood, urine, feces and tissues will be collected for biobanking as appropriate.

 

Implementation of the WHO ICOPE model: the INSPIRE Clinical Care Cohort

The ICOPE model proposed by the WHO(26) is a function-centered, instead of disease-centered, framework for the care management of people during aging, which focuses on the clinical domains of intrinsic capacity: locomotion, cognition, vitality/nutrition, psychological, and sensorial capacities. The ICOPE framework is structured into five steps, and the care pathway can be briefly described as follows: In ICOPE step-1, IC clinical domains are screened; people with normal IC levels will receive general health advices (usual care), whereas those with low levels in at least one IC domain will go to ICOPE step-2 and will receive in-depth assessments; if the low IC levels are confirmed, their causes will be investigated; then, in step-3, a patient-centered personalized care plan will be established and monitored; referrals (ICOPE step-4) and caregiver and public health support (ICOPE step-5) may be part of the care plan. The WHO has recently launched the ICOPE app for tablet/smartphone (https://www.who.int/ageing/health-systems/icope/en/), in which data (ie, step 1 and step 2 measurements) and information for the whole ICOPE care pathway can be recorded.
In collaboration with local (Regional Health Agency in the Region Occitanie) and national (French Ministry of Health) health authorities, the INSPIRE program will implement the ICOPE model in the clinical care. For this, primary care providers, in particular community nurses, in the Occitanie Region (South-Western, France), will use the ICOPE app for tablet/smartphone to implement the ICOPE pathway for each older adult they care. ICOPE step-1 (ie, screening for low IC levels) will be performed in all individuals; the other steps of the model may be performed according to available resources in the local care services/facilities. Moreover, a slightly modified version of the step-1 (with discrete/continuous IC variables) of the ICOPE app will be used for the remote monitoring of IC levels over time. All the data will be automatically transferred to a secured database and will be used to investigate the overtime evolution of IC domains.
Once discoveries (eg, biomarkers of aging) emerge from the INSPIRE human/animal cohorts and from other investigators around the world, some of them can be easily and quickly tested and validated in this real-life population composed of people followed by the healthcare professionals who implemented the ICOPE care pathway.

 

What are the novelties brought about by the INSPIRE research program?

One of the hallmarks of INSPIRE relates to the fact this program will cover longitudinally a large age range, from young to very old individuals, and the whole spectrum of the disablement cascade, from robustness (healthy people) to frail to disabled individuals. Moreover, by running two research cohorts in parallel, one with humans the other with animals, we expect to facilitate the cross-talks between human research and experimental models, what will probably speed up the discovery process. It is noteworthy that the animal cohort will mimic this diversity in functional status by providing both healthy and frail animal models to investigations.
Furthermore, in the INSPIRE Human Translational Cohort, clinical assessments and biological material (eg, blood) will be obtained at several time-points and immediately after IC declines are detected; to the best of our knowledge no study has collected biospecimens at the moment clinical symptomatology related to loss of function appears. Indeed, several past and/or on-going studies on aging and biomarkers, including very large studies, such as the UK biobank(27), the All of Us research program (https://allofus.nih.gov/) or still the Canadian Longitudinal Study on Aging(28) (http://www.clsa-elcv.ca/), have collected blood, urine, and/or saliva. However, biospecimens’ collection was done only at baseline or at regular pre-defined time intervals, such as every three years. The issue with these procedures is that clinically meaningful declines in IC as well as the onset of other clinical conditions may happen between two consecutive data collection visits and the biological changes determining the eruption of phenotypic manifestations may, then, be lost because the clinical condition may have been treated before the next wave of data collection. Moreover, the regular remote monitoring of IC will allow us to investigate the overtime fluctuations in IC domains more closely as well as to find out the predictors (eg, biological, behavioral, environmental, and others) of resilience during aging.
Another major innovative aspect of INSPIRE relates to digital markers of aging, which have been only modestly investigated in previous studies. In the INSPIRE human cohort, we are planning to equip initially 30 homes (projections are to equip up to 100 homes) with original sensors that collect daily life data in a continuous flow; this will allow us to investigate their associations with both biological markers of aging and well-established clinical assessments. Another innovative digital approach we are planning to undertake is automated video analysis of mobility as well as 3D facial images; by employing big data techniques, we expect to be able to detect digital markers of mobility decline and biological aging, respectively.
Finally, by implementing the ICOPE model in routine care, the INSPIRE program will be contributing to the change in the care approach to aging, replacing the current disease-centered model by a function-centered healthcare pathway. Furthermore, by following the patients overtime through the ICOPE app, this real-life population may constitute a pool of potential participants for the activities of the INSPIRE program (eg, feasibility of the remote monitoring of IC levels, validation of biomarkers, etc). See Box 1 for a summary of the main novelties of the INSPIRE program.

