TYPE Brief Research Report PUBLISHED 15 September 2022 DOI 10.3389/fnagi.2022.969381 Crosstalk of Alzheimer’s OPEN ACCESS disease-phenotype, HPA axis, EDITED BY Anamitra Ghosh, Wave Life Sciences Ltd., United States splenic oxidative stress and REVIEWED BY Hailong Song, frailty in late-stages of University of Pennsylvania, United States Qingwei Ruan, dementia, with special concerns Fudan University, China Stefania Schiavone, University of Foggia, Italy on the effects of social isolation: Luigia Trabace, University of Foggia, Italy A translational neuroscience approach *CORRESPONDENCE Lydia Giménez-Llort lidia.gimenez@uab.cat SPECIALTY SECTION This article was submitted to Aida Muntsant1,2 and Lydia Giménez-Llort1,2* Alzheimer’s Disease and Related 1 Institut de Neurociències, Universitat Autònoma de Barcelona, Barcelona, Spain, 2 Department of Dementias, Psychiatry and Forensic Medicine, School of Medicine, Universitat Autònoma de Barcelona, a section of the journal Barcelona, Spain Frontiers in Aging Neuroscience RECEIVED 14 June 2022 ACCEPTED 23 August 2022 PUBLISHED 15 September 2022 CITATION Coping with emotional stressors strongly impacts older people due to their Muntsant A and Giménez-Llort L age-related impaired neuroendocrine and immune systems. Elevated cortisol (2022) Crosstalk of Alzheimer’s levels seem to be associated with an increased risk of cognitive decline disease-phenotype, HPA axis, splenic oxidative stress and frailty and dementia. In Alzheimer’s disease (AD), alterations in the innate immune in late-stages of dementia, with special system result in crosstalk between immune mediators and neuronal and concerns on the effects of social isolation: A translational neuroscience endocrine functions. Besides, neuropsychiatric symptoms such as depression, approach. anxiety, or agitation are observed in most patients. Here, we studied Front. Aging Neurosci. 14:969381. doi: 10.3389/fnagi.2022.969381 the psychophysiological response to intrinsic (AD-phenotype) and extrinsic (anxiogenic tests) stress factors and their relation to liver, kidneys, heart, COPYRIGHT © 2022 Muntsant and Giménez-Llort. and spleen oxidative status in 18-months-old female gold-standard C57BL/6 This is an open-access article mice and 3xTg-AD mice model for AD. The emotional, cognitive, and distributed under the terms of the Creative Commons Attribution License motor phenotypes were assessed under three different anxiogenic conditions. (CC BY). The use, distribution or Survival, frailty index, and immunoendocrine status (corticosterone levels reproduction in other forums is permitted, provided the original and oxidative stress of peripheral organs) were evaluated. Genotype author(s) and the copyright owner(s) differences in neuropsychiatric-like profiles and cognitive disfunction in are credited and that the original publication in this journal is cited, in 3xTg-AD females that survived beyond advanced stages of the disease accordance with accepted academic persisted despite losing other behavioral and hypothalamic–pituitary– practice. No use, distribution or adrenal (HPA) physiological differences. A secondary analysis studied the reproduction is permitted which does not comply with these terms. impact of social isolation, naturally occurring in 3xTg-AD mice due to the death of cage mates. One month of isolation modified hyperactivity and neophobia patterns and disrupt the obsessive-compulsive disorder- like digging ethogram. Frailty index correlated with spleen organometrics Frontiers in Aging Neuroscience 01 frontiersin.org Muntsant and Giménez-Llort 10.3389/fnagi.2022.969381 in all groups, whereas two AD-specific salient functional correlations were identified: (1) Levels of corticosterone with worse performance in the T-maze, (2) and with a lower splenic GPx antioxidant enzymatic activity, which may suppose a potent risk of morbidity and mortality in AD. KEYWORDS Alzheimer’s disease, neuroimmunoendocrine crosstalk, aging, 3xTg-AD mice, cognition, neuropsychiatric symptoms, translational neuroscience, social isolation Introduction Experimental gerontologists highlight the relevance of using aged animals to mimic the complexity and multi- Alzheimer’s disease (AD) is a complex and multi- factorial aging processes in humans (Turner et al., 2012; factorial disorder caused by the interaction of biological Mitchell et al., 2015), so that the translational approach and environmental factors, where age-related changes play a provides immediate clues to fast human neuroscience research. determinant role (Llewellyn et al., 2019; Knopman et al., 2021). Among the animal models of AD, we have proposed long- The complex aging process leads to old age being the most term female survivors of the widely used 3xTg-AD mice heterogeneous period of life. In AD, the complexity of patients’ as a model for mortality selection bias and heterogeneity clinical profiles (Kaufer et al., 1998; Kamiya et al., 2014) and in end-of-life dementia (Torres-Lista et al., 2017). Based on the heterogeneity in the temporal progression of the disease the familial AD mutations PS1/M146V and APPSwe, and highlight the relevance of personalized-based interventions, harboring tauP301L human transgene, this model progressively especially in the advanced stages of the disease (Komarova and develops temporal- and regional-specific neuropathological Thalhauser, 2011). patterns observed in the human brain of AD patients (Oddo Dementia is associated with increased mortality (van et al., 2003; Belfiore et al., 2019). Cognitive deficits (Billings Dijk et al., 1991) and although it is not yet well-understood, et al., 2005; Sterniczuk et al., 2010), a broad spectrum of experimental, clinical, and epidemiological evidence suggest NPS-like symptoms (Giménez-Llort et al., 2007; España et al., that crosstalk interactions between the brain and the peripheral 2010), and impairment in neuro-immunoendocrine status that or systemic abnormalities might have a crucial role in correlate with worse NPS-like behavioral profiles (Giménez- the development and progression of AD (Wang et al., Llort et al., 2008; Gimenez-Llort et al., 2014) have been 2017). According to the AD neuroimmunomodulation described. hypothesis, alterations in the innate immune system result in Since the establishment of 3xTg-AD and non-transgenic a dysregulation of the crosstalk between immune mediators (NTg) mouse colonies in our laboratory, we have repeatedly and neuronal function both at central and peripheral levels observed that the animals overcoming 15 months of age show (Giménez-Llort et al., 2008, 2012; Maccioni et al., 2009). Coping milder impairment of cognitive and NPS-like behaviors than with emotional stressors has also a stronger impact on older expected for their neuropathological status. Sex differences people due to their age-related impaired neuroendocrine and in mortality rates (Giménez-Llort et al., 2008; Arranz et al., immune systems (Kiecolt-Glaser et al., 2002). In the last years, 2011; Blázquez et al., 2014), made females survivors a the relevance of social relationships’ effects on morbidity singular scenario (Torres-Lista et al., 2017). The present and mortality outcomes has been reported, taking particular work further explores previously observed behavioral and importance during the COVID-19 pandemic (Gerst-Emerson functional phenotype convergence in long-term 3xTg-AD