Introduction
Alzheimer’s disease (AD) is the most common cause of dementia, affecting six billion people in USA alone [
1]. This progressive disease is characterized by gradual memory loss, and the most common form is late-onset AD, which, in most cases, has no clear inheritable cause. Thus, for many, the risk of developing AD is modulated by an amalgamation of genetic and environmental factors, but the exact mechanisms leading to AD remain elusive. One such environmental risk factor is exposure to adversity during childhood, which can contribute to early-life stress (ELS) and alter brain development [
2]. The changes due to ELS may lead to long-lasting, harmful effects, including an increased risk of developing AD (reviewed in [
2]). In addition, ELS increases the likelihood of developing major depressive disorder (MDD) [
3], which in itself is a risk factor for AD along with other stress-related disorders (for reviews, refer to [
4,
5]).
Sex-specific effects have been observed both in response to ELS and the development of AD [
6]. Of those who have been exposed to ELS, women tend to be more susceptible to developing a stress-related disorder compared to men. Among those with AD, women are also disproportionately affected compared to men [
7]. At a cellular level, sex-related differences are also present in microglia, the resident immune cells of the brain. These differences have been noted as differences in the structure, function, and transcriptomic and proteomic profiles of microglia between males and females [
8]. This suggests that there are sex-dependent microglial responses in the context of different disorders and diseases. Within the last decade, neuroinflammation and microglial dysfunction have been identified as key drivers of AD [
9]. In addition, more recent evidence has shown altered crosstalk between the brain and peripheral immune systems, which may play a role in the development of neurodegenerative diseases [
10]. Therefore, inflammatory alterations may serve as the bridge between ELS-related alterations and increased risk of developing AD [
11]. Still, the mechanisms linking ELS and AD remain largely unknown.
Here, we modeled ELS in wild-type and 5xFAD AD-model mice by using a protocol for maternal separation (MS) (reviewed in [
12]) and looked for MS- and sex-specific alterations, both as separate main effects and as an interaction effect. We find that MS impacts the inflammatory system in the brain and periphery in a sex-specific manner. In the long-term, MS leads to increased depressive-like behavior and impaired novel object recognition memory depending on genotype and sex. In 5xFAD mice, AD pathology is exacerbated by MS differentially based on sex. Altogether, we provide molecular and cellular clues that may help explain the link between ELS and AD risk and highlight the importance of considering sex-specific responses.
Materials and methods
Animals
All experiments were performed in accordance with the guidelines on experimental animal research approved by the Malmö-Lund Ethical Committee for Animal Research in Sweden (Dnr. 5.8. 18-01107/2018). The experiments were conducted on age-matched B6SJL 5xFAD Tg6799 transgenic mice and their wild-type (WT) littermates using the following breeding strategy: two WT female mice were paired with one heterozygous 5xFAD male mouse (9–12 weeks old) per cage. Thus, from the same breeding cage, we obtained both WT and 5xFAD heterozygous male and female littermates. Pups were weaned at postnatal day 30 (P30). Age- and sex-matched littermates were group-housed (4–5 animals/cage) in standard cages on a 12 h light/dark cycle, and water, food and nesting material were provided ad libitum.
Genotyping
Mice were genotyped for WT or 5xFAD status by PCR as previously described [
13]. Briefly, DNA from ear punches collected at P30 was extracted using a kit (Extract-N-Amp™, Sigma-Aldrich) and amplified for PCR using the 2× PCR Bio HS Taq Mix Red enzyme (PCR Biosystems) with the following primers (5′–3′): APP forward: AGGACTGACCACTCGACCAG; APP reverse: CGGGGGTCTAGTTCTGCAT; PSN1 forward: AATAGAGAACGGCAGGAGCA; PSN1 reverse: GCCATGAGGGCACTAATCAT; WT APP forward: CTAGGCCACAGAATTGAAAGATCT; WT APP reverse: GTAGGTGGAAATTCTAGCATCATCC.
Maternal separation
Maternal separation (MS) was performed as recently reported [
14]. Briefly, pups were separated from their dams every day from P2 to P14, 3 h per day (09.00 AM–12.00 PM), and placed in a different room to avoid vocalized communication with their dams. Extra nesting material (cotton pieces) was added to keep them warm. After 180 min, pups were returned to their home cage and left undisturbed until the following MS session. Control litters (non-MS) were handled similarly to the MS pups from P2 to P14 without separation from their dams. No differences in body weight were found at the end of the MS manipulation.
