ORIGINAL RESEARCH published: 24 November 2020 doi: 10.3389/fpsyt.2020.572583 Impact of Social Isolation on the Behavioral, Functional Profiles, and Hippocampal Atrophy Asymmetry in Dementia in Times of Coronavirus Pandemic (COVID-19): A Translational Neuroscience Approach Aida Muntsant 1,2 and Lydia Giménez-Llort 1,2* 1 Department of Psychiatry and Forensic Medicine, School of Medicine, Universitat Autònoma de Barcelona, Barcelona, Spain, 2 Institut de Neurociències, Universitat Autònoma de Barcelona, Barcelona, Spain The impact of COVID-19 on the elderly is devastating, and nursing homes are struggling Edited by: to provide the best care to the most fragile. The urgency and severity of the pandemic Amit Anand, Case Western Reserve University, forces the use of segregation in restricted areas and confinement in individual rooms United States as desperate strategies to avoid the spread of disease and the worst-case scenario Reviewed by: of becoming a deadly trap. The conceptualization of the post–COVID-19 era implies Laura Orsolini, University of Hertfordshire, strong efforts to redesign all living conditions, care/rehabilitation interventions, and United Kingdom management of loneliness forced by social distance measures. Recently, a study of Raquel Luiza Santos, gender differences in COVID-19 found that men are more likely to suffer more severe Federal University of Rio de Janeiro, Brazil effects of the disease and are over twice as likely to die. It is well-known that dementia *Correspondence: is associated with increased mortality, and males have worse survival and deranged Lydia Giménez-Llort neuro-immuno-endocrine systems than females. The present study examines the impact lidia.gimenez@uab.cat of long-term isolation in male 3xTg-AD mice modeling advanced stages of Alzheimer’s Specialty section: disease (AD) and as compared to age-matched counterparts with normal aging. We This article was submitted to used a battery of ethological and unconditioned tests resembling several areas in nursing Mood and Anxiety Disorders, a section of the journal homes. The main findings refer to an exacerbated (two-fold increase) hyperactivity and Frontiers in Psychiatry emergence of bizarre behaviors in isolated 3xTg-AD mice, worrisome results since Received: 15 June 2020 agitation is a challenge in the clinical management of dementia and an important cause Accepted: 28 September 2020 of caregiver burden. This increase was consistently shown in gross (activity in most of the Published: 24 November 2020 tests) and fine (thermoregulatory nesting) motor functions. Isolated animals also exhibited Citation: Muntsant A and Giménez-Llort L re-structured anxiety-like patterns and coping-with-stress strategies. Bodyweight and (2020) Impact of Social Isolation on kidney weight loss were found in AD-phenotypes and increased by isolation. Spleen the Behavioral, Functional Profiles, and Hippocampal Atrophy Asymmetry weight loss was isolation dependent. Hippocampal tau pathology was not modified, in Dementia in Times of Coronavirus but asymmetric atrophy of the hippocampus, recently described in human patients Pandemic (COVID-19): A Translational with dementia and modeled here for the first time in an animal model of AD, was Neuroscience Approach. Front. Psychiatry 11:572583. found to increase with isolation. Overall, the results show awareness of the impact of doi: 10.3389/fpsyt.2020.572583 isolation in elderly patients with dementia, offering some guidance from translational Frontiers in Psychiatry | www.frontiersin.org 1 November 2020 | Volume 11 | Article 572583 Muntsant and Giménez-Llort Impact of Isolation in Dementia neuroscience in these times of coronavirus and post–COVID-19 pandemic. They also highlight the relevance of personalized-based interventions tailored to the heterogeneous and complex clinical profile of the individuals with dementia and to consider the implications on caregiver burden. Keywords: isolation, confinement, risk factors, neuropsychiatric symptoms in dementia, gender, nursery homes, animal models-rodent, brain asymmetry INTRODUCTION the incorrect use of the term “social distancing” to refer to a number of measures to increase physical space between people The impact of COVID-19 on the elderly is devastating despite to prevent the spread of COVID-19 (9). It also highlights the nursing homes and residential care homes struggling to provide relevance of referring instead to “physical distancing” and to the best care to the most fragile of the elderly population, who conceptualize the policies to avoid disconnection from family due to their physical and/or mental conditions already needed and loved ones because in the current situation there is a continuous surveillance and/or professional care. Between 70 and pressing need to stay emotionally and socially connected (9). 80% of nursing home residents are affected by dementia (1), a Social vulnerability, frailty, and mortality are a dangerous triad mental disease that, per se, is associated with increased mortality for the elderly as well (4). Social isolation is considered a risk in comparison with aged control populations (2, 3). In these factor for morbidity and mortality since it strongly contributes scenarios, the severe constraints that all other parts of the society to comorbid conditions such as hypertension, cardiovascular are facing not only exist but also may have a greater impact on disease, cognitive decline, depression, and early mortality or the the elderly due to their vulnerability, frailty, and increased risk of disabilities related to them (10–14). Social isolation not only mortality (4). increases the risk of dementia but it is also associated with an Official data on the numbers of death linked to COVID-19 in increased memory decline and exacerbation of the symptoms care home residents are still difficult to ascertain. Differences in in dementia patients (15, 16). Thus, the singular features of testing and the policies of different countries make international this pandemic will also generate collateral effects in people’s comparisons difficult. Despite these difficulties, it is obvious that health, mostly in patients (those already ill) without COVID-19 nursing home residents have a higher risk of dying from COVID- (17). Recent reports suggest that a pandemic can be a traumatic 19. The most recent report in Europe (Amsterdam) shows that experience for some individuals and lead to posttraumatic stress mortality is higher in residents with confirmed COVID-19 with disorder (PTSD). Although it may depend on several other risk more typical symptoms (fever, dyspnea, coughing), reaching factors, the COVID-19 pandemic can be a trigger for mental 37%, but even when no typical symptoms but sore throat and health problems (18, 19). The emergence of psychiatric problems delirium are present, mortality in residents with confirmed and/or deterioration of mental health are getting attention, and COVID-19 is 27% (5). Early international evidence, with the technical guides for interventions focusing on the individual actual official data, indicates that about half of all people who have needs are being developed urgently. New adaptations, such as died from COVID-19 were care home residents (6). telecare, are playing a very important role (20, 21). Telepsychiatry The urgency and severity of this pandemic has forced care is becoming a useful tool for patients with mental disorders institutions to use segregation in restricted areas (floors) and (22). For example, a current report on older adults with mild confinement in individual rooms as desperate strategies, first, cognitive impairment or mild dementia performed by Telehealth to avoid the spread of disease, and thereafter to hamper the home support shows that those who were living alone during the worst-case scenario of becoming a deadly trap. With visits to confinement reported decreased well-being, greater anxiety, and nursing homes and long-term care facilities banned, residents more sleeping problems (23). Coping with stress and negative have no face-to-face contact with their families. Group activities and sad emotions have a stronger impact on the elderly due are no longer possible in care homes or nursing homes. to their age-related immunosenescence and the neuro-immune Residents have become more socially isolated, lacking the dysregulation associated with these psycho-social processes (24). social and environmental enrichment that are key rehabilitation A current study of gender differences in COVID-19 found that factors to counteract their progressive physical and/or mental in spite of males and females