and Jayawardhana, 2015; Donovan and Blazer, 2020). Social female survivors (Torres-Lista et al., 2017). Here, we studied isolation and loneliness are particularly relevant among the cognitive, motor and NPS-like phenotypes of 18-month- older adults due to functional declines, health morbidities, old females assessed under different anxiogenic conditions. or relationship losses (Steptoe et al., 2013; Gerst-Emerson This age corresponds to old age in NTg mice and late stages and Jayawardhana, 2015). Regarding AD, social isolation of the disease in 3xTg-AD mice. Survival and indicators not only increases the risk of developing dementia (Kuiper of frailty phenotype and neuro-immunoendocrine status et al., 2015; Read et al., 2020), but it is also associated (as determined by corticosterone levels, oxidative stress of with an increased memory decline, negatively affecting peripheral organs, and organometrics) were also measured. cognitive performance, and worsening neuropsychiatric In a secondary analysis, we studied the impact of short-term symptoms (NPS) (Friedler et al., 2015; Porcelli et al., 2019; social isolation in a subgroup of age-matched female 3xTg-AD Curelaru et al., 2021). mice. Frontiers in Aging Neuroscience 02 frontiersin.org Muntsant and Giménez-Llort 10.3389/fnagi.2022.969381 Materials and methods the test. Distance, time in the center/periphery, and immobile time were evaluated by video-tracking analysis (ANY-Maze Animals 5.14). Defecation boli and urination were also recorded. Day 2: Context and Object Recognition Test (OF2OR)- A total of twenty-six 18-month-old (18.7 ± 0.16) female The day after, the animals were re-tested in the open field mice from the Spanish colonies of homozygous 3xTg-AD (OF2) to evaluate their response when confronting the same (n = 17) and NTg (n = 9) mice on a C57BL/6J background (after anxiogenic environment again. Analysis of activity was done embryonic transfer and backcrossing at least 10 generations) during the first minute of the test, the period where neophobia established in the Universitat Autònoma de Barcelona (Baeta- is more manifested, and we have previously described AD- Corral and Giménez-Llort, 2014) were used in this study. The genotype differences (Torres-Lista et al., 2014). Immediately animals were maintained under standard laboratory conditions after, animals were moved to a standard home cage, where of food and water ad lib, 22 ± 2◦ C, 12-h light/dark cycle with they remained for 1 min before being reintroduced in the OF lights on at 8:00 a.m., and 50–60% relative humidity. (a known environment) and were assessed for their ability to recognize a familiar object (S, sample) from a new one (N) with the novel object recognition test (OR) (Giménez-Llort et al., Social conditions 2021). In the sample trial, the animals were let explore two identical objects, S1 and S2 (glass bottles, 15 cm × 12 cm, Animals of the same genotype were maintained in groups 5 cm diameter), equally spaced until they reached the 20 s of 3–4 mice per cage (Macrolon, 35 cm × 35 cm × 25 cm). exploration criteria until a maximum time of 600 s. Two hours Since at these advanced stages of the disease, increased mortality thirty minutes later, animals were reintroduced to the apparatus rates in 3xTg-AD mice occur (Giménez-Llort et al., 2008), in for 5 min (test trial), where two different non-explored objects a secondary analysis, a set of 3xTg-AD mice living in social were located: an identical copy of the sample objects (S3) isolation (n = 9, one animal living alone in one cage for and a completely new object (N, rectangular aluminum can, 1 month) was also studied. When isolated, animals still were 15 cm × 10 cm × 4 cm). Preference for the new object was socially connected through olfaction and audition, and as in the measured. group-housed conditions, the cages were enriched with nesting Day 3: Marble test (MB)—Mice were placed individually materials. The study complies with the ARRIVE guidelines for a 30-min period in a standard home cage containing nine developed by the NC3Rs (Kilkenny et al., 2010). glass marbles (1 cm × 1 cm × 1 cm) evenly spaced, making a square. The number of marbles intact (untouched), moved or half-buried, and buried (completely hidden) were counted as Behavioral assessment previously described (Torres-Lista et al., 2015). Day 4: T-maze (TM)—Working memory was assessed A behavioral battery of tests evaluates locomotion/ in a black T-shaped maze (woodwork; two short arms of exploration, anxiety-like behaviors, emotionality, and cognitive 30 cm × 10 cm and a long arm of 50 cm × 10 cm). One forced- functions under three different anxiogenic conditions: choice (acquisition trial) was followed by one free-choice (recall anxiogenic direct exposure to an illuminated field with/without trial) with a 60 s intertrial interval. Latencies of turn, reach objects, mild neophobia in a new home cage, and black the intersection, crossed the intersection (four paws criteria), corridors of a maze resembling burrows (Giménez-Llort et al., and the time elapsed until the mice completed the 20 s in 2007). Behavioral assessments were performed blind to the the forced arm (time to reach the criteria) were recorded. experiment, in a counterbalanced manner, in the light cycle, The animals that completed the trial in less than the cut- from 09:00 to 13:00 h. off time (10 min) were allowed to explore the maze (both Day 1: Corner Test (CT) and Open Field Test (OF)—The arms accessible) in the recall trial for 5 min. The choice of animal was placed in the center of a clean standard home cage, the already visited arm in the previous trial was considered and neophobia was evaluated for 30 s through the number of an error, and the total number was calculated. The time corners visited, rearings, and latency to perform the first rearing. spent reaching the correct arm, defecation boli, and urination Immediately after the CT, mice were placed in the center of were also recorded. an open field (beige metal drawer, 42 cm × 38 cm × 15 cm) and observed for 5 min. The sequence of behavioral events that define the animal’s ethogram was recorded as follows: duration Survival, body weight, and mouse of freezing (latency to move), latency to leave the central square clinical frailty index and enter the peripheral ring, latency, and total duration of self- grooming behavior. Horizontal (distance covered) and vertical Survival was recorded continuously with a daily cadence. (rearings) locomotor activities were recorded for each minute of After the behavioral assessment, the body weight was recorded Frontiers in Aging Neuroscience 03 frontiersin.org Muntsant and Giménez-Llort 10.3389/fnagi.2022.969381 to monitor the physical status of the animals. Frailty was Salient behavioral alterations despite assessed using an adaptation of the Mouse Clinical Frailty Index phenotypic convergence (Whitehead et al., 2014), including 30 “clinically” assessed non- invasive items. Figures 1A,B illustrates the temporal curves (left) and total accumulated counts (right) of horizontal and vertical exploratory activity during the 5 min in the OF and the first Immunoendocrine status and oxidative min in the repeated test. A decrease in vertical exploration was stress of liver, kidneys, heart, and observed in 3xTg-AD mice compared to NTg mice (Figure 1B, spleen G∗ , p = 0.036). In the horizontal component, the