Social Nesting, Animal Welfare, and Disease Monitoring.

The assessment of welfare and disease progression in animal models is critical. Most tools rely on evaluating individual subjects, whereas social behaviors, also sensitive to acute illness, chronic diseases, or mental health, are scarcely monitored because they are complex and time-consuming. We propose the evaluation of social nesting, a species-typical behavior naturally occurring in standard housing conditions, for such behavioral monitoring. We provide an example of its use to evaluate social deficits and the long-term effects of neonatal tactile-proprioceptive sensorial stimulation from postnatal day 1 to 21, in male and female adult 3xTg-AD mice for Alzheimer's disease compared to sex- and age-matched non-transgenic (NTg) counterparts with normal aging. Social nesting was sensitive to genotype (worse in 3xTg-AD mice), sex (worse in males), profile, and treatment (distinct time to observe the maximum score and incidence of the perfect nest). Since social nesting can be easily included in housing routines, this neuroethological approach can be useful for animal welfare, monitoring the disease's progress, and evaluating potential risk factors and effects of preventive/therapeutical strategies. Finally, the noninvasive, painless, simple, short time, and low-cost features of this home-cage monitoring are advantages that make social nesting feasible to be successfully implemented in most animal department settings.

Pain in Older Adults With Dementia: A Survey in Spain.

The risk of suffering pain increases significantly throughout life, reaching the highest levels in its latest years. Prevalence of pain in nursing homes is estimated to range from 40 to 80% of residents, most of them old adults affected with dementia. It is already known that pain is under-diagnosed and under-treated in patients with severe cognitive impairment and poor/absent verbal communication, resulting in a serious impact on their quality of life, psychosocial, and physical functioning. Under-treated pain is commonly the cause of behavioral symptoms, which can lead to misuse of antipsychotic treatments. Here, we present two Regional and National Surveys in Spain (2015-2017) on the current practices, use of observational tools for pain assessment, guidelines, and policies. Results, discussed as compared to the survey across central/north Europe, confirm the professional concerns on pain in severe dementia, due to poor standardization and lack of guidelines/recommendations. In Spain, observational tools are scarcely used because of their difficulty and low reliability in severe dementia, since the poor/absent verbal communication and comprehension are considered limiting factors. Behavioral observation tools should be used while attending the patients, in a situation including rest and movement, should be short (3-5 min) and scored using a numeric scale. Among the pain items to score, "Facial expression" and "Verbalization" were considered essential and very useful, respectively. This was in contrast to "Body movements" and "Vocalizations," respectively, according to the survey in central/north Europe. Scarce time availability for pain assessment and monitoring, together with low feasible and time-consuming tools, can make pain assessment a challenge. The presence of confounding factors, the low awareness and poor knowledge/education of specific tools for this population are worrisome. These complaints draw future directions to improve pain assessment. More time available, awareness, and involvement of the teams would also benefit pain assessment and management in cognitive impairment. The experiences and opinions recorded in these surveys in Spain and other E.U. countries were considered sources of knowledge for designing the "PAIC-15 scale," a new internationally agreed-on meta-tool for Pain Assessment in Impaired Cognition and the "Observational pain assessment" in older persons with dementia.

Vibrating Tail, Digging, Body/Face Interaction, and Lack of Barbering: Sex-Dependent Behavioral Signatures of Social Dysfunction in 3xTg-AD Mice as Compared to Mice with Normal Aging.

Modeling of Alzheimer's disease (AD), classically focused on the subject-environment interaction, foresees current social neuroscience efforts as improving the predictive validity of new strategies. Here we studied social functioning among congeners in 13-14-month-old mice with normal aging in naturalistic and experimental conditions and depicted behavioral signatures of dysfunction in age-matched 3xTg-AD mice. The most sensitive variables were vibrating tail, digging, body/face and self-grooming, that can be easily used in housing routines and the assessment of strategies. Sex-specific signatures (vibrating tail, digging, and grooming) defined female 3xTg-AD mice ethogram. All animals sleep huddled while barbering was only found in females with normal aging.

Impact of Chronic Risperidone Use on Behavior and Survival of 3xTg-AD Mice Model of Alzheimer's Disease and Mice With Normal Aging.

