High-fat diet exacerbates cognitive decline in mouse models of Alzheimer's disease and mixed dementia in a sex-dependent manner

Olivia J. Gannon; Lisa S. Robison; Abigail E. Salinero; Charly Abi-Ghanem; Febronia M. Mansour; Richard D. Kelly; Alvira Tyagi; Rebekah R. Brawley; Jordan D. Ogg; Kristen L. Zuloaga

doi:10.1186/s12974-022-02466-2

High-fat diet exacerbates cognitive decline in mouse models of Alzheimer's disease and mixed dementia in a sex-dependent manner

Gannon, Olivia J.; Robison, Lisa S.; Salinero, Abigail E.; Abi-Ghanem, Charly; Mansour, Febronia M.; Kelly, Richard D.; Tyagi, Alvira; Brawley, Rebekah R.; Ogg, Jordan D.; Zuloaga, Kristen L. 2022-05-14 00:00:00 Background: Approximately 70% of Alzheimer’s disease (AD) patients have co‑morbid vascular contributions to cognitive impairment and dementia ( VCID); this highly prevalent overlap of dementia subtypes is known as mixed dementia (MxD). AD is more prevalent in women, while VCID is slightly more prevalent in men. Sex differences in risk factors may contribute to sex differences in dementia subtypes. Unlike metabolically healthy women, diabetic women are more likely to develop VCID than diabetic men. Prediabetes is 3× more prevalent than diabetes and is linked to earlier onset of dementia in women, but not men. How prediabetes influences underlying pathology and cognitive outcomes across different dementia subtypes is unknown. To fill this gap in knowledge, we investigated the impact of diet‑induced prediabetes and biological sex on cognitive function and neuropathology in mouse models of AD and MxD. Methods: Male and female 3xTg‑AD mice received a sham (AD model) or unilateral common carotid artery occlusion surgery to induce chronic cerebral hypoperfusion (MxD model). Mice were fed a control or high fat (HF; 60% fat) diet from 3 to 7 months of age. In both sexes, HF diet elicited a prediabetic phenotype (impaired glucose tolerance) and weight gain. Results: In females, but not males, metabolic consequences of a HF diet were more severe in AD or MxD mice compared to WT. In both sexes, HF‑fed AD or MxD mice displayed deficits in spatial memory in the Morris water maze (MWM). In females, but not males, HF‑fed AD and MxD mice also displayed impaired spatial learning in the MWM. In females, but not males, AD or MxD caused deficits in activities of daily living, regardless of diet. Astrogliosis was more severe in AD and MxD females compared to males. Further, AD/MxD females had more amyloid beta plaques and hippocampal levels of insoluble amyloid beta 40 and 42 than AD/MxD males. In females, but not males, more severe glucose intolerance (prediabetes) was correlated with increased hippocampal microgliosis. Olivia J. Gannon and Lisa S. Robison contributed equally to this work *Correspondence: zuloagk@amc.edu Department of Neuroscience & Experimental Therapeutics, Albany Medical College, 47 New Scotland Avenue; MC‑136, Albany, NY 12208, USA Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Gannon et al. Journal of Neuroinflammation (2022) 19:110 Page 2 of 20 Conclusions: High‑fat diet had a wider array of metabolic, cognitive, and neuropathological consequences in AD and MxD females compared to males. These findings shed light on potential underlying mechanisms by which pre ‑ diabetes may lead to earlier dementia onset in women. Keywords: Sex, Vascular, Dementia, Alzheimer’s disease, High‑fat diet, Inflammation, Obesity, Cerebral hypoperfusion, Metabolic, Glucose intolerance, Diabetes Background have shown that HF diet impairs adult hippocampal neu- Diabetes increases the risk of developing dementia by rogenesis in female, but not male mice [18]. Further, we twofold [1–5]. While diabetes is increasingly common, recently found that HF diet in middle-aged mice causes prediabetes is estimated to affect 1 out of every 3 Ameri - a wider array of cognitive deficits in females compared cans [6], and most people are unaware of their status. to males [19]. Others have found that a HF diet in the Like diabetes, prediabetes is characterized by impaired 3xTg-AD model of AD exacerbates cognitive impairment glucose tolerance; however, those with prediabetes show [20–25] and AD pathology, such as brain atrophy [20], slight elevations in insulin and fasting blood glucose, inflammation [22, 26], and Aβ load [26–28]. Examina- rather than hyperglycemia. Diabetes and prediabetes tion of sex differences in the cognitive effects of HF diet are shared risk factors and common