having the same prevalence of the deterioration. Moreover, redistribution and relocation of rooms disease, males are more at risk for worse outcomes and death can increase the stress and behavioral problems as the new independent of age (25). According to the morbidity/mortality arrangements become a new environment for the patient (7, 8). paradox, also in dementia males show worse survival and While we wait for the vaccine to arrive, the conceptualization deranged neuro-immuno-endocrine system than females despite of elderly care in the post–COVID-19 era implies strong efforts their less bad neuropathological status (2, 3). AD, as many to redesign all living conditions and lifestyles, to find new care other forms of dementia, is one of the principal causes of and rehabilitation strategies, and to provide better management disability in old adulthood. It is defined as an accumulation of loneliness forced by physical distance. Due to the impact of amyloid-β (Aβ) plaques and tau-containing neurofibrillary of isolation on mental well-being, the WHO cautions about tangles (NFTs), accompanied by a progressive memory decline Frontiers in Psychiatry | www.frontiersin.org 2 November 2020 | Volume 11 | Article 572583 Muntsant and Giménez-Llort Impact of Isolation in Dementia (26). Neuropsychiatric symptoms (NPS) (27), also called MATERIALS AND METHODS behavioral and psychological symptoms of dementia (BPSD), are observed in 90% of patients and may include agitation, anxiety, Animals verbal or physical aggression, sundowning behavior, wandering, A total of 29 11-month-old (11.1 ± 0.16) male mice from the depression, challenging and disruptive behaviors, hallucinations, Spanish colonies of homozygous 3xTg-AD (n = 20) and non- etc. For instance, the prevalence of aggression is between 33 transgenic (NTg, n = 9) mice in a C57BL/6 background were and 46%, with males especially affected (28, 29). These NPS are used. The 3xTg-AD mice were genetically engineered at the highly associated with caregiver burnout (30), frequently leading University of California, Irvine as described previously (51). to institutionalization (31) as their management usually needs Animals were housed three or four per cage or isolated pharmacological treatment supported by non-pharmacological and maintained in Macrolon cages (35 × 35 × 25 cm) under interventions. However, despite efficacy in the reduction of standard laboratory conditions of food and water ad libitum, 22 most of these challenging symptoms by antipsychotics (32), they ± 2◦ C, a 12-h light/dark cycle, and relative humidity of 50–60%. should not be used routinely for the treatment of aggression When isolated, animals still were socially connected through and psychosis in these patients because they have an associated olfaction and audition, and as in the group-housed conditions, increased risk of cerebrovascular accidents, respiratory diseases, the cages were enriched with nesting materials. Behavioral and mortality (33–35). Unfortunately, at a practical level, the assessments were performed blind to the experiment, in a medication reviews are infrequent, and antipsychotics are used counterbalanced manner, in the light cycle, from 09:00 to 13:00 h. on a long-term basis despite their higher mortality risk (36). It All procedures were in accordance with the Spanish legislation also has been noted that care homes usually present a higher on the “Protection of Animals Used for Experimental and Other prevalence of antipsychotic prescription, and this seems to be Scientific Purposes” and the EU Directive (2010/63/UE) on this increased in those with fewer staff (37, 38), important data to be subject. The protocol CEEAH 3588/DMAH 9452 was approved taken into account within the pandemic consequences. by Departament de Medi Ambient i Habitatge, Generalitat de The present report aims to address these questions from Catalunya. The study complies with the ARRIVE guidelines a translational approach, studying the impact of long-term developed by the NC3Rs and the aim to reduce the number of isolation in male 3xTg-AD mice modeling advanced stages of animals used. AD and as compared to age-matched group-housed 3xTg-AD mice and non-transgenic (NTg) counterparts with normal aging. Behavioral Assessments This mice model of AD, created in the laboratory of Prof. The impact of social isolation was measured in 11-month-old Frank M. LaFerla at the University of California, Irvine, harbors 3xTg-AD mice after a long-term, naturally occurring housed PS1/M146V, APPswe, and tau P301L human transgenes and isolation (8.5 ± 0.3 months). The battery of behavioral tests uniquely mimics various symptoms of the disease in temporal consisted of a series of classical unconditioned tasks measuring and neuroanatomical patterns like those observed in patients locomotion and exploratory activity, anxiety-like behaviors, (39, 40). Moreover, 3xTg-AD male mice have shown increased cognitive functions, and daily life activities (39). The results were mortality as compared to females and NTg counterparts (41– compared to age-matched, group-housed 3xTg-AD mice and 44), which means many 3xTg-AD males arrived at old age in non-transgenic counterparts with normal aging. socially isolated housing conditions. The impact of individual housing conditions at different ages of the rodents’ lifespan is Day 1: Corner Test and Open Field Test used as an animal model for some mental health disorders. Post- Neophobia to a new home-cage was assessed by a 30-s trial. weaning social isolation of mice is a well-established animal Animals were placed individually in the center of a clean standard model for developmental disorders, such as attention-deficit home-cage, filled with wood shaving bedding. The number of hyperactivity disorder, autism spectrum disorder, and specific corners visited was recorded (52). The latency to realize the first learning disability (45). In the case of adolescent rodents, social rearing (vertical displacement) and the number of rearings were isolation is regarded as a model of heightened vulnerability also registered (39). to comorbid alcoholism and anxiety disorders (46), while in Immediately after the corner test, mice were placed in the adults it is used for pharmacological treatments for PTSD (47). center of an illuminated (20 lx) open field (metalwork, white box, Yet, only a few laboratories have studied the effects of poor 42 × 38 × 15 cm) and exploratory and anxiety-like behaviors social housing, and they show a worsening of the cognitive and were evaluated during 5 min (53). First, the ethogram of action beta-amyloid pathology in animal models for dementia (48–50). programs described by the temporal profile of the following Here, for the first time, we investigated the impact of long- sequence of behavioral events was recorded: duration of freezing term social isolation in the elder 3xTg-AD male mice, focusing behavior, latency to leave the central square and enter the on several cognitive, BPSD-like, and daily-life-activity functions, peripheral ring, and performing first wall rearing as well as physical status (body, liver, kidney, and spleen weights) as well latency and total duration of self-grooming behavior. Second, the as the neuropathological AD-phenotype. Thus, the animals were time course and total levels of exploratory activity were measured confronted with a battery of tests that included enclosures of as horizontal (number of crossings of 10 × 10-cm squares) differing levels of anxiogenic conditions that, for the purpose of and vertical (rearings with a wall support) locomotor activity. the present work, could resemble or translate into several areas in Third, variables of emotionality (54) included the number of nursing homes. defecations, the presence of urine, and the grooming behavior, Frontiers in Psychiatry | www.frontiersin.org 3 November 2020 | Volume 11 | Article 572583 Muntsant and Giménez-Llort Impact of Isolation in Dementia their number, latency, and total time. Finally, as described Statistics previously (55), we evaluated the presence of bizarre behaviors Results are expressed as mean ± SEM. SPSS 20.0 software such as stereotyped rearings without wall support. was used. Differences were studied using multivariate general linear model analysis, followed by post hoc Duncan’s tests Day 2: Spontaneous Activity Test when possible. Repeated measures ANOVA was used to study The mice were individually tested in a new home-cage containing differences in the time course of behaviors. Student’s t-test was a small amount (25 ml) of clean sawdust on the floor. Here, used to compare two independent