sensitive variable was the walking speed, with 3xTg-AD groups walking Blood samples and organs tissue were collected during slower than NTg mice (Figure 1C, G∗ , p = 0.016). In the the euthanasia. Serum was obtained by centrifugation and repeated test, a burst of initial locomotor performance was stored at −80◦ C. Immunoendocrine status was monitored observed in 3xTg-AD animals, reaching statistical significance through spleen weight and index (relative weight, % in the horizontal component (Figure 1A, G∗ , p = 0.031). of body weight). Corticosterone content (ng/mL) was The immediate freezing response (neophobia) during the first analyzed using a commercial kit (Corticosterone EIA kit seconds of the test was analyzed (Figure 1D). No differences from Immunodiagnostic Systems) and read at 450 nm were observed on the first day. However, compared to NTg mice, of absorbance with Varioskan LUX ESW 1.00.38. The this second test recorded a significant decrease in 3xTg-AD mice antioxidant capacity was studied from liver, kidneys, (G∗∗∗ , p = 0.000). heart, and spleen homogenates. Total glutathione (GSH) The subgroup of isolated 3xTg-AD did not differ from levels were assayed by the enzymatic recycling method by their counterparts housed in groups (Table 1 and Figure 1). monitoring the change in absorbance at 412 nm adapted Still, a slight increase in activity and speed (Figures 1A–C) to 96-well plates. Glutathione reductase (GR) enzymatic counteracted the low activity phenotype characteristic of 3xTg- activity was assessed following the oxidation of NADPH AD mice, reducing the changes to discriminate alterations spectrophotometrically at 340 nm for 300 s. For the GPx compared to NTg animals. Their freezing response in the re- activity, the reaction was followed spectrophotometrically test (Figure 1D) was statistically significant as compared to their by the decrease of the absorbance at 340 nm for 300 s previous neophobia response in the open-field test (TT p = 0.002) (Muntsant and Giménez-Llort, 2021). and differed from that of NTg mice with higher significance than did group-housed 3xTg-AD mice (∗∗∗ p = 0.000). As detailed in Table 1, all the other variables measured in Statistics the CT and the OF test showed striking similarities between genotypes. Concerning the OR, most animals were unsuccessful Statistical analyses were performed using SPSS in reaching the acquisition criteria (rate of success: NTg: 1/9; 15.0 software. Data are presented as mean ± SEM 3xTg-AD: 3/8; isolated 3xTg-AD: 4/9). However, the ethogram or percentage. Differences between two different concerning the latencies to touch the objects for the first time, genotypes were evaluated with Student’s t-test while Chi- and Time 1 (acquisition criteria, 600 s for those failing to do so) square or Fisher’s exact test for incidences. Subgroups can be reported. In all the groups, a high individual variability comparisons were conducted with ANOVA followed that resulted in a high statistical variance in most variables was by Bonferoni post-hoc test. In temporal courses paired noticeable. t-tests were used for within-subject analysis. Pearson’s Figure 2A depicts the ethograms performed in the two trials correlation analyzed the pathological/behavioral correlates. of the TM. As detailed in Figure 2B, only 6/9 NTg mice achieved The survival curve was analyzed with the Kaplan- the learning criteria, while the coping-with-stress strategy of the Meier test. In all the tests, p < 0.05 was considered other three was to rest with their back protected at the starting statistically significant. point. In contrast, all the 3xTg-AD mice (8/8) completed the first free trial. The proactive strategy of isolated 3xTg-AD mice resulted in only 2/9 mice being unable to complete the test. In Results the recall test (Figure 2C), animals that completed the first trial were evaluated. Again, all 3xTg-AD mice completed the test, The results, depicted in Figures 1–3, and Table 1, show whereas 4/6 NTg mice achieved the test completion criteria (1/6 the psychophysiological response to intrinsic and extrinsic did not even start) (Figure 2D). The number of spatial working stress factors, hypothalamic–pituitary–adrenal (HPA) axis, and memory errors (revisiting an explored area) was recorded in peripheral status (organometrics and oxidative stress). those animals able to perform the task. Albeit the sample size Frontiers in Aging Neuroscience 04 frontiersin.org Muntsant and Giménez-Llort 10.3389/fnagi.2022.969381 FIGURE 1 Salient behavioral alterations in 18-month-old female 3xTg-AD mice in the open field tests compared to NTg females with normal aging. Results are expressed as mean ± SEM. (A) OF: Open field test Horizontal activity: left, time course; and right, total distance; (B) vertical activity: left, time course; and right, total number; (C) speed; (D) freezing response. Statistics: Students t-test: G, genotype effect, G*p < 0.05, G**p < 0.01, G***p < 0.001. ANOVA for comparisons between all the groups of mice followed by Bonferroni’s post-hoc test: *p < 0.05 and ***p < 0.01 vs. the NTg-group. Paired t-test, TT p < 0.01 vs. the same group on Day 1. was not enough to reach statistical significance, an increased correlations, specific of 3xTg-AD mice in their ethogram in the number of errors in spatial alternation was noted in the isolated acquisition trial with corticosterone levels (©CORT) were found 3xTg-AD mice (Figure 2E). Statistically significant Pearson’s (T-intersection, r = 0.691, p = 0.002; T-intersection four paws, Frontiers in Aging Neuroscience 05 frontiersin.org Muntsant and Giménez-Llort 10.3389/fnagi.2022.969381 r = 0.695, p = 0.002; Learning criterion, r = 0.761, p = 0.000). organs (Figures 3F–H). However, we performed a secondary In the MB, isolation broke the habitual digging ethogram with segregation analysis since a correlation between corticosterone an increased number of buried marbles compared to the other levels and spleen GPx was found in 3xTg-AD mice. Animals groups (Figure 2F, # p = 0.047). with corticosterone levels above the mean (HC, high corticosterone, n = 9) showed a significant decrease in splenic GPx activity compared to their low corticosterone (LC, Survival, physical health, and frailty below the mean, n = 8) counterparts (∗ p = 0.016). index Figure 3A illustrates the survival curves of an initial Discussion sample of 57, 24 NTg, and 33 3xTg-AD mice from birth to euthanasia. Log-rank analyses showed genotype differences [G∗ , Exclusion or under-representation of older individuals is χ2 (1) = 4.319, p = 0.038]. In this cohort, the survival drops not unusual in clinical trials despite being the most significant for NTg females started at adulthood (5 months) and decreased health care resource (Zulman et al., 2011). At the preclinical continuously until the end of the experiment (19 months), with a level, most experimental designs are performed in young survival rate of 32.47%. In contrast, 3xTg-AD females exhibited adulthood, some in middle age. The reduced sample size of an early 10% mortality rate before young adulthood (2 months), survivors, with the concomitant increase in laboratory costs and but their survival rate at the end of the experiment was 67.76%. the complex heterogeneity of age-related scenarios, produce a Two isolated 3xTg-AD females died during the isolation period scarcity of research in older