Experimental design
5xFAD and WT littermate mice were randomly assigned to the Non-MS and MS conditions between P2 and P14. At P15, two cohorts of animals (
n = 5 animals/group) were sacrificed to analyze microglia and cytokine levels. Behavioral characterization started at 4 months old and was performed in five different cohorts (5–13 animals/group). Following three consecutive days of handling, mice were tested for
anxiety-like behavior in the open field test (
n = 6–13 animals/group), learning and memory using the novel object recognition memory test (5–7 animals/group) and
depressive-like behavior in the forced swim test (6–12 animals/group) (see Fig.
2A). Three days after the last behavioral session, a random subset of animals from each group was perfused, and their brains were collected for subsequent molecular analysis (≥ 4 animals/group). Flow cytometry analyses were performed on the spleens from 25 animals included in the behavioral experiments and 42 animals that did not undergo behavioral testing for a minimum of 5 animals/group.
Behavioral testing
Behavioral tests were performed in specific behavioral rooms during the light phase (09.00 AM–3.00 PM) by the same researcher. First, mice were habituated to the behavioral room for at least 30 min before starting the test. All equipment was thoroughly cleaned with 70% ethanol at the beginning of the session and between trials to remove olfactory cues. The testing and analyses of videotaped sessions were performed by a blinded, experienced researcher.
Open field test
To evaluate
anxiety-like behavior, the open field test was performed as previously described [
15] with minor modifications. Mice were placed facing the wall in an arena (55 × 40 × 40 cm) and allowed to freely explore for 5 min. The session was recorded, and the percentage of the time spent in the center was manually scored and represented.
Novel object recognition memory test
To test cognition, we performed the novel object recognition (NOR) memory test as described previously [
16]. Briefly, mice were placed in an arena located in the behavioral room with dim lighting and background noise. During the training session, mice were allowed to explore two identical objects for 15 min. One-hour post-training short-term memory was tested by returning the mice to the arena with one object from the training session (familiar object) and one novel object for 10 min. The time spent exploring each object was recorded and manually scored to evaluate the relative exploration of the novel object compared with the familiar object and was calculated as the discrimination index (DI = (
tnovel −
tfamiliar)/(
tnovel +
tfamiliar)). Only active exploration was considered, defined as direct interaction with the nose towards the object within a 1.5 cm range and/or touching the object with the nose or vibrissae. Sitting on the object or circling around it was not considered exploratory behavior and not included in the analysis.
Forced swim test
To test depressive-like behavior, the forced swim test was performed as previously described [
17]. Briefly, mice were individually placed in a cylindrical glass tank filled with water at 23 ± 1 ºC. Each mouse was recorded for 6 min and, at the end of the test, was immediately dried and warmed using a dry paper towel before being returned to their home cage. The time spent immobile, defined as the time spent not moving and floating passively, was manually analyzed from video recordings and represented as a percentage encompassing the last 2 min of the test.
Sample collection and tissue processing
At P15 and 4 months of age, mice were anesthetized using isoflurane (5%) in oxygen (Virbac) and transcardially perfused with 0.9% saline solution.
Brain
Brains were hemisected to enable several means of downstream analysis per mouse. One hemisphere of the brain from P15 and 4-month-old mice was transferred to a 4% paraformaldehyde (PFA, Histolab) solution and fixed overnight at 4 °C. The brains were then transferred to a 30% sucrose solution and stored at 4 °C for 48 h. Coronal sections (40 μm) were sliced using a freezing microtome (Leica SM2000DR) and preserved in a cryoprotective solution (30% sucrose [Sigma-Aldrich], 30% ethylene glycol [Sigma-Aldrich], 40% phosphate-buffered saline) at − 20 °C.
With the other hemisphere, hippocampus and prefrontal cortex were isolated and quickly frozen at − 80 °C for further processing.
Spleen
Spleens from 4-month-old mice were harvested and manually dissociated into single cell suspensions in FACS buffer (PBS containing 10% FBS and 0.2 mM EDTA). After erythrocyte lysis with ACK (ammonium-chloride-potassium) lysing buffer for 2 min at room temperature (R/T), total cellularity was determined by direct counting using a Sysmex KX-21 N analyzer (Sysmex).