groups, per genotype or (horizontal) spontaneous locomotor activity was continuously isolation. For categorical variables, chi square or Fisher’s exact test recorded during a 45-min period and short-term spatial with 2 × 2 were used. Pearson’s correlation analysis evaluated the habituation was determined by means of the VideoTrack analysis pathological/behavioral correlates. In all the tests, p < 0.05 was system (ViewPoint Behavior Technology, Lion, France) (56). considered statistically significant. Day 3: T-Maze Coping-with-stress strategies, risk assessment, and working RESULTS memory were assessed in a spontaneous alternation task (57) in a black T-shaped maze. The apparatus consisted of a woodwork, The results, depicted in Figures 1–7 and Table 1, show the two short arms of 30 × 10 cm, and a long arm of 50 × 10 cm. impact of social isolation on the behavioral, functional, and The animal was placed inside the long arm of the maze with its neuropathological phenotype of 11-month-old male 3xTg-AD head facing the end wall, and it was allowed to explore the maze mice, an age mimicking advanced stages of the disease and as for a maximum of 5 min. The goal latencies, namely to move compared to age-matched NTg mice. and turn (freezing behavior), to reach the intersection, the time elapsed until the animal crossed (4 paws criteria) the intersection Corner Test of the three arms, and the total time invested to explore the three The horizontal and vertical behaviors in the corner test are arms of the maze (test completion criteria) were recorded. The illustrated in Figure 1. Increased neophobia as compared to entry of an already visited arm in the trial before completing the NTg mice was found in 3xTg-AD mice shown as a statistically test was considered an error. Defecation boli and urination were significant reduction in the number of visited corners and also noted. rearings (p < 0.05). In the single-housed 3xTg-AD mice, genetic differences with NTg mice were lost as a result of increased Days 4–6: Nesting horizontal and vertical activities. Although their rearing latencies In a regular manner, for animal well-being, the cages were did not reach statistical significance, in both 3xTg-AD groups, enriched with nesting material. However, in order to specifically they were indicative of increased neophobia response. assess this daily life activity, our 3-day protocol for nesting behavior using tissue paper was used (58). One paper towel tissue Open Field Test (70 × 30 mm of “paper towel”) (Sudelab, Rubí, Barcelona, Spain) Figures 2A–C illustrates the temporal curves (left) and total was introduced in the home-cage. On the next day, 48 and 72 h accumulated counts (right) of horizontal and vertical exploratory later, the nests were assessed according to the Deacon 5-point activity during the 5 min in the open field test performed scale (59) from 1 to 5 as follows: 1 = not noticeably touched, 2 = immediately after the corner test. The ethogram was developed partially torn up, 3 = mostly shredded but often no identifiable similarly in the three groups of mice, and the presence of site, 4 = identifiable but flat, 5 = perfect or nearby. Pictures were thigmotaxis indicated the preference for peripheral protected taken prior to evaluation for documentation. areas as compared to the open and central ones (see Table 1). However, the time course of the horizontal (Figure 2A left, p Day 6: Pathological Status < 0.001) and vertical (Figure 2B left, p < 0.001) activities also Bodyweight was recorded to monitor the physical status of indicated that isolated 3xTg-AD mice exhibited a hyperactive animals. The size (weight in mg) and relative size (% vs. pattern. Thus, a two-fold increase in horizontal activity (groups bodyweight) of liver, kidneys, and spleen were also recorded, as p < 0.05, post hoc isolated 3xTg-AD p < 0.05 vs. NTg and an indirect measure of the physiological status of the metabolic, vs. 3xTg-AD) and increasing elicitation of vertical exploration excretory, and peripheral immune organs, respectively (41). through the test (groups p < 0.05, post hoc isolated 3xTg-AD Tissue samples were stored at −80◦ C for further biochemical p < 0.05 vs. NTg). These enhanced activity levels resulted in analysis (see below). For biochemical analysis of the level of tau statistically significant increased total levels of both horizontal pathology, right and left hippocampus were dissected, frozen and vertical activities (Figures 2A,B right, respectively; p < 0.05 in dry ice, and kept at −80◦ C. Thereafter, frozen samples were vs. NTg and vs. 3xTg-AD mice). In contrast, the levels of lysed in cold lysis buffer containing protease and phosphatase horizontal activity were very low in both middle-aged NTg and inhibitors (Sigma). Protein content was quantified with the 3xTg-AD mice housed in standard conditions, and their patterns BCA Protein Assay Kit (Thermo Scientific), resolved with SDS- were regular, with poor habituation to the test. As illustrated in polyacrylamide gel electrophoresis, and detected by Western Figure 2C, the presence of stereotyped vertical rearing without blotting using the mouse anti-phosphorylated Ser396/404 (PHF- wall support, absent in NTg mice, was late (210 s), exhibited 1; 1:1000) antibody. Quantification was performed using the in only 1/10 group-housed 3xTg-AD mice. In contrast, 4/10 ImageStudio Lite 5.2 software. isolated 3xTg-AD mice exhibited this bizarre behavior with an Frontiers in Psychiatry | www.frontiersin.org 4 November 2020 | Volume 11 | Article 572583 Muntsant and Giménez-Llort Impact of Isolation in Dementia were found among groups in the other variables of the test (see Table 1). Spontaneous Activity Test As illustrated in Figure 3, the spontaneous activity assessed during a long period of 45 min demonstrated a statistically significant sustained hyperactivity pattern induced by isolation in 3xTg-AD mice in contrast to the poor activity levels shown in animals housed in standard conditions. Here, although the locomotor activity levels of 3xTg-AD mice did not reach statistical significance, they were below those of NTg mice. The initial 5-min period reproduced the results found in the 5-min session in the open field test. The hyperactive pattern was evolving during the different periods of habituation to the test, but still with significantly higher levels of activity as compared to 3xTg-AD mice housed in standard conditions (time p < 0.001; groups, p < 0.05, post hoc isolated 3xTg-AD mice vs. 3xTg-AD mice). T-Maze Figure 4A depicts the fast ethogram performed by isolated 3xTg- AD animals as compared to NTg and 3xTg-AD mice in a spontaneous alternation paradigm in the T-maze. Although all animals showed similar latencies to turn from the “facing the wall” starting position, the latency to cross the intersection of the three arms and the latency to complete the task were significantly shorter as compared to the long delays usually exhibited by 3xTg- AD mice (p < 0.05 vs. 3xTg-AD). Decision making, measured as the difference between latency to intersection and latency to cross it with 4 paws criteria, was longer in both 3xTg-AD groups, while NTg mice made the decision in a short gap of 10 s (genotype effect, p < 0.05). As in the previous tests, the increase in locomotor activity of the isolated 3xTg-AD mice resulted in completing the test faster than the other groups (p < 0.05 vs. NTg and vs. 3xTg-AD). As detailed in Figure 4B, a number of animals (3/9) exhibited the NTg coping-with-stress strategy of resting with their backs protected in the starting point. Another two of them spent the time but did not cross the intersection (2/9) or did not complete the test successfully (1/9). Among 3xTg-AD mice, a similar total number of animals did not even start (4/10) or complete (1/10) the test. The proactive strategy of isolated 3xTg-AD mice resulted only in mice resting (1/10), and two were not able to complete the test (2/10). In this small representation (NTg: 6/9; 3xTg-AD: 6/10; isolated 3xTg-AD: 9/10), the number of spatial working memory errors (revisiting an explored area) were recorded in those animals able to initiate the task. Although the sample size was not enough to reach statistical significance, FIGURE 1 | Effects of isolation on 11-month-old male 3xTg-AD mice in the Corner test. Results are expressed as mean ± SEM. (A) Horizontal activity; (B) an increased number of errors in spatial alternation was noted in Vertical activity. Groups: NTg, group-housed non-transgenic mice; 3xTg-AD, the isolated 3xTg-AD mice (Figure 3C). group-housed 3xTg-AD mice; Isolated 3xTg-AD, single-housed 3xTg-AD