animals, even more in models of compared to one group-housed. The day before euthanasia, two neurodegenerative disease (Fahlström et al., 2011; Shoji et al., NTg females died. Four NTg and seven 3xTg-AD mice were 2016; Jackson et al., 2017; Torres-Lista et al., 2017). used for other experiments. Our laboratory described for the first time the convergence No differences were found in the body weight (Figure 3B) of behaviors of 3xTg-AD and NTg mice as a part of the complex and the frailty index scores (Figure 3C) despite a slight increase old age-related scenarios and the heterogeneity regarding in the index in 3xTg-AD females and its enhancement in those physical, cognitive, and anxiety-like patterns (Torres-Lista et al., with isolation. As detailed below, frailty index correlated with 2017). In the present work, the convergence was confirmed in the splenic organometrics (weight and relative weight) in all almost all variables analyzed, and we also showed that the poor cases (©spleen weight: all mice, r = 0.416; only 3xTg-AD mice, performance of the aged NTg animals also contributed to it. r = 0.545; ©spleen index, all mice, r = 0.407; only 3xTg-AD mice, Despite the convergence, the 2-days open field paradigm and r = 0.452; p < 0.05 in all cases). the MB test allowed to detect salient behavioral alterations in the 3xTg-AD mice and/or the effects of isolation. Also, AD- specific salient functional correlations were identified among Convergence but Alzheimer’s the enhanced anxious-like profile and HPA axis and the worse disease-dependent crosstalk between splenic oxidative stress, pointing at AD-derangement in this HPA axis, behavior, and peripheral brain-peripheral psychophysiological triad as a potent risk of organs morbidity and mortality in AD. Cognitive alterations were shown under anxiogenic No genotype differences were found in corticosterone levels conditions when animals were re-tested in the anxiogenic (Figure 3D), but a slight (n.s.) increase in isolated 3xTg- OF, agreeing with previous reports in 14-month-old animals AD females. In the 3xTg-AD mice, corticosterone levels were (Muntsant et al., 2021). In this 2-days open field paradigm correlated to the ethogram in the TM (Figure 2A, as detailed (Torres-Lista et al., 2014), the ethogram and time course of before) and the splenic GPx activity (Figure 3G, r = −0.547, the first experience allows to measure a reduced activity in the p = 0.028). 3xTg-AD. In contrast, in the first minute of the second session The spleen size (weight) and index (relative size, % vs. shows an altered response in the 3xTg-AD mice characterized body weight) were recorded as an indirect measure of their by a need to explore. This is agreement with results in physiological/healthy status. No differences were found, but scenarios mimicking end-of-life dementia (Muntsant et al., a slight (n.s.) increase of splenomegaly incidence in the AD 2021). In the case of isolated 3xTg-AD females, there was an genotype (NTg: 2/9, 3xTg-AD: 5/8, isolated 3xTg-AD: 6/9). hyperactive effect in agreement with recently described increase Statistically significant Pearson’s correlations indicated that both of fine and gross motor function in male sex (Muntsant and variables correlated with the frailty score (Figure 3C,(© FI, as Giménez-Llort, 2020), and non-purposed behaviors mimicking detailed before). obsessive-compulsive disorder (OCD) (Gimenez-Llort and No differences between the experimental groups were Alveal-Mellado, 2021). High activity with low freezing response found in the antioxidant parameters of four different was present in 3xTg-AD females and enhanced in the isolated Frontiers in Aging Neuroscience 06 frontiersin.org Muntsant and Giménez-Llort 10.3389/fnagi.2022.969381 FIGURE 2 Salient behavioral alterations in 18-month-old female 3xTg-AD females in the T-maze and marble test compared to NTg females with normal aging. Results are expressed as mean ± SEM or incidence (%). (A) Ethogram in the acquisition trial (TM1) in the T-Maze; (B) rate of success in TM1; (C) ethogram in the recall trial (TM2) in the T-Maze; (D) rate of success in TM2; (E) spatial Working memory in TM2; (F) MB: Marble test, Interaction with marbles. Inset of bars, number of animals exhibiting the behavior. Statistics: Students t-test: # , isolation effect, # p < 0.05. Pearson’s correlation analysis, ©©p < 0.01; ©©p < 0.001; CORT: corticosterone levels. subgroup. Working memory deficits were enhanced in isolated studied the effects of poor social housing in AD models, females, albeit they did not reach statistical significance. Similar despite reporting worsened cognition and neuropathology results were recently reported on isolated males (Muntsant and (Pietropaolo et al., 2009; Huang et al., 2015; Muntsant and Giménez-Llort, 2020). However, their increased burying was Giménez-Llort, 2020; Peterman et al., 2020), enhancement of noticeable and disrupted the ethogram signature, an effect we olfactory patterns (Marín-Pardo and Giménez-Llort, 2021), recently reported in isolated 3xTg-AD males (Gimenez-Llort body and organs weight loss, and increased asymmetric atrophy and Alveal-Mellado, 2021) with translational value regarding of the hippocampus (Muntsant and Giménez-Llort, 2020). the impact of isolation OCD. Thus, like in humans (Gerst- Interestingly, the present work shows that even short isolation Emerson and Jayawardhana, 2015), housing/social conditions could increase the activity in 3xTg-AD mice and counteract can significantly impact animal behavior, morbidity, and the low activity levels associated with the AD-genotype. This mortality outcomes. However, only a few laboratories have also warns of potential confounding factors and false negatives Frontiers in Aging Neuroscience 07 frontiersin.org Muntsant and Giménez-Llort 10.3389/fnagi.2022.969381 FIGURE 3 Survival, physical health, HPA axis, and peripheral oxidative stress in 18-month-old 3xTg-AD females compared to NTg females with normal aging. (A) Survival; (B) body weight; (C) frailty index scores; (D) HPA axis measured by Corticosterone levels; (E) organometrics of spleen: weight and spleen index (relative weight, % vs. body weight); Oxidative Stress of liver, kidney, heart, and spleen: (F) GSH levels; (G) GPx enzymatic activity and (H) GR enzymatic activity. Results are expressed as the mean ± SEM. HC (high corticosterone levels), LC (low corticosterone levels). Statistics: Kaplan-Meier and Log-Rank test; G, genotype effect, G*p < 0.05. Student t-test, *p < 0.05 vs. LC counterparts. Pearson’s correlation analysis, ©p < 0.05; ©©p < 0.01; TM, T-maze test; FI, frailty index. when experimental groups of mutant animals include isolated mortality rates in the NTg mice, but a survival bias in 3xTg- animals, a common practice due to the high incidence of this AD was associated with a severe initial survival drop before housing condition due to several reasons (i.e., aggressivity and adulthood (2 months of age). In APP23 females, we recently mortality). reported also the relevance of survival bias to define the very- We have consistently reported a sex-dependent increase in old age experimental scenarios (Giménez-Llort et al., 2021). the vulnerability of 3xTg-AD mice (Giménez-Llort et al., 2010; As mentioned, the study of the age-matched NTg female with Torres-Lista et al., 2017) and a survival bias (Giménez-Llort this survival scenario also provides