Immunofluorescence
Brain sections from P15 and 4-month-old mice were labeled as previously described [
14]. Briefly, free-floating coronal sections (40 µm) were permeabilized with 1% (v/v) Triton X-100 in PBS (PBS-T 1%, Sigma-Aldrich) for 1 h and then incubated in blocking solution with 5% normal donkey serum (NDS) in PBS-T 1% for Gal-3, 6E10 and Iba1 co-labeling. For Iba1 and CD68 co-labeling, sections were blocked in 3% NDS in PBS-T 0.3% for 1 h. Sections were then incubated overnight at 4ºC with primary antibodies for microglia (Iba1, Wako, 1:500), Gal-3 (R&D, 1:300), CD68 (BioRad, 1:1000) and APP/amyloid-beta (Aβ) (6E10, Covance, 1:500). The following day, the tissue was rinsed for 1 h in PBS-T 0.1%, incubated with the corresponding secondary antibody (1:500, donkey-anti-rabbit 647 for Iba1, donkey-anti-goat 488 for Gal3, donkey-anti-rat conj. Cy2 for CD68 and donkey-anti-mouse 555 for 6E10, Invitrogen) for 1 h and then mounted with Diamond Antifade Mountant (ThermoScientific, Sweden) for visualization. Images of Iba1 labeling of P15 mice and Iba1, Gal3, and 6E10 co-labeling of 4-month-old mice were taken using a Nikon A1RHD laser-scanning confocal microscope using a 20 × air objective (numerical aperture 0.5). CD68-Iba1 co-labeled sections were visualized with a Nikon Eclipse 80i upright microscope using a 20 × objective (numerical aperture 0.75). All acquisition parameters were kept constant for a given experiment and were taken by the same researcher, blinded to the genotype, sex, and ELS exposure. The fluorescently labeled structures were analyzed using Fiji Image J software (W. Rasband, National Institutes of Health). For analysis, image background was first subtracted before a brightness threshold was set for the measurement of each marker. Confocal images were analyzed from a maximum intensity projection. At P15, hippocampal microglia were morphologically classified into three morphological phenotypes: with ramified processes, with stout processes, and round/ameboid like previously reported [
18]. At least 2–3 brain sections per animal and brain region were analyzed at the following Bregma coordinates: hippocampus (− 2.0, − 2.5 mm), prefrontal cortex (+ 2.22, + 1.98 mm), and amygdala (− 1.7, − 2.0 mm).
Congo red staining
To measure plaque burden, coronal brain sections (40 µm) from 4-month-old 5xFAD mice were mounted onto SuperFrost Plus slides (Thermo Scientific) 24 h before staining with Congo red. Sections were then incubated for 3 min in an alcoholic sodium chloride solution (0.03% (w/v) NaCl in 100% ethanol) with 20% Congo red (Sigma Aldrich) before being dehydrated for visualization. Slides were scanned using a 40 × objective in brightfield mode on a digital scanner (Nanozoomer 2H). Densitometric analysis of the labeled plaques was performed using NDP2-viewer software (Hammamatsu, JP) by a researcher blinded to the sex and ELS exposure.