mice. Statistics: G, genotype effect, *p < 0.05. Student t-test comparisons are Nesting shown in the graphs as # p < 0.05 vs. the NTg-group. Representative nest buildings at 72 h are shown in Figure 5. The effect of isolation was consistently shown since the first day of assessment. While nests of NTg and 3xT-AD mice were partially early appearance as part of the fearful response during the first torn up (mean score 2), those of isolated 3xTg-AD mice were minute of the test (statistics on the mean latency, p < 0.05 vs. NTg mostly shredded although not yet in an identifiable site (mean and vs. 3xTg-AD mice). No statistically significant differences score 3). The progressive improvement in nest building allowed Frontiers in Psychiatry | www.frontiersin.org 5 November 2020 | Volume 11 | Article 572583 Muntsant and Giménez-Llort Impact of Isolation in Dementia FIGURE 2 | Effects of isolation on 11-month-old male 3xTg-AD mice in the Open field test. Results are expressed as mean ± SEM or incidence (%). (A) Horizontal activity: Left, time course; right, total; (B) Vertical activity: Left, time course; right, total; (C) Bizarre behavior: Incidence (left), latency (center), latency when present. Groups: NTg, group-housed non-transgenic mice; 3×Tg-AD, group-housed 3xTg-AD mice; Isolated 3xTg-AD, single-housed 3xTg-AD mice. Inset of bars, number of animals exhibiting the behavior. Statistics: G, genotype effect, *p < 0.05. Student t-test comparisons are shown as # p < 0.05, ## p < 0.01 vs. the NTg-group; *p < 0.05 vs. the 3×Tg-AD mice. Frontiers in Psychiatry | www.frontiersin.org 6 November 2020 | Volume 11 | Article 572583 Muntsant and Giménez-Llort Impact of Isolation in Dementia FIGURE 3 | Effects of isolation on 11-month-old male 3xTg-AD mice in the Spontaneous activity test. Results are expressed as mean ± SEM. Horizontal activity: left, time course; right, total distance covered. Groups: NTg, group-housed non-transgenic mice; 3xTg-AD, group-housed 3xTg-AD mice; Isolated 3xTg-AD, single-housed 3xTg-AD mice. Statistics: Student t-test comparisons are shown as # p < 0.05 and vs. the NTg-group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. the 3xTg-AD mice. us to see a delay in 3xTg-AD mice. Thus, the mean score 3 for nest AD-genotype (mean weight; right hippocampus: 12.0 ± 0.57 mg construction was achieved by NTg at 48 h, but 3xTg-AD mice vs. left hippocampus 16.5 ± 0.83 mg, p < 0.001). The effects of needed an extra day (72 h). At the endpoint, identifiable but flat isolation were seen as a trend of the lower mean weight of the nests (mean score 4) were only seen in the isolated group, with hippocampus. Western blot analysis showed a significant AD- nests of very few animals considered perfect or nearly reaching genotype-dependent (p < 0.01) increase in phosphorylated tau the maximum score of 5 (1/9 NTg, 0/10 3xTg-AD but 3/10 at Ser396/404 (PHF1) residues in hippocampal lysates of both isolated 3xTg-AD mice). groups of 3xTg-AD mice as compared with NTg mice (both, p < 0.01). The levels of tau did not differ between the right and left Pathological Status hemispheres. Isolation induced in 3xTg-AD mice showed a slight Bodyweight of animals was monitored during the battery of increase in tau, but it did not reach statistical significance. behavioral tests as a measure of their physical/healthy status. Meaningful correlation analysis with behavioral variables Figures 6, 7 illustrate the results of a representative sample sensitive to genotype and/or isolation are indicated as insets in of animals used for pathological evaluation. As detailed in Figures 7A,B. The weight of the hippocampus was found to be Figure 6A, genotype effects in the bodyweight indicated a positively correlated with the activity cage variables (p < 0.05), decrease in the groups of 3xTg-AD mice (p < 0.05). This effect kidney weight (p < 0.05), and bodyweight (p < 0.05), while it was mostly due to the notorious weight loss in isolated 3xTg- was found to be negatively correlated with the nest building end AD mice, which reached statistical significance as compared to point at 72 h (p < 0.05). The left hemisphere was correlated the normal weight of NTg mice (p < 0.05). In Figure 6B, the with the activity test (p < 0.05), while the right hippocampus size (weight in mg) and relative size (% vs. bodyweight) of liver, was the one involved with the correlates in body (p < 0.05) and kidneys, and spleen were also recorded as an indirect measure of kidney (p < 0.01) weights, as well as nesting behavior at 24 h (p their physiological/healthy status. Similarly, the bodyweight loss < 0.05). Conversely, the levels of tau pathology were negatively was reflected in the liver, kidneys, and spleen but only reached correlated with most time points and total activity developed in statistical significance in the excretory (genotype effects, p < 0.01) the activity-cages and the kidney weight (all, p < 0.05). and immune (isolation effect, p < 0.001) organs. The analysis of relative weight indicates that the weight loss induced by isolation may refer to a general reduction in somatic size in transgenic DISCUSSION animals. Still, the spleen was the most sensitive organ to show the effects of isolation. In the assessment of the long-term effects of Figure 7 shows the weights and biochemical analysis of the preventive/therapeutic interventions, risk factors, and hazards levels of tau pathology in the left and right hippocampus, the through different periods of our lives, the translational AD-target area. The size (weight in mg) of L/R hippocampus is neuroscience approach benefits of the shorter life span of depicted in Figure 7A. Differences in L/R hippocampus weight rodents as compared to humans is a key time frame that can were seen in all the groups (less weight in the right hippocampus) be critical in this respect. Like in humans, housing conditions although the hippocampal asymmetry was only attributable to can have a significant impact on animal behavior, and this Frontiers in Psychiatry | www.frontiersin.org 7 November 2020 | Volume 11 | Article 572583 Muntsant and Giménez-Llort Impact of Isolation in Dementia more than twice as likely to die (25). On the other hand, it is well-known that dementia is associated with increased mortality, and according to the morbidity/mortality paradox, males show worse survival and deranged neuro-immuno-endocrine system than females despite their decreased bad neuropathological status (2, 3). In the present work, at a translational level, we studied the effects of social isolation in the behavioral, functional, and neuropathological profiles of male 3xTg-AD mice, one of the most used models mimicking the AD shown in humans, at 11 months of age mimicking advanced stages of AD (40), and as compared to age-matched NTg and 3xTg-AD mice housed in standard (grouped) conditions. Fear, anxiety, and agitation are the most common neuropsychiatric symptoms (NPS) associated with dementia that can be studied in mice models of AD (39). They are also called behavioral and psychological symptoms associated with dementia (BPSD) since they also include behavioral disruptive behaviors, they have an early onset, and they worsen with the progression of the disease. Since most behavioral tests assess the animals in behavioral paradigms, the analysis of the actions program (61) allows us to dissect several behavioral domains and constructs including not only the hallmark cognitive dysfunction of AD but also NPS-like behaviors to be recorded (62). For that purpose, a battery of classical unconditioned and ethological tests with the purpose of recording the quantitative and qualitative features of cognitive dysfunction as well as BPSD-like behaviors modeled in 3xTg-AD mice was used (39, 63). The tests evaluated cognition, locomotion/exploration, emotionality, and anxiety- like behaviors in four enclosures differing in the levels of anxiogenic conditions and that, for the purpose of the present work, could resemble or translate into several areas in nursing homes: the mild neophobia response in a cleaned home-cage (Corner test); the direct exposure to an open and illuminated area, which is aversive to nocturnal species like rodents (Open- field); the spontaneous activity and habituation in an activity cage during a long 45-min period (Activity test); and the coping- with-stress strategies and spontaneous alternation in black corridors of a maze resembling burrows (T-maze test). Finally, an ethological nesting behavior was included as a daily life activity involving executive but also protective/thermoregulatory functions. The health status, inferred from their physical