co-morbidities for in the 3xTg-AD mouse have been mixed, with some stud- the two most common forms of dementia: Alzheimer’s ies finding greater cognitive impairment in females [20], disease (AD) and vascular contributions to cognitive while others have found no sex differences [21]. Previ - impairment and dementia (VCID) [1–5]. Obesity, which ously, we reported that HF diet results in greater meta- is often co-morbid with prediabetes or type 2 diabetes, bolic impairment (weight gain, visceral fat, and glucose increases AD risk threefold and VCID fivefold [7–9]. AD intolerance) in female compared to male 3xTg-AD mice is characterized by amyloid plaques, tau tangles, and neu- [29]. HF-fed females also showed increased astrogliosis in rodegeneration culminating in brain atrophy and cogni- the hypothalamus, a brain region that controls metabolic tive impairment. VCID is caused by deficits in cerebral function. Whether these greater metabolic disturbances blood flow and/or damage to cerebral vessels. In reality, in females would also lead to more severe cognitive defi - the distinction between AD and VCID is less clear-cut. cits and neuropathology in brain regions associated with Dementia pathologies often overlap, with more than half learning and memory had not yet been tested. In the cur- of dementia patients having multiple pathologies [10], a rent study, using mouse models of both AD and MxD, we condition known as mixed dementia (MxD). The most found that diet-induced obesity with prediabetes led to a common form of MxD is a mix of AD and VCID, as this wider array of cognitive deficits and neuropathology in occurs in ~ 70% of AD patients [11–13]. MxD is under- females compared to males. represented in animal research despite its high clinical prevalence. Understanding the interaction between AD Methods and VCID risk factors will provide insight into MxD. Animals and experimental design Dementia risk and prevalence vary by sex: women are This study was conducted in accordance with the more likely to develop AD[10], while men are slightly National Institutes of Health Guidelines for the Care and more likely to develop VCID [14, 15]. Discrepancies in Use of Laboratory Animals, and protocols were approved risk factors may drive these sex differences. In patients by the Institutional Animal Care and Use Committee at with diabetes, the sex difference observed in the non- Albany Medical College (Albany, NY, USA). Temperature diabetic population is reversed: diabetic women are at a and humidity were set at 72 °F, 30–70% humidity, with a 19% greater risk of VCID than diabetic men [3]. Among 12-h light/dark cycle (7 a.m. on/7 p.m. off ). Mice were those who have VCID, women are also more likely to fed a standard chow diet (Purina Lab Diet 5P76) until the have diabetes [16]. Prediabetes is associated with cogni- start of this study. They were housed in Allentown cages tive impairment and earlier onset of dementia in women, at a density of 2–5 mice. Mice were provided with envi- but not men, suggesting it may be a sex-specific risk fac - ronmental enrichment (Nestlets and Shepherd Shacks) tor [17]. However, it is unknown how prediabetes affects and were group housed at all times, except during the MxD, and whether these effects differ by sex. nest building test. Male and female wild-type (WT) High-fat (HF) diet is commonly used to induce meta- B6129SF2/J mice (#101045) and 3xTg-AD (#34830-JAX) bolic disease in rodents, as it causes both obesity and breeding pairs were purchased from Jackson Labora- prediabetes. HF diet can have profound effects on the tories (Bar Harbor, ME) and used to maintain a colony brain, some of which are sex dependent. For example, we at Albany Medical Center’s Animal Resource Facility. G annon et al. Journal of Neuroinflammation (2022) 19:110 Page 3 of 20 The 3xTg-AD mice, which are on a C57BL/6;129X1/ sites were closed with Vetbond, and the mice were given SvJ;129S1/Sv background, have three mutations that are 100µL 0.03 mg/mL buprenorphine via subcutaneous associated with AD in humans: APPSwe, tauP301L, and injection twice per day for 3 days as an analgesic. tm1Mpm Psen1 [30]. A timeline of the experiment is shown in Fig. 1A. At ~ 3 months of age, 3xTg-AD mice under- Glucose tolerance test went a sham surgery (AD group) or a unilateral com- As previously described [19, 29, 33], mice were given a mon carotid artery occlusion surgery (MxD group). WT glucose tolerance test (GTT) to assess diabetic status controls