Flow cytometry
Spleens were collected and homogenized as described in Sect. "
Spleen". Subsequently, a fraction of each sample was divided into two different tubes for labeling with specific antibody cocktails: one for identifying mainly lymphoid cells and the other for identifying mostly myeloid cells. Cells were labeled in FACS buffer for 30 min at 4 °C with the corresponding master mix containing the following antibodies: lymphoid master mix containing CD4-APC Cy7 (GK1.5), NK1.1- Biotin (PK136), CD45.2-FITC (104), CD45.1-FITC (A20), Streptavidin-BV605 (Biolegend), CD8a- PeCy5 (53-6.7), CD11b-APC (M1/70), and CD62L-BV421; and Myeloid master mix containing B220- Biotin (RA3-6B2), NK1.1- Biotin (PK136), CD4-Biotin (GK1.5), CD8a-Biotin (53-6.7), CD45.2-FITC (104), CD45.1-FITC (A20), CD11b-APC (M1/70), CD115-BV605 (AF598), Ly6G-APC/Fire750 (1A8), CD11c-BV570 (N418) (Biolegend), Ly6c-PE (AL-21) and streptavidin-PerCP-Cy5.5 (BD bioscience). After labeling and prior to FACS processing, cells were stained using propidium iodide (Life Technologies) diluted 1:1000 in FACS buffer to determine viability. All samples were processed in an LSR Fortessa analyzer (Becton Dickinson), wherein compensation controls with singly labeled anti-rat/anti-hamster or anti-mouse compensation beads (BD Bioscience) were set up prior to the acquisition. Fluorescence minus one (FMO) controls with unfractionated splenocytes were run for proper gating definition. Acquired data were analyzed using FlowJo software (TreeStar). Immunophenotypic descriptions of analyzed populations include the following: inflammatory monocytes CD45 + Lin-(CD4; CD8; Nk1.1; B220) CD11bhiLy6ChiLy6G-/loCD115 + ; T cytotoxic cells CD45 + CD8 + CD4-Nk1.1-CD11b-; dendritic cells CD45 + CD11c + ; eosinophils CD45 + Lin-(CD4; CD8; Nk1.1; B220) CD115-CD11bhiSSChi; neutrophils CD45 + Lin-(CD4; CD8; Nk1.1; B220) CD115- CD11bhiSSCloLy6G + , activated T helper cells CD45 + CD4 + CD8-Nk1.1-CD11b-CD62L- and Activated T cytotoxic cells CD45 + CD8 + CD4-Nk1.1-CD11b-CD62L-.
RNA extraction and RT-qPCR analysis
Total hippocampal RNA from 4-month-old mice was extracted using TRI-reagent (Sigma-Aldrich) following manufacturer’s instructions. RNA concentrations were measured using a NanoDrop (2000C, Thermo), and 1 μg of total RNA was converted to cDNA using an iScript™ cDNA synthesis kit (BioRad). Real-time RT-qPCR was performed using the following primer sequences (5′–3′):
Bdnf [
19] (forward: GCGGACCCATGGGACTCT; reverse: CTGCTGCTGTAGTGACCGA) and
Arc [
19] (forward: GCTGAGCTCTGCTCTTCTTCA; reverse GGTGAGCTGAAGCCACAAAT). Amplification was done using a CFX96™ Real-Time System-C1000™ Thermal Cycler (BioRad). Relative gene expression was normalized to mRNA levels of the housekeeping gene
Gapdh (forward: ACCCAGAAGACTGTGGATGG; reverse: ACACATTGGGGGTAGGAACA).
Sequential protein extractions of prefrontal cortex (PFC) samples from 4-month-old mice were performed as previously described [
20]. Briefly, to obtain the S1 fraction, tissue was homogenized with a dounce homogenizer in PBS (1 ml/ 100 µg of tissue) and ultracentrifuged for 1 h at 40,000 rpm. The supernatant (S1 fraction) was aliquoted and stored at – 80 ºC. The pellet was dissolved in RIPA buffer (Sigma-Aldrich) and centrifuged at 30,000 rpm for 1 h. The resulting supernatant (S2 fraction) was aliquoted and stored at – 80 ºC. S1 and S2 fractions contained soluble amyloid. RIPA and PBS solutions were prepared with a protein inhibitor (Protein Inhibitor Cocktail, ThermoScientific; PhosphoStop, Roche). To obtain the S3 fraction, which contained protofibrils, the pellet obtained in the S2 fraction was re-dissolved in buffered-SDS (2% SDS, 20 mM Tris–HCl, pH 7.4, 140 mM NaCl) and centrifuged at 30,000 rpm for 1 h, and the supernatant (S3 fraction) was stored at – 80 ºC. To obtain the P3 fraction, which included dense plaques, the pellet obtained in the S3 fraction was dissolved in SDS-urea (20 mM Tris–HCl, pH 7.4, 4% SDS and 8 M urea) and stored at – 80 ºC.