status, FIGURE 4 | Effects of isolation on 11-month-old male 3xTg-AD mice in the was monitored by means of the bodyweight and the weight of T-maze. Data are expressed as the mean ± SEM or incidence (%). liver, kidneys, and spleen, organs that show severe complications (A) Ethogram; (B) Incidence to the goal; (C) Spatial working memory. Groups: associated with the COVID-19 scenario (64, 65). Brain pathology NTg, group-housed non-transgenic mice; 3xTg-AD, group-housed 3xTg-AD of the left and right hippocampus, the hallmark target area in AD, mice; Isolated 3xTg-AD, single-housed 3xTg-AD mice. Inset of bars, number of animals exhibiting the behavior. Statistics: G, genotype effect, *p < 0.05. measured by weight and hallmark tau pathology, was performed Student t-test comparisons are shown as *p < 0.05 vs. the 3xTg-AD mice. and correlated with behavior. The main findings refer to an exacerbated (two-fold increase) hyperactivity and emergence of bizarre behaviors in isolated 3xTg-AD mice, worrisome results since agitation is a challenge in the clinical management of can also be important in those modeling diseases where social dementia and an important cause of caregiver burden. This behavior is already impoverished (60). Using these advantages, increase was consistently shown in gross (activity in most of we can explore in rodents the impact of social isolation or social the tests) and fine-motor (thermoregulatory nesting) function. restrictions due to social distance imposed by COVID-19 and Isolated animals also exhibited re-structured anxiety patterns in post-COVID scenarios in the most fragile elder population. (negative valence system), emergence of bizarre behaviors, and Recently, gender differences in COVID-19 found that men flight coping-with-stress strategies. Worse risk assessment was are likely to suffer more severe effects of the disease and are dependent on AD-genotype. Bodyweight and kidney weight loss Frontiers in Psychiatry | www.frontiersin.org 8 November 2020 | Volume 11 | Article 572583 Muntsant and Giménez-Llort Impact of Isolation in Dementia FIGURE 5 | Effects of isolation on 11-month-old male 3xTg-AD mice in the Nesting behavior. Data are expressed as the mean ± SEM. (A) Representative images of nest buildings: (B) Nesting score at 24, 48, and 72 h. Groups: NTg, group-housed non-transgenic mice; 3xTg-AD, group-housed 3xTg-AD mice; Isolated 3xTg-AD, single-housed 3xTg-AD mice. Statistics: Student t-test comparisons are shown as *p < 0.05, **p < 0.01, ***p < 0.001 vs. the 3xTg-AD mice. were found in the AD-phenotype and increased with isolation. Here, we benefit from the use of a clean standard home- Isolation-dependent spleen weight loss was observed. Tau cage to transport the animal to the area where the open pathology in the hippocampus, a target area in AD, was not field will be performed, to assess this immediate fearful modified, but asymmetric atrophy of hippocampus, recently response. As shown, both horizontal and vertical components described in human patients with dementia and modeled here confirmed that house-grouped 3xTg-AD mice exhibited for the first time in an animal model of AD, was found to increased levels of fear in this natural neophobia response increase with isolation. These results are discussed in detail in the as compared to age-matched NTg mice. This was shown next paragraphs. as a two-fold decrease in horizontal and vertical activities and a longer time to elicit the first rearing. The delay in Effects of Isolation in the Innate Neophobia vertical exploratory activity was also present in isolated Changes in the innate neophobic response of animals 3xTg-AD animals, indicating an increased fearful response. immediately elicited when being held and put in a new However, differences with NTg counterparts were lost since place, before they can recognize it as novel or familiar, is one of the coping strategy exhibited was a fight-to-flight behavior that the most sensitive ethological behaviors of the early phenotype counteracted the low activity levels due to the AD-genotype. in the 3xTg-AD mice modeling AD (39, 41, 55, 66, 67). This was similar to the psychostimulant effect that our laboratory Frontiers in Psychiatry | www.frontiersin.org 9 November 2020 | Volume 11 | Article 572583 Muntsant and Giménez-Llort Impact of Isolation in Dementia FIGURE 6 | Effects of isolation on 11-month-old male 3xTg-AD mice in the Physical status. Results are expressed as mean ± SEM. (A) Body weight; (B) Organometrics of liver, spleen, and kidney. Groups: NTg, group-housed non-transgenic mice; 3xTg-AD, group-housed 3xTg-AD mice; Isolated 3xTg-AD, single-housed 3xTg-AD mice. Statistics: G, genotype effect, *p < 0.05. Student t-test comparisons are shown as # p < 0.05, ## p < 0.01 vs. the NTg-group; *p < 0.05, ***p < 0.001 vs. the 3xTg-AD mice. Frontiers in Psychiatry | www.frontiersin.org 10 November 2020 | Volume 11 | Article 572583 Muntsant and Giménez-Llort Impact of Isolation in Dementia FIGURE 7 | Effects of isolation on 11-month-old male 3xTg-AD mice in Neuropathology, R/L hippocampal asymmetry, and Behavioral correlates. Data are depicted and also expressed as mean ± SEM. (A) Hippocampal asymmetry; Left, R/L weights, Individual data of the corresponding area in the left (black) and right (red) hemispheres; Right, mean hippocampal weight and behavioral correlates. (B) Tau pathology; Left, Relative levels of tau (PHF1); Right, Western blots of right and left hippocampus. Groups: NTg, group-housed non-transgenic mice; 3xTg-AD, group-housed 3xTg-AD mice; isolated 3xTg-AD, single-housed 3xTg-AD mice. Statistics: Paired t-test: G, genotype effect in asymmetry between R/L hippocampus, @@@ p < 0.001. Paired t-test R/L asymmetry in each group; @ p <0.05, @@ p < 0.01. G, genotype effect, **p < 0.05. Student t-test comparisons are shown as ## p < 0.01 vs. the NTg-group. Positive (black) and negative (blue) Pearson’s correlation analysis evaluating hippocampus weight or the hippocampal relative levels of tau/behavioral correlates *p < 0.05; ACT min x, Activity test in the minute x; Total, Total activity in the activity test; BW, Bodyweight; Kidney W, Kidney weight. Frontiers in Psychiatry | www.frontiersin.org 11 November 2020 | Volume 11 | Article 572583 Muntsant and Giménez-Llort Impact of Isolation in Dementia TABLE 1 | Effects of isolation on 11-months-old male 3xTg-AD mice in the Open field test. Males, 11-month-old NTg 3xTg-AD mice n = 20 Statistics Ethogram and Emotionality (n = 9) Open field test 3xTg-AD Isolated 3xTg-AD AD Isolation (n = 10) (n = 10) Genotype Initial freezing (latency, s) 16.44 ± 12.40 17.80 ± 6.08 9.00 ± 2.61 n.s n.s Exit of the center (latency, s) 45.22 ± 19.39 59.30 ± 31.08 22.30 ± 12.65 n.s n.s Entrance to the periphery (latency, s) 68.67 ± 33.11 60.60 ± 30.96 64.00 ± 28.66 n.s n.s Vertical activity (latency, s) 146.89 ± 41.68 131.90 ± 34.67 116.40 ± 27.22 n.s n.s Thigmotaxis (total crossings 3.20 ± 0.95 3.70 ± 0.88 3.80 ± 1.18 n.s n.s periphery/center) Self-grooming (latency, s) 204.00 ± 32.24 199.10 ± 36.21 162.40 ± 22.27 n.s n.s Self-grooming (number) 1.00 ± 0.29 1.20 ± 0.20 1.20 ± 0.20 n.s n.s Defecation boli (number) 4.22 ± 0.52 3.90 ± 0.58 4.50 ± 0.45 n.s n.s Ethogram, thigmotaxis, self-grooming and defecation. Results are expressed as mean ± SEM. Groups: NTg, group-housed non-transgenic mice; 3xTg-AD, group-housed 3xTg-AD mice; Isolated 3xTg-AD, single-housed 3xTg-AD mice. All n.s., p>0.05. has shown in this animal model after chronic treatment with scarcely seen in 3xTg-AD mice (only one animal, late-onset) caffeine (68). but was exhibited early in half of the 3xTg-AD mice housed in isolation conditions. This result reinforces the above-mentioned Effects of Isolation in the BPSD-Like and increased active fearful response induced by isolation. Cognition in the Open Field and Activity In contrast to the open field test, the low anxiogenic levels of activity cages facilitate the expression of hyperactive patterns Tests as already shown in our laboratory in 12-month-old female When confronting an open and illuminated environment such 3xTg-AD mice (39). Therefore, the activity levels recorded here, as the open field, both group-housed mice exhibited a poor with a maximum of 10 m in group-housed NTg and 3xTg- exploratory activity pattern. This result was confirmed during AD mice but 20 m in isolated 3xTg-AD mice, emphasized the first 5 min of the activity test, where equal performances the noticeable hyperactivity pattern induced by isolation of were also observed in NTg and 3xTg-AD mice. In this other individuals. Thus, the results in spontaneous locomotor activity