Psychosis and/or aggression are common problems in dementia, and when severe or persistent, cause considerable patient distress and disability, caregiver stress, and early institutionalization. In 2005, the Food and Drug Administration (FDA) determined that atypical antipsychotics were associated with a significantly greater mortality risk compared to placebo, which prompted the addition of an FDA black-box warning. The American College of Neuropsychopharmacology (ACNP) White Paper, 2008, reviewed this issue and made clinical and research recommendations regarding the use of antipsychotics in dementia patients with psychosis and/or agitation. Increased mortality risk has also been described in cerebrovascular adverse events in elderly users of antipsychotics. In the present work, at the translational level, we used male 3xTg-AD mice (PS1M146V, APPSwe, tauP301L) at advanced stages of the disease reported to have worse survival than females, to study the behavioral effects of a low chronic dose of risperidone (0.1 mg/kg, s.c., 90 days, from 13 to 16 months of age) and its impact on long-term survival, as compared to mice with normal aging. Animals were behaviorally assessed for cognitive and BPSD (behavioral and psychological symptoms of dementia)-like symptoms in naturalistic and experimental conditions (open-field test, T-maze, social interaction, Morris water maze, and marble test) before and after treatment. Weight, basal glucose levels, and IPGTT (i.p. glucose tolerance test) were also recorded. Neophobia in the corner test was used for behavioral monitoring. Survival curves were recorded throughout the experiment until natural death. The benefits of risperidone were limited, both at cognitive and BPSD-like level, and mostly restricted to burying, agitation/vibrating tail, and other social behaviors. However, the work warns about a clear early mortality risk window during the treatment and long-lasting impact on survival. Reduced life expectancy and life span were observed in the 3xTg-AD mice, but total lifespan (36 months) recorded in C57BL/6 × 129Sv counterparts with normal aging was also truncated to 28 months in those with treatment. Sarcopenia at time of death was found in all groups, but was more severe in wild-type animals treated with risperidone. Therefore, the 3xTg-AD mice and their non-transgenic counterparts can be useful to delimitate critical time windows and for studying the physio-pathogenic factors and underlying causal events involved in this topic of considerable public health significance.

Survival Curves and Behavioral Profiles of Female 3xTg-AD Mice Surviving to 18-Months of Age as Compared to Mice with Normal Aging.

New evidence reveals a high degree of heterogeneity in Alzheimer's disease (AD) clinical and temporal patterns, supporting the existence of several subgroups of patients. Prognosticators of end-of-life dementia specific to elderly patients are necessary to address this heterogeneity. Among 3xTg-AD mice, a widely-used model for AD, a very small number of animals overcome advanced neuropathological stages of disease beyond 18 months of age. They are usually females, which reach longevity in spite of worse neuropathological status as compared to males (the morbidity/mortality paradox). We posit that 3xTg-AD long-term survivors could serve to model end-of-life dementia but also aware about the mortality selection bias. In the present study, we performed behavioral and functional phenotype in long-term survivors, 18-month-old female 3xTg-AD mice and age-matched wildtype undergoing normal aging. Animals were followed up until natural death to correlate survival with phenotype assessments. Strong similarity of their behavioral profiles in all the variables analyzed (e.g. reflexes, sensorimotor functions, locomotion, exploration, emotionality, and anxiety-like behaviors) was found, with the exception of memory impairment, which was a salient trait in old 3xTg-AD survivors. The two groups showed similar mean life expectancy and had behavioral correlates among lifespan, neophobia and long-term memory in common, with some distinctions in 3xTg-AD, supporting recent studies in end-of-life patients. In spite of the small sample size, this brief report presents an interesting scenario to further study heterogeneity and survival in Alzheimer's disease. 3xTg-AD survivors may be a model to gain insight into the frailty/survival paradigm in normal and pathological aging.

Early postnatal handling and environmental enrichment improve the behavioral responses of 17-month-old 3xTg-AD and non-transgenic mice in the Forced Swim Test in a gender-dependent manner.

Forced Swimming Test (FST) models behavioural despair in animals by loss of motivation to respond or the refusal to escape. The present study was aimed at characterizing genetic (genotype and gender) and environmental factors (age/stage of disease and rearing conditions: C, standard; H, early postnatal handling; EE, environmental enrichment consisting in physical exercise as well as social and object enrichment) that may modulate the poor behavioural and cognitive flexibility response we have recently described in 12-month-old male 3xTg-AD mice in the FST. The comprehensive analysis of the ethogram shown in the FST considered the intervals of the test (0-2 and 2-6min), all the elicited behavioural responses (immobility, swimming and climbing) and their features (total duration and frequency of episodes). The long persistence of behaviours found in 17-month-old (late-stages of disease) 3xTg-AD mice was comparable to that recently described in males at 12 months of age (beginning of advanced stages) but also suggested increased age-dependent frailty in both genotypes. The poor behavioral flexibility of 3xTg-AD mice to elicit the behavioural despair shown by the NTg mice, was also found in the female gender. Finally, the present work demonstrates that early-life interventions were able to improve the time and frequency of episodes of immobility, being more evident in the female gender of both old NTg and 3xTg-AD mice. Ontogenic modulation by early-postnatal handling resulted in a more effective long-term improvement of the elicited behaviours in the FST than that achieved by environmental enrichment. The results talk in favor of the beneficence of early-life interventions on ageing in both healthy and disease conditions.