Western blotting
Total protein concentrations were estimated using the BCA assay following the manufacturer’s guidelines (BCA Protein Assay-Kit, ThermoScientific, Sweden). Proteins were separated out using SDS-PAGE with pre-cast gels (4–20%, Bio-Rad) and transferred to nitrocellulose membranes using the TransBlot Turbo system (Bio-Rad). The membranes were blocked in a blocking solution (3% skim milk in PBS) for 1 h R/T and incubated with anti-human amyloid precursor protein (APP)/amyloid-β (6E10, 1:3000, Covance) overnight at 4 ºC. The following day, the membranes were washed three times in PBS-T20 0.25% followed by incubation with the secondary antibody (peroxidase-conjugated anti-immunoglobulin, 1:10,000, DAKO) and a conjugated actin antibody for 2 h R/T. Blots were developed using the ECL Clarity kit (Bio-Rad) and visualized using a ChemiBlot XRS + (Bio-Rad). Bands were analyzed by densitometry using Fiji ImageJ software (W. Rasband, National Institutes of Health) and normalized to actin signal (arbitrary units).
ELISA plates
Cytokine levels in hippocampal extracts of P15 mice were measured using a Meso Scale Discovery V-Plex Plus Kit (MSD Mesoscale Discovery, USA) with proinflammatory mouse panels (IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, TNF-α and CXCL1) following manufacturer’s protocol as done previously [
21]. Cytokines below the detection limit were removed from the analysis.
Statistics
All statistical analyses were performed using GraphPad Prism 8.0 Software for Macintosh (GraphPad Software, San Diego, CA, USA). In graphs, individual dots represent single mice. Two-way ANOVA followed by Tukey’s multiple comparisons test was performed and the main effects due to sex, MS and the interaction effect between those factors were reported. Statistical 2-way ANOVA differences between groups are stated in Additional file
3: Tables S1–S5. Additionally, three-way ANOVA followed by Tukey’s multiple comparisons test was performed and the main effects due to sex, MS, genotype and the interaction effect between those factors are reported in Additional file
4: Tables S1.1–S5.1. Data are presented as mean ± SD.
P values ≤ 0.05 were considered statistically significant and are indicated in the figure legends.
Discussion
We sought to elucidate the mechanisms that link ELS to the increased risk of developing AD in vivo, focusing on inflammatory alterations and brain regions particularly affected in AD. To that end, we used MS to induce ELS in WT and 5xFAD AD-model mice. First, we found that at P15, MS alters microglia in a sex-, genotype, and brain region-specific manner as shown by the analysis of Iba1 + area, which increased between WT MS mouse groups only. The microglial morphology in prefrontal cortex was altered with more amoeboid microglia in MS females, but the levels of CXCL1 and TNF-α were unaltered due to MS. Interesting to note, during brain development, microglia density peaks in the mouse hippocampus at P15 [
28]. In addition, sex is a crucial factor in microglial maturation as the number of microglial cells begins to differ dramatically between female and male rats from P4 to P30 [
18]. We previously characterized the neuroinflammatory profile of untreated 5xFAD mice at timepoints preceding plaque development and reported no significant differences in the cytokine levels of pre-plaque 5xFAD mice compared to age-matched WT male mice [
13]. In light of this and the current study, we observed that sex and MS might predispose the brain to be in a reactive, primed state for future challenges. Therefore, though the effects observed at P15 are relatively immediate effects, they likely set the stage for longer-term alterations.
We recently reported that MS altered the neuroinflammatory profile and behavior in adolescent WT mice in a sex-specific manner [
14]. In the present study, we furthered this investigation and evaluated neuroinflammatory status and behavioral outcomes due to MS in adult WT and 5xFAD mice and considered sex as a possible factor in our results. Clinical data show that exposure to maternal abuse or neglect is associated with the development of mood disorders and cognitive disturbances in adulthood [
3,
29]. These findings have been somewhat recapitulated in rodent models, wherein MS promoted the development of depressive-like behavior [
30]. In addition, the development of stress-related disorders is also linked to the risk of developing AD [
5] though ELS-induced mood disorders can also be an early manifestation of AD. For example, even before Aβ plaque deposition, depressive-like behavior and memory impairment are already present in male APP/PS1 mice [
31]. This sex-specificity is reflected in a study showing that MS induced significantly increased immobility time in the forced swim test in male but not female rats [
32]. However, this may need to be reconsidered based on genotype as, in our study, 5xFAD females were the most affected by MS. Regardless, MS overall has been associated with altered behavior, specifically a lower discrimination index in the novel object recognition memory test in rats [
33] and other AD mouse models [
27]. Although our data suggest that MS may induce behavioral abnormalities in the adulthood regarding depression (as shown by increased immobility time in the forced swim test) and cognition (as shown by decreased discrimination index in the novel object recognition memory test), a larger behavioral phenotyping is needed to further characterize the effect of MS in the adulthood. Nevertheless, these findings are interesting in light of the fact that women are more likely to develop MDD and AD and that ELS may predispose women to both disorders [
6,
34].