test, which did not reach statistical significance, the subsequent not only confirmed the hyperactive fearful response of isolated analysis of the long-term habituation in the activity cages showed 3xTg-AD mice already shown in the previous tests (corner test a worse (lower) pattern in the 3xTg-AD mice. Our laboratory has for neophobia and the 5 min of the open field test) but also previously described the convergence of profiles in the context demonstrated that the effect of isolation was sustained on time, of poor aging and related physical and cognitive declines in despite habituation to the enclosure. Differences were maintained NTg mice as part of the complexity of age-related scenarios statistically significant during 40 of the 45 min duration of the and heterogeneity among all populations, including wild-type test. This is important because habituation is considered the mice (not just 3xTg-AD mice) (69). Interestingly, these smooth simplest form of learning and memory (70, 71). In fact, the results patterns in middle-aged mice housed in standard conditions also would also be in agreement with the psychostimulant effects emphasized the impact of isolation shown as prominent levels induced by chronic treatment with a low dose of caffeine in this of both horizontal and vertical activities. This effect was more animal model of AD as measured in a spatial learning cognitive noticeable in the horizontal locomotor component of activity as task (68). they were sustained from the very first minute of the test, in agreement with the flight-to-fight response shown also in the corner test and all the subsequent behavioral tests. As in the Effects of Isolation in the BPSD-Like and other two groups, the levels of vertical exploratory activity of Cognition in the T-Maze isolated 3xTg-AD mice were mostly null at the beginning of The spontaneous alternation in the T-shaped maze with black the test, in agreement with the immediate fear response in an and narrow corridors resembling burrows is a paradigm mostly anxiogenic open and illuminated enclosure. However, according used to evaluate spatial working memory in a naturalistic to the action program described by Lát (61), as soon as this manner. More recently, some authors have dissected other initial fear was managed, the level of vertical exploration rose and cognitive aspects (72). The test also involves emotionality/anxiety progressively increased during the test until its end. Isolation not and exploratory activity as we have shown extensively, in both only increased the levels of standard locomotor and exploratory male and female mice, from young to old age under healthy behaviors but also elicited the emergence of bizarre actions not and diverse neuropathological conditions (39, 62, 63, 66, 73). shown in wild-type mice. Here, the presence of vertical activity This neuropsychiatric component was also pointed out here by without wall support, indicative of an escape behavior (55), was the correlation analysis. Thus, the T-maze ethogram correlated Frontiers in Psychiatry | www.frontiersin.org 12 November 2020 | Volume 11 | Article 572583 Muntsant and Giménez-Llort Impact of Isolation in Dementia with that shown in the open-field test, the horizontal and vertical The impact of isolation in these questions was addressed, activities in the first minute where a fearful response is elicited, at the translational level, by means of nesting behavior, an and the total horizontal and vertical activities developed. innate ethological inference to assess well-being and daily Interestingly, the latency to cross the intersection of the maze life activity in normal mice and models for neuropsychiatric allows us to assess the coping-with-stress strategies of animals. diseases (58, 59, 84). Conversely, nesting material is considered Alterations in this parameter, as compared to the standard a tool for the improvement of the animal’s welfare. Nesting wild-type performance to cope, have been found to correlate behavior is observed in the context of maternal affiliative with a worse neuro-immunoendocrine function, indicators of behaviors, facilitating family structures, maternal interaction, and accelerated aging and premature death in mice (74). Although protecting the offspring from outsiders and predators. However, no significant differences were observed in the number of errors nesting behavior also extends to males and non-pregnant females in the spontaneous alternation behavior, most 3xTg-AD mice driven by protection and thermoregulation needs. We first revisited areas of the maze previously explored. The 3xTg-AD described deficiencies of 3xTg-AD mice in this instrumental mice and those isolated exhibited opposite performances since task involving executive functions and as dependent on the the latency to reach the intersection and the time to complete protocol and nesting material. As we demonstrated, a 3-day the maze were delayed or fastened, respectively, in a statistically assessment with paper towel instead of standard protocols (using significant manner. In previous studies, we have shown that cotton, assessing only at 24 h) revealed genotype-, gender- and young 3xTg-AD mice mimicking prodromal stages of disease age-dependent differences in 3xTg-AD mice under different exhibit a fast-speed performance to reach the intersection in social settings (58). In the present work, the mean nesting comparison with age-matched NTg mice, in a typical flight scores and 3-day patterns for NTg and 3xTg-AD mice are behavior in the fight-or-flight strategy. At old or middle ages, in agreement with those observed in 12-month-old NTg and like those of the animals used here, a slow performance involving 3xTg-AD males (isolated during the 3 days of nest-building petrification is the most common behavioral response (75). assessment) as reported in our previous work (58). Here, we In the present work, the fast pattern in isolated animals, demonstrate that long-term isolation of 3xTg-AD mice not only resembling that of chronically psycho-stimulated 3xTg-AD mice resulted in hyperactive locomotor patterns but also higher nest- (68), suggests that the hyperactive pattern also involves a switch building scores and smaller nests. This can be understood in in their coping-with-stress strategies. The impaired decision- the context of “thermoregulatory nesting,” a behavioral construct making process is shown as a delay to cross the intersection, demonstrated by geographic cline in nest-building behavior (85). a function that depends on prefrontal cortical areas, and Thus, the lower core body temperature of aged 3xTg-AD mice was not modified by isolation. We have previously reported (86), the worse thermoregulation shown also in other models similar delays in the decision to move from one side to (87), and the loss of group-sleeping behavior in isolated 3xTg- another in the dark-light box test, suggesting that the AD AD animals may have enhanced the utility of nests to cover genotype models differences in risk assessment (39, 62, 63, increased metabolic, thermoregulation, and protective needs. 66). Sensorial tactile stimulation known to modulate cognitive Regarding their hyperactive phenotype, it is interesting to note and anxiety-like behaviors hampered this phenomenon in the that several studies have identified a negative genetic correlation 3xTg-AD mice (76, 77) and infantile cerebral palsy model (78). between thermoregulatory nesting and wheel-running activity Impairment in working memory in other spatial tasks has with increased wheel running in mice selected to build small also been reported in APP/PS1 and Tg2576 mice after social nests (88, 89). Inverse relation of nesting with body mass isolation (79, 80). The conspicuous neuropsychiatric-like profile and litter size (85) and body temperature (90) has also been of 3xTg-AD mice models the complexity of the advanced stages demonstrated and would be in agreement with the trend to a of the disease where behavioral alterations disrupt cognitive lower bodyweight of isolated 3xTg-AD mice found here. Overall, function and cause the most important burden of disease for the the present results point out that isolation induces increases in patients and caregivers. In fact, in addition to cognitive aspects, both gross (hyperactivity) and fine (nesting) motor function. pharmacological management of dementia targets agitation, Therefore, maximal aerobic capacity enabling such sustained irritability, aggressiveness, disorientation, and wandering among activities, resting metabolic rates, thermoregulatory abilities, and other neuropsychiatric symptomatology. In this