data on a scarcely reported et al., 2021). Therefore, due to the higher variability of the aging normal aging process in gold-standard C57BL/6J strain. process, the survival curves are a must to understand in which The frailty index is becoming a common tool to measure scenario the results are referred. More recently, we have reported health status as it seems sensitive to predicting disease outcomes cohorts that offered a distinct survival scenario (Muntsant et al., and mortality (Zeng et al., 2015). At the translational level, the 2021; Castillo-Mariqueo and Giménez-Llort, 2022). In this case, Mouse Clinical Frailty Index (Whitehead et al., 2014; Feridooni the survival analysis showed genotype-dependent increased et al., 2015) is also a valuable tool in longevity and aging studies Frontiers in Aging Neuroscience 08 frontiersin.org Muntsant and Giménez-Llort 10.3389/fnagi.2022.969381 in mice. In these 18-month-old cohorts, no differences were the corticosterone levels correlated with worse performance in found, but a trend to increase in 3xTg-AD females and the the TM and a poor splenic GPx capacity, which would agree with isolated subgroup correlated with those in spleen organometrics. the HPA axis as one of the major pathways involved in stress The higher frailty scores were observed in the integument and response (Nelson, 1972) and changes in the glucocorticoid’s muscular-skeletal systems, such as alopecia, loss of whiskers, levels favoring alterations in the immune response (Costa-Pinto and dermatitis, which are the more common frailty clinical and Palermo-Neto, 2010). Despite the convergence in the HPA presentations in aged mice (Pettan-Brewer and Treuting, 2011; axis and oxidative stress profiles, HC 3xTg-AD females showed Muntsant and Giménez-Llort, 2021). lower splenic GPx antioxidant activity than LC counterparts. Mice isolation has been proposed as an animal model for This data would also agree with findings suggesting that the self-induced body weight loss (van Leeuwen et al., 1997), being effects of stressful events on the immune system depend on the males significantly affected (Guo et al., 2004) as we also recently individual anxiety response produced by the situation (Cruces demonstrated in 3xTg-AD mice after a naturalistic long-term et al., 2014). social isolation (Muntsant and Giménez-Llort, 2020). In the Regarding the convergence, it would agree with our present work, the sexual dimorphism of body size and the first report on 15-month-old animals, where a significant shorter period of isolation could explain the lack of differences. increase in plasma corticosterone was observed only in Crosstalk between endocrine abnormalities of the HPA axis males (Giménez-Llort et al., 2008) associated with their and patients with AD has been repeatedly described (Ouanes higher vulnerability/mortality ratios. The possible existence and Popp, 2019). Interestingly, in these old 3xTg-AD females, of protective or regulatory mechanisms in 3xTg-AD TABLE 1 Convergence of 18-month-old NTg and 3xTg-AD females’ phenotypes in the corner test, open field test and object recognition test. NTg mice (n = 9) 3xTg-AD (n = 8) 3xTg-AD-ISO (n = 9) Mean ± SEM Mean ± SEM G Mean ± SEM G ISO Corner test (30 s) Visited corners (n) 7.22 ± 0.88 5.88 ± 1.27 n.s 5.56 ± 0.85 n.s n.s Rearing (latency, s) 17.89 ± 3.02 16.63 ± 3.81 n.s 13.11 ± 3.32 n.s n.s Rearings (number) 1.44 ± 0.29 1.88 ± 0.58 n.s 2.56 ± 0.41 n.s n.s Open field test (Day 1, 5 min) Initial freezing (latency, s) 20.56 ± 13.79 3.00 ± 0.63 n.s 5.00 ± 0.87 n.s n.s Exit of the center (latency, s) 28.33 ± 13.98 9.75 ± 1.94 n.s 44.78 ± 31.96 n.s n.s Entrance to the periphery (latency, s) 78.33 ± 37.12 64.75 ± 31.60 n.s 94.33 ± 41.35 n.s n.s Vertical activity (latency, s) 161.78 ± 38.45 175.38 ± 41.39 n.s 210.22 ± 33.49 n.s n.s Bizarre, vertical rearing (latency, s) 265.33 ± 26.1 207.88 ± 42.5 n.s 295.89 ± 4.1 n.s n.s Self-grooming (latency, s) 161.00 ± 26.49 147.50 ± 27.36 n.s 161.33 ± 28.44 n.s n.s Self-grooming (n) 1.33 ± 0.29 1.25 ± 0.25 n.s 1.00 ± 0.24 n.s n.s Defecation boli (n) 2.78 ± 0.46 3.63 ± 0.84 n.s 3.00 ± 0.50 n.s n.s Context and object recognition test Open field test (Day 2, 1 min) Initial freezing (latency, s) 20.11 ± 8.12 9.71 ± 2.53 n.s 7.00 ± 2.15 n.s n.s Exit of the center (latency, s) 23.44 ± 7.80 17.29 ± 3.99 n.s 13.38 ± 4.39 n.s n.s Entrance to the periphery (latency, s) 23.44 ± 8.52 24.57 ± 6.22 n.s 23.50 ± 7.77 n.s n.s Vertical activity (latency, s) 50.67 ± 6.72 53.86 ± 4.09 n.s 40.50 ± 7.62 n.s n.s Bizarre, vertical rearing (latency, s) 60.00 ± 0.00 60.00 ± 0.00 n.s 60.00 ± 0.00 n.s n.s Self-grooming (latency, s) 56.67 ± 3.33 60.00 ± 0.00 n.s 59.00 ± 0.94 n.s n.s Self-grooming (n) 0.33 ± 0.17 0.00 ± 0.00 n.s 0.14 ± 0.13 n.s n.s Defecation boli (n) 1.22 ± 0.46 0.29 ± 0.17 n.s 0.14 ± 0.13 n.s n.s Object recognition test (Day 2, 10 min) Latency 1st object (s) 142.67 ± 76.52 60.29 ± 42.74 n.s 60.13 ± 19.48 n.s n.s Latency 2nd object (s) 235.00 ± 65.45 362.57 ± 85.02 n.s 238.00 ± 80.77 n.s n.s Time 1, acquisition criteria (s) 551.00 ± 40.01 490.71 ± 50.53 n.s 408.88 ± 73.64 n.s n.s Total touch duration (s) 10.50 ± 2.58 12.14 ± 2.95 n.s 14.12 ± 2.35 n.s n.s Animals to reach acquisition criteria 1/9 3/8 n.s 4/9 n.s n.s Statistics: Results are expressed as mean ± SEM or incidence. Student t-test, G, genotype effect (vs. NTg mice), ISO, isolation effect in 3xTg-AD mice (vs. 3xTg-AD mice housed in groups). All n.s., p > 0.05. Frontiers in Aging Neuroscience 09 frontiersin.org Muntsant and Giménez-Llort 10.3389/fnagi.2022.969381 female survivors could also explain the convergence with (Holt-Lunstad and Smith, 2016; Valtorta et al., 2016), and it is controls observed in the present work. A trend of increased considered a risk factor for all-cause mortality (Holt-Lunstad corticosterone levels in the isolated subgroup of 3xTg-AD et al., 2015). females was observed but did not reach statistical significance, At translational level, in accordance with the data presented, probably due to the short isolation period. In fact, the duration several systemic abnormalities have been also described, of the isolation is an important influence on the neuroendocrine reflecting underlying disease processes (Wang et al., 2017). response (Cacioppo et al., 2015). Concerning oxidative stress Identifying and better understanding this crosstalk might status, we have described early peripheral alterations in male offer new chances for develop diagnostic biomarkers or new and female 3xTg-AD mice, with a decrease in antioxidant treatments against AD. defenses in the spleen, kidney, and liver (de la Fuente et al., 2012). In the present work, a decrease in antioxidants associated with the normal aging process, as described in 15- and 18- Conclusion months-old animals (Martínez De Toda et al., 2020) could also explain the convergence in the oxidative stress profiles The scarcity of research in older animals caused by found here when comparing old NTg females with 3xTg-AD the complex age-related scenario and the high individual survivors. heterogeneity mirrors the clinical research scenarios, and it is Spleen and liver dysfunction reflected as splenomegaly a strong limitation to addressing critical gerontological/geriatric (Giménez-Llort et al., 2008; Marchese et al., 2014; Yang issues. Further phenotypical and behavioral studies in normal et al., 2015) and