While behavioral measurements help to serve as a readout for dysfunction at a systems level, behavioral alterations are essentially underlain by changes at the synaptic and cellular levels. Therefore, we assessed hippocampal gene expression of
Arc and
Bdnf, which are genes involved in activity-dependent synaptic plasticity, and learning and memory processes [
35,
36]. Dysregulation of these genes has been reported in both AD [
5] and MDD [
37]. Here, we observed decreased
Arc gene expression together with a lower discrimination index in the novel object recognition memory test in MS 5xFAD male mice.
Arc expression is regulated by activating glucocorticoid receptors, which are key receptors in the stress response [
38]. In aged MS rats [
39] and AD-transgenic mice [
40], impaired
Arc expression was associated with deficits in spatial cognition. Moreover,
Arc has been shown to interact with presenilin-1 and regulate γ-secretase trafficking, which, if altered, may contribute to AD pathogenesis [
41]. Regarding
Bdnf gene expression, similar to previous studies conducted in rodents [
42‐
44], we found genotype-independent significantly lower levels of
Bdnf transcript in MS males compared to non-MS males. Decreased hippocampal levels of this factor have been associated with AD [
45] and depression [
46] as well as those with a history of childhood abuse [
47].
Another important factor to consider in the ELS response is microglial status. We and others have reported microglial and neuroinflammatory alterations due to MS in adolescent and young adult mice [
14,
30]. Sex-differences have been described for female and male microglia that range from cell density and morphology to different functions and regional distribution [
48]. For instance, ELS has the ability to activate hippocampal microglia in young male rats [
49,
50] but not in females [
51], similar to our findings. Additionally, in early developmental stages, similar microglial density is found in cortex independently of sex [
8], which could explain the lack of differences in our findings. Moreover, MS has also been shown to induce microglial activation, specifically in male rat pups [
49]. These findings led us to consider sex as a crucial factor in response to ELS and its link to AD and associated neuroinflammatory alterations.
Neuroinflammatory processes are important in AD pathogenesis and can be activated by central and peripheral stimuli, such as Aβ plaques and neurofibrillary tangles [
52]. In this study, we focused our AD characterization on the PFC area, since we did not find MS-induced differences in microglial activation nor Aβ plaques in hippocampus or amygdala. Specifically, in the 5xFAD MS females, we found increased immunoreactivity of Iba1, Gal-3, and Aβ, which is associated with higher levels of plaque burden and microglial activation, suggesting that ELS experiences could be driving the sex-specific early apparition and/or development of AD in females. However, contrary to what Hui et al. observed [
27], we did not see differences between 5xFAD male groups due to MS, which could be explained by the different mouse strain and age that we used. The exposure to ELS during childhood has been associated with a higher incidence of AD and depression [
53‐
55]. Thus, it is tempting to speculate that ELS possibly modulates or “primes” microglia in an early developmental stage. As a result, these cells are unable to respond to pathological Aβ levels in the same way as “non-stressed” microglia, leading to an altered Aβ burden that might be related to the behavioral abnormalities found in this study.
Finally, we evaluated effects on the peripheral immune system by flow cytometry analysis of the spleen, wherein we reported significant sex and MS effects on the myeloid and lymphoid cell populations. Interestingly, genome-wide association studies have pointed out alterations in the transcription factor PU.1, a critical factor for myeloid and B-lymphoid cell development and function as a risk factor for AD [
56]. In addition, higher levels of neutrophils and leukocytes have been found in MDD [
57], highlighting the importance of peripheral inflammation in mood disorders. Another important factor that may impact microglial activation is the infiltration of immune cells into the brain. In fact, macrophage infiltration is associated with increased inflammation in APP/PS1 AD-model mice [
58] and stress-model rodents (reviewed in [
59]). Our findings suggest that ELS triggers a long-lasting impact on the immune system and the inflammatory response, which may promote the infiltration of immune cells into the brain. This, in turn, may enhance the neuroinflammatory and microglial responses that could lead to the observed AD phenotype and ELS-induced behavioral disturbances that we observed in MS mice.
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