Marble-burying is enhanced in 3xTg-AD mice, can be reversed by risperidone and it is modulable by handling.

Translational research on behavioural and psychological symptoms of dementia (BPSD) is relevant to the study the neuropsychiatric symptoms that strongly affect the quality of life of the human Alzheimer's disease (AD) patient and caregivers, frequently leading to early institutionalization. Among the ethological behavioural tests for rodents, marble burying is considered to model the spectrum of anxiety, psychotic and obsessive-compulsive like symptoms. The present work was aimed to study the behavioural interactions of 12 month-old male 3xTg-AD mice with small objects using the marble-burying test, as compared to the response elicited in age-matched non-transgenic (NTg) mice. The distinction of the classical 'number of buried marbles' but also those left 'intact' and those 'changed' of position of marbles or partially buried (the transitional level of interaction) provided new insights into the modelling of BPSD-like alterations in this AD model. The analysis revealed genotype differences in the behavioural patterns and predominant behaviors. In the NTg mice, predominance was shown in the 'changed or partially buried', while interactions with marble were enhanced in 3xTg-AD mice resulting in an increase of marble burying. Besides, genotype-dependent meaningful correlations were found, with the marble test pattern of 3xTg-AD mice being directly related to neophobia in the corner tests. In both genotypes, the increase of burying was reversed by chronic treatment with risperidone (1mg/kg, s.c.). In 3xTg-AD mice, the repetitive handling of animals during the treatment also exerted modulatory effects. These distinct patterns further characterize the modelling of BPSD-like symptoms in the 3xTg-AD mice, and provide another behavioural tool to assess the benefits of preventive and/or therapeutic strategies, as well as the potential action of risk factors for AD, in this animal model.

Secreted and Transmembrane αKlotho Isoforms Have Different Spatio-Temporal Profiles in the Brain during Aging and Alzheimer's Disease Progression.

The Klotho protein is a ß-glucuronidase, and its overexpression is associated with life extension. Its mechanism of action is not fully understood, although it has been recently reported that αKlotho improves synaptic and cognitive functions, and it may also influence a variety of structures and functions during CNS maturation and aging. The αKlotho gene has two transcripts, one encoding a transmembrane isoform (m-KL), and the other a putative secreted isoform (s-KL). Unfortunately, little is known about the secreted αKlotho isoform, since available antibodies cannot discriminate s-KL from the KL1 domain cleaved from the transmembrane isoform. This study shows, for the first time, that the klotho transcript produced by alternative splicing generates a stable protein (70 kDa), and that in contrast to the transmembrane Klotho isoform, it is ten times more abundant in the brain than in the kidney suggesting that the two isoforms may have different functions. We also studied whether klotho expression in the CNS was influenced by aging, Alzheimer's disease (AD), or a healthy lifestyle, such as voluntary moderate continuous exercise. We observed a strong correlation between high expression levels of the two klotho transcripts and the healthy status of the animals. Expression of Klotho in brain areas decayed more rapidly in the 3xTg-AD model of AD than in healthy animals, whilst moderate continuous exercise in adulthood prevents the decline in expression of both klotho transcripts.

Persistence of behaviours in the Forced Swim Test in 3xTg-AD mice at advanced stages of disease.

Forced Swimming Test (FST) models behavioural despair in animals by loss of motivation to respond or the refusal to escape. The present study characterizes the behavioural responses of 12-month-old male 3xTg-AD mice in FST as compared to age-matched no-transgenic (NTg) mice. Paradoxical results were consistently found from what would be expected from their BPSD (Behavioural and Psychological Symptoms of Dementia)-like profile. The comprehensive analysis of the ethogram shown in the FST considered the intervals of the test (0-2 and 2-6min), all the elicited behavioural responses (immobility, swimming and climbing) and their features (total duration, frequency of episodes and mean duration). Both genotypes showed equal number of swimming episodes and climbing attempts during the first interval, that resulted in high swimming times, short climbing and scarce immobility. Thereafter, the NTg mice showed a behavioural shift over time and the immobility response showed up. In contrast, all the measures consistently evidenced that 3xTg-AD persisted with the previous behavioural pattern. Genotype differences consisted in less number of episodes of immobility and swimming, and a low immobility time in favour of swimming. No differences were found in 'climbing' attempts. The behavioural response observed is discussed as a lack of ability of 3xTg-AD mice to shift behaviour over time that may result of poorest cognitive flexibility and copying with stress strategies more than behavioural despair per se.