hepatomegaly (Fraile-Ramos and Giménez- aging and AD animal models may help to understand better this Llort, 2021) respectively, have been reported in this animal complex scenario and identify vulnerability/resilience factors. model. We proposed organometry as an early indicator The main findings of this work refer to cognitive, physical, and of peripheral immunological system aging and their easy anxiety-like profiles in 3xTg-AD females that survived beyond weight measurement by echography as a clinical translation advanced stages of the disease but were hardly distinguishable (Giménez-Llort et al., 2012; Gimenez-Llort et al., 2014). In from normal aging counterparts in almost all variables analyzed. the present work, the size and index of the spleen and Despite this convergence, AD-salient behaviors and crosstalk liver [as an immunological organ (Racanelli and Rehermann, with the HPA axis and spleen support the relevance of studying 2006)] were recorded as an indirect measure of animals’ brain-periphery interactions. On the other hand, in a subgroup immunological/physiological status. of 3xTg-AD females, short-term isolation modified anxiety Interestingly, in a longitudinal study in 3xTg-AD females, patterns and cope with stress strategies. The correlations from 2 to 15 months of age, we reported function and redox and segregation analysis according to corticosterone levels state of peritoneal leukocytes as preclinical and prodromic revealed a negative impact on the antioxidant GPx enzymatic markers, but convergence of many immune-oxidative stress activity in the spleen, which may suppose a potential risk of markers with those of NTg females at the oldest studied age morbidity and mortality. Further studies should investigate (Maté et al., 2015). Thus, the results indicated changes in the relationship between the immunoendocrine status, and CD25 + B and naïve CD8 T cells percentage, NK percentage the underlying neuroanatomical substrates related to AD, and cytotoxic activity, GSSG/GSH ratio, and GSH content especially in emotion-related brain regions, such as amygdala or already at asymptomatic stages (2 months of age). Premature ventral hippocampus. immunosenescence at 4 months of age, when intraneuronal β- amyloid immunoreactivity emerges, with reduced chemotaxis, phagocytosis, and lymphoproliferation and also lower reduced glutathione and higher xanthine oxidase activity. Alterations Data availability statement in lymphoproliferation, phagocytosis, xanthine oxidase activity and CD5+ B1 cells continued at 15 months of age. The raw data supporting the conclusions of this article will However, CD11b+, CD335+, CD19+, CD25+, macrophage and be made available by the authors, without undue reservation. lymphocyte chemotaxis, basal lymphoproliferation, natural killer activity, GSH, GSSG/GSH ratio and catalase activity of 3xTg-AD mice and NTg mice converged at this old age. Ethics statement Despite the organometrics and oxidative status of spleen in 3xTg-AD females did not reach statistical significance, The study was conducted according to the guidelines of the their correlation with frailty score and HPA axis suggests Declaration of Helsinki and approved by the Ethics Committee that even a short isolation in 3xTg-AD female survivors may of Departament de Medi Ambient i Habitatge, Generalitat de increase their risk of morbidity and mortality. In fact, various Catalunya (CEEAH 3588/DMAH 9452) on March 8, 2019. The meta-analyses have described that social isolation is associated study complies with the ARRIVE guidelines developed by the with an increased risk of cardiovascular disease and stroke NC3Rs and the aim to reduce the number of animals used. Frontiers in Aging Neuroscience 10 frontiersin.org Muntsant and Giménez-Llort 10.3389/fnagi.2022.969381 Author contributions California, Irvine, CA, United States, for kindly providing the progenitors of the Spanish colonies of homozygous 3xTg-AD LG-L: conceptualization, funding acquisition, and writing— and NTg mice. Thanks to Javier Carrasco, The INc Molecular review and editing. AM: performance and analysis of behavior, Biology Unit, for his help in corticosterone and oxidative statistical analysis, illustrations, and writing—original draft, stress determinations. review, and editing. Both authors have read and agreed to the published version of the manuscript. Conflict of interest Funding The authors declare that the research was conducted in the absence of any commercial or financial relationships that could This work was funded by the UAB-GE-260408 and be construed as a potential conflict of interest. ArrestAD H2020 Fet-OPEN-1-2016-2017-737390, European Union’s Horizon 2020 research and innovation program under grant agreement No. 737390 to LG-L, and Envellir bé-Saber envejecer—Healthy Aging.org Memorial Mercedes Llort Sender Publisher’s note 2022/81/24091941/02. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated Acknowledgments organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or We thank Frank M. LaFerla, Institute for Memory claim that may be made by its manufacturer, is not guaranteed Impairments and Neurological Disorders, University of or endorsed by the publisher. References Arranz, L., de Castro, N. M., Baeza, I., Giménez-Llort, L., and de La Fuente, de la Fuente, M., Manassra, R., Mate, I., Vida, C., Hernanz, A., and Gimenez- M. (2011). Effect of environmental enrichment on the immunoendocrine aging of Llort, L. (2012). Early oxidation and inflammation state of the immune system in male and female triple-transgenic 3xTg-AD mice for Alzheimer’s disease. JAD 25, male and female triple transgenic mice for Alzheimer’s Disease (3xTgAD). Free 727–737. doi: 10.3233/JAD-2011-110236 Rad. Biol. Med. 53:S165. doi: 10.1016/J.FREERADBIOMED.2012.08.346 Baeta-Corral, R., and Giménez-Llort, L. (2014). Bizarre behaviors and risk Donovan, N. J., and Blazer, D. (2020). Social Isolation and Loneliness in Older assessment in 3xTg-AD mice at early stages of the disease. Behav. Brain Res. 258, Adults: Review and Commentary of a National Academies Report. Am. J. Geriatr. 97–105. doi: 10.1016/J.BBR.2013.10.017 Psych. 28:1233. doi: 10.1016/J.JAGP.2020.08.005 Belfiore, R., Rodin, A., Ferreira, E., Velazquez, R., Branca, C., Caccamo, España, J., Giménez-Llort, L., Valero, J., Miñano, A., Rábano, A., Rodriguez- A., et al. (2019). Temporal and regional progression of Alzheimer’s disease- Alvarez, J., et al. (2010). Intraneuronal β-Amyloid Accumulation in the Amygdala like pathology in 3xTg-AD mice. Aging Cell 18:e12873. doi: 10.1111/ACEL. Enhances Fear and Anxiety in Alzheimer’s Disease Transgenic Mice. Biol. Psych. 12873 67, 513–521. doi: 10.1016/J.BIOPSYCH.2009.06.015 Billings, L. M., Oddo, S., Green, K. N., McGaugh, J. L., and LaFerla, F. M. (2005). Fahlström, A., Yu, Q., and Ulfhake, B. (2011). Behavioral changes in Intraneuronal Abeta causes the onset of early Alzheimer’s disease-related cognitive aging female C57BL/6 mice. Neurobiol. Aging 32, 1868–1880. doi: 10.1016/J. deficits in transgenic mice. Neuron 45, 675–688. doi: 10.1016/J.NEURON.2005.01. NEUROBIOLAGING.2009.11.003 040 Feridooni, H. A., Sun, M. H., Rockwood, K., and Howlett, S. E. (2015). Reliability Blázquez, G., Cañete, T., Tobeña, A., Giménez-Llort, L., and Fernández-Teruel, of a Frailty Index Based on the Clinical Assessment of Health Deficits in Male A. (2014). Cognitive and emotional profiles of aged Alzheimer’s disease (3×TgAD) C57BL/6J Mice. Biol. Sci. 70, 686–693. doi: 10.1093/gerona/glu161 mice: Effects of environmental enrichment and sexual dimorphism. Behav. Brain Fraile-Ramos, J., and Giménez-Llort, L. (2021). Sex-dependent hepatomegaly, Res. 268, 185–201. doi: 10.1016/J.BBR.2014.04.008 and increased hepatic oxidative stress in old male and female 3xTg-AD mice as Cacioppo, J. T., Cacioppo, S., Capitanio, J. P., and Cole, S. W. (2015). The compared to mice with physiological aging. Proc. Int. Electr. Conf. Brain Sci. 8, 8. Neuroendocrinology of Social Isolation. Annu. Rev. Psychol. 