Box 1
New aspects of the INSPIRE program

 

Although there are Institutions (eg, the Buck Institute ; https://www.buckinstitute.org/) dedicated to investigate biological aging and projects devoted to study biomarkers of aging, such as the European Mark-Age Consortium(29), although animal cohorts dedicated to investigations on biological aging (eg, National Institute on Aging rodent cohort : https://www.nia.nih.gov/research/dab/aged-rodent-colonies-handbook) and Gerosciences(30) exist, and although currently available longitudinal human studies with several years of follow-up may be used to examine the biomarkers of aging and provide metrics of human biological age(31), to the best of our knowledge the INSPIRE program is one of the first research initiatives on Gerosciences to:
1. Provide a comprehensive approach to Gerosciences and healthy aging, going from basic science to clinical research, and then to the healthcare system. For this, INSPIRE includes animal models (mice and fish), human bioresources and clinical and digital outcomes, and the implementation of integrated care for older people (ICOPE), a function- and person-centred approach, in the healthcare system.
2. Collect both clinical and biological data at the moment declines on intrinsic capacity are detected and confirmed, which will contribute to improving the understanding about molecular and physiological mechanisms involved in healthy aging

 

Discussion

To the best of our knowledge, the INSPIRE program is one of the major international research and care initiatives on Gerosciences and healthy aging. INSPIRE has the potential to strongly contribute to the discovery of biomarkers and mechanisms of aging, paving the way for the development of pharmacological and non-pharmacological approaches targeting biological aging. Moreover, by associating the healthcare pathway of older adults to research (32), INSPIRE will inform about both the feasibility of evaluating IC levels in primary care services and the remote overtime monitoring of IC in this population; investigations of the associations between the ICOPE healthcare pathway and clinical outcomes (33, 34) (in particular, the prevention of disability and dependency) and health economics may also be explored.
The expected impact of the INSPIRE program relates to both research and care on aging and health. First, due to its innovative procedures, characterized by clinical and biological data collection at the moment IC declines are detected and confirmed, this project will contribute to improving our understanding about molecular and physiological mechanisms involved in healthy aging; such mechanisms may interact or not with disease-specific biological changes. Ultimately, INSPIRE may contribute to establish a set of biomarkers of healthy aging. Second, by following and monitoring the IC levels of participants over time, this research and care program will provide invaluable information about a new, function-centered healthcare pathway, which would be in agreement with WHO recommendations for an integrated care for older people (26). Third, the digital part of the study may lead to the identification of markers of healthy/unhealthy aging; when validated, such measures have the major advantage of being non-invasive and easily assessable, which means they have the potential to be translated into practice.
In sum, aging is complex and is a major risk factor for most chronic diseases and dependency in late life; we believe its underpinning biological processes can be manipulated and, then, contribute to healthy aging (35, 36). The exponential growth of the aging population, the potential explosion of disabling conditions in the coming decades and the associated catastrophic increase in healthcare costs, ask for urgent solutions. The INSPIRE program will give rise to a unique center gathering expertise from basic and translational science, clinical research, and geriatric care, with the ultimate goal of preventing adverse health consequences of aging, delaying their onset or reducing their severity. The challenges for the INSPIRE program are multiple and reflect challenges faced by the fields of Gerosciences and healthy aging, including: the coordination among researchers from different research areas, from basic science, translational, and clinical research to public health and clinical care; contributing to changing the disease-based healthcare culture, which has been established for decades; identifying biomarkers of aging that are (or that can be in the near future) easily assessable and cheap for facilitating its use in routine clinical care. Although these and other challenges cannot be overcome in a short time interval, we consider that applying the principles of Gerosciences to foster discoveries that can contribute to healthy aging and that can be translated into clinical practice is an exciting and worthy initiative.

 

Acknowledgments: The Inspire Platform was supported by grants from the Region Occitanie/Pyrénées-Méditerranée (Reference number: 1901175), the European Regional Development Fund (ERDF) (Project number: MP0022856), and the Inspire Chairs of Excellence funded by: Alzheimer Prevention in Occitania and Catalonia (APOC), EDENIS, KORIAN, Pfizer, Pierre-Fabre.
Conflcit of interest: All authors of the paper “The INSPIRE research initiative: a program for GeroScience and healthy aging research going from animal models to humans and the healthcare system” declare no Conflicts of Interest related to this manuscript
Open Access: This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, duplication, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

 

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