Crosstalk between behavior and immune system during the prodromal stages of Alzheimer's disease.

The crosstalk in the functional interplay of the neuroimmune system is essential to ensure homeostasis preservation and health. Alzheimer's disease (AD) can be understood in the context of aging of this neuroimmune communication. AD has an important genderdependent component and is benefitted by lifestyle strategies such as physical exercise, enriched environments and nutrition. Recently, the functional and redox state of peripheral immune cells has been proposed as a useful tool for measuring the progression of AD. The present review summarizes the relevance of the disruption of crosstalk among neurons, glial cells, immune mediators and cells from the very beginning of the prodromal stages of AD, when early BPSD symptoms have already started but cognitive function still seems apparently normal. The study of the role of neuroimmune system and how its disruption contributes to the onset of disease may help in understanding its biological mechanisms and in finding behavioral parameters and immunological biomarkers for the prodromic phases. Here we present results of 3xTg-AD mice from pre-morbid to early-stages of AD and how early BPSD-like symptoms correlate with changes in the organometrics of thymus and spleen that are indirect indicators of the immunological status. These functional relationships between behavioral and peripheral system also revealed the existence of differences between biological and chronological ages (an advanced biological age) since the prodromal stages. Overall, the data available suggest that the crosstalk between behavior (nervous) and immune system plays an important role since prodromal stages of AD.

Neophobia, NQO1 and SIRT1 as premorbid and prodromal indicators of AD in 3xTg-AD mice.

Increased oxidative stress seems to be a key factor underlying natural processes of aging, but also to occur prior to neuropathological hallmarks of neurodegenerative diseases. The present work studied the temporal variation of three key antioxidant enzymes in cortex and hippocampus during the development of behavioral and cognitive symptoms in 3xTg-AD mice, and as compared to age-matched controls. At 2 months of age, when no intraneuronal Aß immunoreactivity has been reported, increased neophobia shown as a delayed and reduced rearing, evidenced the onset of BPSD-like symptoms at premorbid stages of disease. In these animals, NQO1 was found increased in both the hippocampus (800%) and cortex (400%) and progressively diminished at older ages. SOD1 was increased in the hippocampus at 4 months of age, when neuronal Aß accumulation has been established. These hippocampal increases of antioxidants before the prodromal emergence of cognitive symptoms support their role as defense mechanisms. SIRT1 levels showed opposite age-dependent changes in cortex (increase) and hippocampus (decrease) relative to controls. Prodromal cognitive deficits emerged at 6 months of age, concomitantly to cortical overexpression of SIRT1 but down-regulation of NQO1 and SIRT1 in the hippocampus, suggesting inadequate antioxidative protection to prevent or delay the subjacent neuronal damage. The present data further support the link between oxidative status and the anxious profile. Their crosstalk may underline AD-pathological mechanisms that may lead to deranged physiology and selective neuronal degeneration. It also points out increased neophobia and high expression of NQO1 among the first indicators of disease in the 3xTg-AD mice.

Impairment of nesting behaviour in 3xTg-AD mice.

Deterioration in executive functions and daily life activities (DLA) are early signs of Alzheimer's disease (AD) that signal the need for caregiver attention. We have addressed this issue in the 3xTg-AD mice model for AD and studied nesting behaviour as a natural DLA of parental structures as well as at early- (6 month-old) and advanced-stages (12 month-old) of the disease in isolated animals. The results show genetic, gender and age-dependent impairment of nesting behaviour but also aware about the relevance of factors such as the temporal course of nest construction and the nesting material. Paper towel consistently showed the impairment of nesting behavior in 3xTg-AD mice since early stages of the disease and in both social conditions. Their nest construction was slow temporal pattern and of poor quality, especially in females and advanced stages of the disease where the deficits were shown from the first day. In all cases, cotton elicited an intense behaviour that lead to perfect nesting during the first 48 h. Genotype, gender and age differences were found in the onset of nesting behaviour, with a time delay in the 3xTg-AD mice, particularly in females. The reported impairment of nesting behaviour in 3xTg-AD provides another behavioral tool to assess the benefits of preventive and/or therapeutic strategies, as well as the potential action of risk factors of AD, in this animal model.