66:733. doi: 10.1146/ doi: 10.3390/IECBS2021-10668 ANNUREV-PSYCH-010814-015240 Friedler, B., Crapser, J., and McCullough, L. (2015). One is the deadliest Castillo-Mariqueo, L., and Giménez-Llort, L. (2022). Kyphosis and bizarre number: the detrimental effects of social isolation on cerebrovascular diseases and patterns impair spontaneous gait performance in end-of-life mice with cognition. Acta Neuropathol. 129, 493–509. doi: 10.1007/S00401-014-1377-9 Alzheimer’s disease pathology while gait is preserved in normal aging. Neurosci. Gerst-Emerson, K., and Jayawardhana, J. (2015). Loneliness as a Public Health Lett. 767:136280. doi: 10.1016/J.NEULET.2021.136280 Issue: The Impact of Loneliness on Health Care Utilization Among Older Adults. Costa-Pinto, F. A., and Palermo-Neto, J. (2010). Neuroimmune Interactions in Am. J. Publ. Health 105:1013. doi: 10.2105/AJPH.2014.302427 Stress. Neuroimmunomodulation 17, 196–199. doi: 10.1159/000258722 Gimenez-Llort, L., and Alveal-Mellado, D. (2021). Digging Signatures in 13- Cruces, J., Venero, C., Pereda-Pérez, I., and de la Fuente, M. (2014). A higher Month-Old 3xTg-AD Mice for Alzheimer’s Disease and Its Disruption by Isolation anxiety state in old rats after social isolation is associated to an impairment of the Despite Social Life Since They Were Born. Front. Behav. Neurosci. 14:277. doi: immune response. J. Neuroimmunol. 277, 18–25. doi: 10.1016/J.JNEUROIM.2014. 10.3389/FNBEH.2020.611384/BIBTEX 09.011 Giménez-Llort, L., Arranz, L., Maté, I., and de La Fuente, M. (2008). Curelaru, A., Marzolf, S. J., Provost, J. C. K. G., and Zeon, H. H. H. (2021). Social Gender-specific neuroimmunoendocrine aging in a triple-transgenic 3xTg- Isolation in Dementia: The Effects of COVID-19. J. Nurse Practit. 17, 950–953. AD mouse model for Alzheimer’s disease and its relation with longevity. doi: 10.1016/J.NURPRA.2021.05.002 Neuroimmunomodulation 15, 331–343. doi: 10.1159/000156475 Frontiers in Aging Neuroscience 11 frontiersin.org Muntsant and Giménez-Llort 10.3389/fnagi.2022.969381 Giménez-Llort, L., Blázquez, G., Cañete, T., Johansson, B., Oddo, S., Tobeña, A., Aging With Special Concerns on the Effects of Social Isolation. Front. Neurosci. et al. (2007). Modeling behavioral and neuronal symptoms of Alzheimer’s disease 15:733984. doi: 10.3389/FNINS.2021.733984/FULL in mice: a role for intraneuronal amyloid. Neurosci. Biobehav. Rev. 31, 125–147. Martínez De Toda, I., Vida, C., Garrido, A., and de La Fuente, M. (2020). Redox doi: 10.1016/J.NEUBIOREV.2006.07.007 Parameters as Markers of the Rate of Aging and Predictors of Life Span. J. Gerontol. Giménez-Llort, L., García, Y., Buccieri, K., Revilla, S., Suol, C., Cristofol, R., et al. 75, 613–620. doi: 10.1093/GERONA/GLZ033 (2010). Gender-specific neuroimmunoendocrine response to treadmill exercise in Maté, I., Cruces, J., Giménez-Llort, L., and de La Fuente, M. (2015). Function 3xTg-AD mice. Int. J. Alzheimer’s Dis. 2010:128354. doi: 10.4061/2010/128354 and Redox State of Peritoneal Leukocytes as Preclinical and Prodromic Markers Giménez-Llort, L., Marin-Pardo, D., Marazuela, P., and Hernández-Guillamón, in a Longitudinal Study of Triple-Transgenic Mice for Alzheimer’s Disease. M. (2021). Survival Bias and Crosstalk between Chronological and Behavioral J. Alzheimer’s Dis. 43, 213–226. doi: 10.3233/JAD-140861 Age: Age- and Genotype-Sensitivity Tests Define Behavioral Signatures in Middle- Mitchell, S. J., Scheibye-Knudsen, M., Longo, D. L., and de Cabo, R. (2015). Aged, Old, and Long-Lived Mice with Normal and AD-Associated Aging. Animal Models of Aging Research: Implications for Human Aging and Age- Biomedicines 9:636. doi: 10.3390/biomedicines9060636 Related Diseases. Annu. Rev. Anim. Biosci. 3, 283–303. doi: 10.1146/ANNUREV- Giménez-Llort, L., Maté, I., Manassra, R., Vida, C., and de la Fuente, M. (2012). ANIMAL-022114-110829 Peripheral immune system and neuroimmune communication impairment in a Muntsant, A., and Giménez-Llort, L. (2020). Impact of Social Isolation on mouse model of Alzheimer’s disease. Anna. N. Y. Acad. Sci. 1262, 74–84. doi: the Behavioral, Functional Profiles, and Hippocampal Atrophy Asymmetry in 10.1111/J.1749-6632.2012.06639.X Dementia in Times of Coronavirus Pandemic (COVID-19): A Translational Gimenez-Llort, L., Torres-Lista, V., and Fuente, M. (2014). Crosstalk Neuroscience Approach. Front. Psych. 11:1126. doi: 10.3389/FPSYT.2020.572583/ between Behavior and Immune System During the Prodromal Stages of BIBTEX Alzheimer’s Disease. Curr. Pharmaceut. Design 20, 4723–4732. doi: 10.2174/ Muntsant, A., and Giménez-Llort, L. (2021). Genotype Load Modulates 1381612820666140130205500 Amyloid Burden and Anxiety-Like Patterns in Male 3xTg-AD Survivors Guo, M., Wu, C. F., Liu, W., Yang, J. Y., and Chen, D. (2004). Sex difference despite Similar Neuro-Immunoendocrine, Synaptic and Cognitive Impairments. in psychological behavior changes induced by long-term social isolation in mice. Biomedicines 9:715. doi: 10.3390/BIOMEDICINES9070715 Progr. Neuro Psychopharmacol. Biol. Psych. 28, 115–121. doi: 10.1016/J.PNPBP. Muntsant, A., Jiménez-Altayó, F., Puertas-Umbert, L., Jiménez-Xarrie, 2003.09.027 E., Vila, E., and Giménez-Llort, L. (2021). Sex-Dependent End-of-Life Holt-Lunstad, J., and Smith, T. B. (2016). Loneliness and social isolation as Mental and Vascular Scenarios for Compensatory Mechanisms in Mice risk factors for CVD: implications for evidence-based patient care and scientific with Normal and AD-Neurodegenerative Aging. Biomedicines 9, 1–29. inquiry. Heart 102, 987–989. doi: 10.1136/HEARTJNL-2015-309242 doi: 10.3390/BIOMEDICINES9020111 Holt-Lunstad, J., Smith, T. B., Baker, M., Harris, T., and Stephenson, D. (2015). Nelson, D. H. (1972). Regulation of glucocorticoid release. Am. J. Med. 53, Loneliness and social isolation as risk factors for mortality: a meta-analytic review. 590–594. doi: 10.1016/0002-9343(72)90155-6 Perspect. Psychol. Sci. 10, 227–237. doi: 10.1177/1745691614568352 Oddo, S., Caccamo, A., Shepherd, J. D., Murphy, M. P., Golde, T. E., Kayed, Huang, H., Wang, L., Cao, M., Marshall, C., Gao, J., Xiao, N., et al. (2015). R., et al. (2003). Triple-Transgenic Model of Alzheimer’s Disease with Plaques Isolation Housing Exacerbates Alzheimer’s Disease-Like Pathophysiology in Aged and Tangles: Intracellular Aβ and Synaptic Dysfunction. Neuron 39, 409–421. APP/PS1 Mice. Int. J. Neuropsychopharmacol. 18, 1–10. doi: 10.1093/IJNP/ doi: 10.1016/S0896-6273(03)00434-3 PYU116 Ouanes, S., and Popp, J. (2019). High cortisol and the risk of dementia and Jackson, S. J., Andrews, N., Ball, D., Bellantuono, I., Gray, J., Hachoumi, L., et al. alzheimer’s disease: A review of the literature. Front. Aging Neurosci. 