respect, our energy requirements are key aspects to take into account in the most recent translational study on the pharmacological treatment isolation scenarios (89). of these endophenotypes also found efficiency to reach the goals in this T-maze task being compromised in middle-aged male Effects of Isolation in the Physical Status 3xTg-AD mice, a poor performance that was slightly affected by Overweight, a characteristic of the Spanish colony of 3xTg-AD long-term treatment with the antipsychotic risperidone (81). mice (63), was not found in this particular experimental set. However, a trend of reduced weight was found in isolated animals Effects of Isolation in the Daily Life Activity resembling the effects we described previously in male 3xTg-AD Deterioration in daily life activities and executive functions mice receiving long-term treatment with caffeine (68). In that are early functional signs in patients with dementia. They work, we measured the psychostimulant effects of caffeine in a worsen with the progress of disease and demand important whole light/dark cycle. We observed that hyperactivity in their caregiver attention (82). On the other hand, age-related deficits diurnal sleeping period was followed by enhanced expression in thermoregulation are more pronounced in AD patients (83). of hyperactivity in their active nocturnal activity period, which Frontiers in Psychiatry | www.frontiersin.org 13 November 2020 | Volume 11 | Article 572583 Muntsant and Giménez-Llort Impact of Isolation in Dementia could explain their weight loss. In other words, we have also levels and glutathione peroxidase and reductase activities, as reported that 3xTg-AD are more sensitive than NTg mice to well as in terms of pro-inflammatory (IL-10 decreased)/anti- weight loss under the stressful environment of a forced treadmill inflammatory (IL-1beta and TNF-alpha increased) balance exercise (63) but not voluntary wheel running (66). In those in leucocytes in male and female 3xTg-AD mice since the experiments, the decrease in weight was more evident in the prodromal stages of the disease (93, 101). In the present females, both because of their higher corticosterone response and work, weight and relative weight of spleen were found their lower basal weight than males. Therefore, the bodyweight reduced in isolated 3xTg-AD mice, mimicking again the decrease found in the present work in isolated males may be effect of long-term stimulant effects of caffeine (68). The of functional significance. As mentioned previously, the results increase in chronic physiological stress induced by long- of weight and relative weight suggest that isolation may induce term isolation may also support these results since elevated a general weight loss or reduction of somatic size in 3xTg-AD levels of stress have been related to a decreased spleen mass mice. Separation of mice increases metabolic demands and has (102, 103) and models of physical stress combined with been proposed as a new and easy-to-perform animal model for social isolation show a worsening of the immuno-endocrine weight loss (91). Sex differences in the psychobiological effects system (104). in Swiss kumming mice undergoing 1–4 months social isolation With regard to the kidney, acute renal impairment have also been demonstrated (92). There, both isolated males and complicating viral infection was already observed in some females showed less bodyweight gain; however, male mice were patients affected by SARS-CoV (105). Although AKI was more affected by isolation. reported as uncommon and likely to be related to multi- organ failure rather than the viral tropism in the kidney, it was recognized as carrying a risk of high mortality. The Effects of Isolation in the Metabolic, prevalence of kidney disease in patients with COVID-19 on Excretory, and Peripheral Immunity Organs admission and the development of acute kidney injury during Although brain oxidative stress in AD is accepted, the hospitalization are high and are also associated with in-hospital contribution of the disease to peripheral oxidative redox state mortality (64). As a Kidney International editorial noted, has been little studied. In this regard, we reported peripheral early risk identification, rapid and effective treatments, and alterations (41, 63) in early oxidative stress status in male and avoidance of nephrotoxins may help provide a better prognosis female 3xTg-AD mice, with a decrease in antioxidant defenses of patients with COVID-19. A case report also describes the and an increase in xanthine oxidase activity in most peripheral rapid development of collapsing glomerulopathy, which further tissues, among them the liver, kidney, and spleen studied here deteriorated despite the improvement of respiratory symptoms. (93, 94). Interestingly, these genotype differences were more In this sense, the linkage between chronic kidney disease and exacerbated in male than in female mice. In the present and AD may suggest a higher vulnerability in AD patients in the other works, we used the size and relative size of liver, kidneys, COVID-19 pandemic (95). At the translational level, we have and spleen as an indirect measure of their physiological status preliminary observations on severe glomerular affectation of as we have proposed that these parameters can be a tool for kidneys in 3xTg-AD mice. That also would be in agreement sonographic monitoring of patients (41, 75). Other laboratories with the correlation analysis that, in the present work, found have also confirmed in this animal model this liver and spleen a relation between weight loss of the hippocampus and its dysfunction reflected as hepato- and splenomegaly (95, 96) relative levels of tau with the kidneys’ weight loss. Therefore, and fatty liver pathology (97). Consistent associations of liver the AD-genotype effects found in the present work in the function with AD patients has been recently described, indicating weight loss in the kidneys worsen by isolation and may suggest metabolic disturbances (98). In the current pandemic, persistent clinical implications. inflammatory syndrome is an important negative prognostic indicator for survival. A relation with COVID-19 and liver dysfunction has been reported (65), and metabolic-associated Effects of Isolation in the Right/Left Brain fatty liver disease has been related to a worse evolution of Asymmetry COVID-19 (99). Several psychiatric disorders and psychological conditions Since our first report (41) proposing sex-specific immuno- associated with stress and trauma, such as PTSD (106) and major endocrine aging in old 3xTg-AD mice (worse in males), we have depressive disorder (107), are associated with a reduction in consistently reported thymus involution and splenomegaly as hippocampal volume and alterations in hippocampal memory characteristic of the impairment of the neuro-immune-endocrine function as a result of the key role of the hippocampus in the system in this animal model (100). The easy measurement of regulation of the hypothalamic-pituitary-adrenocortical axis in their weight and relative weight (organometrics), with clinical acute and chronic stress (108). In addition, brain hemispheric translation, can be used as early indicators of peripheral left–right asymmetry and lateralization is known to exist immunological system aging (75, 93, 100), as also confirmed at several anatomical levels in the brain, and hippocampal by other laboratories (96). We have shown previously that asymmetry has been suggested to indicate lateralized differences the alterations in the brain redox state (66, 67) develop in in vulnerability to trauma (106). A meta-analysis also shows parallel to the impaired splenic redox state, measured as an that hippocampal atrophy was associated with trauma exposure increased xanthine oxidase activity and lower total glutathione independent of PTSD diagnosis, PTSD-induced additional Frontiers in Psychiatry | www.frontiersin.org 14 November 2020 | Volume 11 | Article 572583 Muntsant and Giménez-Llort Impact of Isolation in Dementia hippocampal reduction, with the right hippocampus being and negatively with nesting behavior. However, the activity smaller in the PTSD group than the trauma-exposed group patterns of the two 3xTg-mice groups were allocated in two without PTSD (109). Also, in the context of the present work, opposed positions concerning the activity shown by NTg mice the whole-brain analysis has revealed increased neuroanatomical (low in 3xTg-AD mice, high in isolated 3xTg-AD mice): the asymmetries in dementia for hippocampus and amygdala (110). selective atrophy in the right hippocampus in AD-genotypes At the translational level, recent studies in rodents have explored and its enhancement by isolation. Altogether, the correlation left–right hippocampal asymmetry, providing further evidence analysis may show the overall relationship between weight at the neurochemical and molecular levels with regard to the and activity but cannot be directly related to the hippocampal glutamatergic system (111). The lateralization of the stress asymmetry found in the AD-genotype or the loss of weight of regulatory system has also been shown, with long-term isolation the right hippocampus and the enhancement of these aspects stress in rats producing right–left asymmetry of the hippocampus by isolation. norepinephrine and changes in catecholamine synthesis and reuptake (112). At the functional level, hippocampal asymmetry Social Isolation in Other Animal Models of is important for the acquisition of spatial reference memory Alzheimer’s Disease and retention of working memory (113) as wells as for certain Finally, studies in Tg2576, APP/PS1, 5xFAD mice have reported characteristics of non-spatial learning (111). Here, in the present effects of social isolation in the AD pathology as related to work, our results show that in all the animals, the weight of the an increase in Aβ peptide (48, 80, 114). However, the work hippocampus from the right hemisphere was lower than that of by Pietropaolo et al. (50) assessing the impact of isolation the left hemisphere and that this asymmetry was increased in the in the 3xTg-AD mice from post weaning (postnatal day animals with AD-genotype, reaching the maximum statistical 21) until the onset of disease (6 months of age) found no significance. To our knowledge, this is the first time that significant changes. Our present report, in a complementary increased asymmetry in the hippocampus of an animal model manner, studied the impact of a similar period of isolation of AD is demonstrated, thus modeling the above-mentioned on advanced stages of the disease, in naturally occurring asymmetry found in AD human patients. This modeling will scenarios (disruptive behaviors or death of partners). The be useful for the translational development and assessment of biochemical analysis of these samples confirmed the increased preventive/therapeutic interventions and those of the risk factors relative levels of phosphorylated tau protein in the hippocampus and hazards, as well as monitoring disease progression. In this of 3xTg-AD mice as measured by PHF1, with a significant respect, it already indicates that isolation induces an increase of genotype effect but no left–right differences. Isolation induced one degree of magnitude in the statistical significance of the left– a slight increase in the AD-pathology but did not reach the right differences since in the 3xTg-AD mice housed in standard statistical significance. conditions the left–right hippocampal asymmetry reached p < 0.05 while in isolated 3xTg-AD mice the significance reached Conclusions the an order of magnitude of p < 0.01. Further quantitative and In summary, the present study shows that single-housed qualitative analysis of regional neuronal density in CA1, CA2, 3xTg-AD for AD showed a prominent hyperactive pattern as and ventral hippocampus to quantify distinct neuron depletion shown in gross motor function in all the tests, the emergence may reveal a regionally gradient neuronal loss, specific also of bizarre behaviors, and intensified nesting behavior (fine- for the hippocampus of each hemisphere. Interestingly, in the motor function), probably responding to increased demands present work, the weight of the left hippocampus was maintained of protection and thermoregulation induced by isolation in an constant, while the right hippocampus decreased with already AD-frail scenario. Anxiogenic patterns were broken and genotype and with isolation, suggesting hemisphere-dependent coping-with-stress strategies were changed (from freeze) to flight cellular vulnerability. behavior in isolated 3xTg-AD male. Although isolation did not exacerbate the hallmark tau pathology in the hippocampus, we could demonstrate, for the first time, hippocampal atrophy and Behavioral Correlates of Pathological left–right asymmetry and lateralization (behavioral correlates). Status Hippocampal atrophy was selective of the right hemisphere and Considering the three groups, the behavioral correlates of related to bodyweight and kidney weight loss and (negatively) neuropathology indicated that the levels of tau were negatively nesting behavior. Isolation-induced weight loss was observed correlated with most time points and total activity developed in peripheral immune organs (spleen). Despite limitations of in the activity-cages and the weight of the kidneys. Conversely, translating animal models into human application, these results the total weight of the hippocampus was found positively point at isolation having an impact on aspects that increase the correlated with the kidneys and bodyweights, as discussed intrinsically AD-associated deficits and vulnerability. It would previously, as well as the performance in the activity, but be interesting to perform a translational approach of these negatively correlated with the nest building. Considering each preliminary findings in a clinical sample of AD patients to hemisphere, lateralization was found since the left hippocampus study the impact of COVID-19–related social isolation from correlated with the activity, while the right hippocampus a psychological and neuroimaging (fMRI) point of view. The positively correlated with bodyweight and kidney weights, present results help make us aware of the severe impact Frontiers in Psychiatry | www.frontiersin.org 15 November 2020 | Volume 11 | Article 572583 Muntsant and Giménez-Llort Impact of Isolation in Dementia of the new physical distancing measures not only on those ETHICS STATEMENT expected in cognition but also on the expression of BPSD. More importantly, the results point to the worsening of The animal study was reviewed and approved by CEEAH the AD-brain hippocampal asymmetry. These results from Universitat Autònoma de Barcelona & Generalitat translational neuroscience research models provide guidance de Catalunya. about the complexity of the new scenarios. This will be important when considering the best preventive/therapeutic AUTHOR CONTRIBUTIONS strategies in times of coronavirus and the post–COVID- 19 pandemic. From our perspective, there is a strong need LG-L: conceptualization. AM: performance, analysis of behavior, for non-pharmacological interventions for managing BPSD and illustrations. Both authors: equally contributed to the as those provided by the last international consensus (i.e., scientific discussions, writing, and approval of the manuscript. caregiver training, environmental adaptations, music therapy, and other person-centered care and activities) before any FUNDING pharmacological approaches (8, 115, 116). The coordinating role of different health system actors (geriatricians, nurses, This work was funded by UAB-GE-260408 and ArrestAD H2020 psychologists, occupational therapists, physiotherapists) will be Fet-OPEN-1-2016-2017-737390 to LG-L. critical for the implementation and success of these interventions (117). Thus, the present work also highlights the relevance of ACKNOWLEDGMENTS personalized-based interventions tailored to the heterogeneous and complex clinical profile of the individuals with dementia as We thank Prof. Frank M. LaFerla, Institute for Memory well as the effects that the worsening of some symptoms have on Impairments and Neurological Disorders, University of the caregiver burden. California, Irvine, USA, for kindly providing the progenitors of the Spanish colonies of homozygous 3xTg-AD and NTg mice. DATA AVAILABILITY STATEMENT Special thanks are due to Prof. Dulce Papy and Dr. Mohand Ou Idir Ouidja, Laboratoire CRRET EA 4397/ERL CNRS 9215 The original contributions presented in the study are included Université Paris-Est Créteil (UPEC), for assistance in the tau in the article/supplementary material, further inquiries can be determination; and Dr. Peter Davies, Albert Einstein College of directed to the corresponding author/s. Medicine, New York, USA, for providing the PHF-1 antibody. REFERENCES 9. COVID-19 advice - Know the facts | WHO Western Pacific. Available online at: https://www.who.int/westernpacific/emergencies/covid-19/information/ 1. Seitz D, Purandare N, Conn D. 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Frontiers in Psychiatry | www.frontiersin.org 19 November 2020 | Volume 11 | Article 572583