11:43. doi: (2017). Does age matter? The impact of rodent age on study outcomes. Lab. Anim. 10.3389/FNAGI.2019.00043/FULL 51, 160–169. doi: 10.1177/0023677216653984 Peterman, J. L., White, J. D., Calcagno, A., Hagen, C., Quiring, M., Paulhus, K., Kamiya, M., Sakurai, T., Ogama, N., Maki, Y., and Toba, K. (2014). Factors et al. (2020). Prolonged isolation stress accelerates the onset of Alzheimer’s disease- associated with increased caregivers’ burden in several cognitive stages of related pathology in 5xFAD mice despite running wheels and environmental Alzheimer’s disease. Geriatr. Gerontol. Int. 14, 45–55. doi: 10.1111/GGI.12260 enrichment. Behav. Brain Res. 379:112366. doi: 10.1016/J.BBR.2019.112366 Kaufer, D. I., Cummings, J. L., Christine, D., Bray, T., Castellon, S., Masterman, Pettan-Brewer, C., and Treuting, P. M. (2011). Practical pathology of aging mice. D., et al. (1998). Assessing the Impact of Neuropsychiatric Symptoms in Pathobiol. Aging Age Relat. Dis. 1:7202. doi: 10.3402/PBA.V1I0.7202 Alzheimer’s Disease: The Neuropsychiatric Inventory Caregiver Distress Scale. Pietropaolo, S., Sun, Y., Li, R., Brana, C., Feldon, J., and Yee, B. K. (2009). J. Am. Geriatr. Soc. 46, 210–215. doi: 10.1111/J.1532-5415.1998.TB02542.X Limited impact of social isolation on Alzheimer-like symptoms in a triple Kiecolt-Glaser, J. K., McGuire, L., Robles, T. F., and Glaser, R. (2002). transgenic mouse model. Behav. Neurosci. 123, 181–195. doi: 10.1037/A0013607 Psychoneuroimmunology and psychosomatic medicine: back to the future. Porcelli, S., van der Wee, N., van der Werff, S., Aghajani, M., Glennon, J. C., and Psychosomat. Med. 64, 15–28. doi: 10.1097/00006842-200201000-00004 van Heukelum, S. (2019). Social brain, social dysfunction and social withdrawal. Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M., and Altman, D. G. Neurosci. Biobehav. Rev. 97, 10–33. doi: 10.1016/J.NEUBIOREV.2018.09.012 (2010). Improving Bioscience Research Reporting: The ARRIVE Guidelines for Racanelli, V., and Rehermann, B. (2006). The liver as an immunological organ. Reporting Animal Research. PLoS Biol. 8:e1000412. doi: 10.1371/JOURNAL.PBIO. Hepatology 43, S54–S62. doi: 10.1002/HEP.21060 1000412 Read, S., Comas-Herrera, A., and Grundy, E. (2020). Social Isolation and Knopman, D. S., Amieva, H., Petersen, R. C., Chételat, G., Holtzman, D. M., Memory Decline in Later-life. J. Gerontol. 75, 367–376. doi: 10.1093/GERONB/ Hyman, B. T., et al. (2021). Alzheimer disease. Nat. Rev. Dis. Primers 7, 1–21. GBZ152 doi: 10.1038/s41572-021-00269-y Shoji, H., Takao, K., Hattori, S., and Miyakawa, T. (2016). Age-related changes in Komarova, N. L., and Thalhauser, C. J. (2011). High degree of heterogeneity in behavior in C57BL/6J mice from young adulthood to middle age. Molecular Brain Alzheimer’s disease progression patterns. PLoS Computat. Biol. 7:11. doi: 10.1371/ 2016 9:1 9, 1–18. doi: 10.1186/S13041-016-0191-9 JOURNAL.PCBI.1002251 Steptoe, A., Shankar, A., Demakakos, P., and Wardle, J. (2013). Social isolation, Kuiper, J. S., Zuidersma, M., Oude Voshaar, R. C., Zuidema, S. U., van den loneliness, and all-cause mortality in older men and women. Proc. Natl. Acad. Sci. Heuvel, E. R., Stolk, R. P., et al. (2015). Social relationships and risk of dementia: U.S.A. 110, 5797–5801. doi: 10.1073/PNAS.1219686110 A systematic review and meta-analysis of longitudinal cohort studies. Ageing Res. Sterniczuk, R., Antle, M. C., Laferla, F. M., and Dyck, R. H. (2010). Rev. 22, 39–57. doi: 10.1016/J.ARR.2015.04.006 Characterization of the 3xTg-AD mouse model of Alzheimer’s disease: part Llewellyn, D. J., Lourida, I., Hannon, E., Littlejohns, T. J., Langa, K. M., 2. Behav. Cogn. Chang. 1348, 149–155. doi: 10.1016/J.BRAINRES.2010.0 Hyppönen, E., et al. (2019). Association of Lifestyle and Genetic Risk With 6.011 Incidence of Dementia. JAMA 322, 430–437. doi: 10.1001/JAMA.2019.9879 Torres-Lista, V., Fuente, M. D., and Giménez-Llort, L. (2017). Survival Curves Maccioni, R. B., Rojo, L. E., Fernández, J. A., and Kuljis, R. O. (2009). The role and Behavioral Profiles of Female 3xTg-AD Mice Surviving to 18-Months of of neuroimmunomodulation in Alzheimer’s disease. Anna. N. Y. Acad. Sci. 1153, Age as Compared to Mice with Normal Aging. J. Alzheimer’s Dis. Rep. 1:47. 240–246. doi: 10.1111/J.1749-6632.2008.03972.X doi: 10.3233/ADR-170011 Marchese, M., Cowan, D., Head, E., Ma, D., Karimi, K., Ashthorpe, V., et al. Torres-Lista, V., López-Pousa, S., and Giménez-Llort, L. (2015). Marble-burying (2014). Autoimmune Manifestations in the 3xTg-AD Model of Alzheimer’s is enhanced in 3xTg-AD mice, can be reversed by risperidone and it is modulable Disease. J. Alzheimer’s Dis. 39, 191–210. doi: 10.3233/JAD-131490 by handling. Behav. Proc. 116, 69–74. doi: 10.1016/j.beproc.2015.05.001 Marín-Pardo, D., and Giménez-Llort, L. (2021). Olfactory Signatures in the Torres-Lista, V., Parrado-Fernández, C., Alvarez-Montón, I., Frontiñán-Rubio, Food Finding Test in Mice With Normal and Alzheimer’s Disease-Pathological J., Durán-Prado, M., Peinado, J. R., et al. (2014). Neophobia, NQO1 and SIRT1 as Frontiers in Aging Neuroscience 12 frontiersin.org Muntsant and Giménez-Llort 10.3389/fnagi.2022.969381 premorbid and prodromal indicators of AD in 3xTg-AD mice. Behav. Brain Res. Wang, J., Gu, B. J., Masters, C. L., and Wang, Y. J. (2017). A systemic view of 271, 140–146. doi: 10.1016/J.BBR.2014.04.055 Alzheimer disease — insights from amyloid-β metabolism beyond the brain. Nat. Rev. Neurol. 13, 612–623. doi: 10.1038/nrneurol.2017.111 Turner, R. C., Seminerio, M. J., Naser, Z. J., Ford, J. N., Martin, S. J., Matsumoto, R. R., et al. (2012). Effects of aging on behavioral assessment Whitehead, J. C., Hildebrand, B. A., Sun, M., Rockwood, M. R., Rose, R. A., performance: implications for clinically relevant models of neurological disease: Rockwood, K., et al. (2014). A Clinical Frailty Index in Aging Mice: Comparisons Laboratory investigation. J. Neurosurg. 117, 629–637. doi: 10.3171/2012.5.JNS11 With Frailty Index Data in Humans. J. Gerontol. Ser. A 69:621. doi: 10.1093/ 2224 GERONA/GLT136 Valtorta, N. K., Kanaan, M., Gilbody, S., Ronzi, S., and Hanratty, B. (2016). Yang, S. H., Kim, J., Lee, M. J., and Kim, Y. (2015). Abnormalities of plasma Original article: Loneliness and social isolation as risk factors for coronary cytokines and spleen in senile APP/PS1/Tau transgenic mouse model. Scientif. Rep. heart disease and stroke: systematic review and meta-analysis of longitudinal 5, 1–9. doi: 10.1038/srep15703 observational studies. Heart 102:1009. doi: 10.1136/HEARTJNL-2015-30 Zeng, A., Song, X., Dong, J., Mitnitski, A., Liu, J., Guo, Z., et al. (2015). Mortality 8790 in Relation to Frailty in Patients Admitted to a Specialized Geriatric Intensive Care van Dijk, P. T., Dippel, D. W., and Habbema, J. D. (1991). Survival of patients Unit. J. Gerontol. Ser. A 70:1586. doi: 10.1093/GERONA/GLV084 with dementia. J. Am. Geriatr. Soc. 39, 603–610. Zulman, D. M., Sussman, J. B., Chen, X., Cigolle, C. T., Blaum, C. S., and van Leeuwen, S. D., Bonne, O. B., Avraham, Y., and Berry, E. M. (1997). Hayward, R. A. (2011). Examining the evidence: A systematic review of the Separation as a new animal model for self-induced weight loss. Physiol. Behav. 62, inclusion and analysis of older adults in randomized controlled trials. J. General 77–81. doi: 10.1016/S0031-9384(97)00144-3 Int. Med. 26, 783–790. doi: 10.1007/S11606-010-1629-X Frontiers in Aging Neuroscience 13 frontiersin.org