also received a sham surgery (WT group). One at ~ 5.75 months of age. The mice were fasted overnight, week following surgery, mice were placed on either a HF and their fasting blood glucose levels were measured diet (60% fat, 5.24 kcal/g; D12492, Research Diets, New (t = 0) using a glucometer (Verio IQ, OneTouch, Sun- Brunswick, NJ) or a low-fat (LF) control diet (10% fat, nyvale CA, USA) from their tail vein. Following an i.p. 3.82 kcal/g; D12450B, Research Diets) for the duration injection of 2 g/kg of glucose, blood glucose levels were of the study. At ~ 5.75 months of age, mice underwent a measured at 15, 30, 60, 90, and 120 min post-injection to glucose tolerance test (GTT) and a 2-week rest period, assess glucose tolerance. behavioral testing, followed by blood flow imaging, euthanasia, and tissue collection (including brains, fat, Behavior testing and reproductive organs) at ~ 7 months of age. A total of Following a 2-week recovery post-GTT, mice were tested 251 mice were used in this study. Experiments were con- for exploratory activity and anxiety-like behavior in the ducted in cohorts of up to 20 animals. A subset of mice open field (day 1), episodic-like memory in the novel (n = 118) was designated for collection of metabolic data, object recognition test (NORT; day 2), spatial learn- behavior testing, plasma assays [reported in [29]], and ing and memory in the Morris water maze (MWM; blood flow imaging, and brains were microdissected for days 8–10 or 10–12), and activities of daily living using use in Western blots. The remaining mice were used for a nest building task (days 15–16). Videos were recorded collecting metabolic data and blood flow imaging, and of behavioral performance for open field, NORT, and brains were used for immunofluorescence. In total, 23 MWM and analyzed using automated tracking software mice were excluded due to premature death or the pres- (ANY-maze 5.1, Stoelting, Wood Dale, IL). For each test, ence of other major health exclusions (hydrocephaly, mice were placed into the procedure room under dim large fighting wounds, tumors). Final group sizes ranged light and allowed to acclimate for 1 h. Each test appara- from 13 to 25 per group for metabolic measures, 8 to 13 tus was cleaned with 70% ethanol between each mouse to for behavioral testing, and 4 to 6 for immunohistochem- remove olfactory cues. istry (IHC). During tests, experimenters were blinded to surgical group. Blinding to diet and sex was not possible Open field due to mouse appearance. During analysis, experiment- The mice were placed in the test apparatus (495 × 495 mm ers were blinded to sex, diet, and dementia group. box) for 10 min. Distance traveled was used to determine the general activity levels of the animal. The percent of Surgical model of MxD time spent in center of the arena was used to determine To model MxD, 3xTg-AD mice underwent a right com- anxiety-like behavior. One mouse was excluded from this mon carotid artery occlusion surgery, as previously test for being a statistical outlier via Grubb’s outlier test described, to elicit chronic cerebral hypoperfusion and leaving group sizes of 8–13/group. model VCID [19, 31, 32]. Briefly, under isoflurane anes - thesia, the right common carotid artery was ligated NORT with two 6–0 silk sutures and cauterized (MxD group). NORT consisted of two, five-minute trials performed The sham surgery (WT and AD groups only) consisted in the same open field arena. In the first trial, mice were of exposing the carotid artery without ligation. Incision placed in the box and allowed to explore two identical (See figure on next page.) Fig. 1 HF diet caused greater metabolic impairment in AD and MxD females compared to males. A Experimental timeline. GTT (glucose tolerance test). B Weight gain was assessed by the % change in body weight from the start of the study to the end of the study. C Visceral adiposity was determined by isolating and weighing the visceral fat pads and normalizing to body weight. D, E Glucose intolerance was assessed with a GTT following a 16 h fast. D Glucose clearance was gauged by concentrations of glucose in the blood measured over time (time 0 = fasting blood glucose). E Blood glucose concentration over time was used to calculate area under the curve. We previously reported metabolic data for the Sham WT and Sham AD, but not the MxD, groups in Robison et al. (2020) in the Journal of Neuroinflammation [29]; licensed under a Creative Commons Attribution 4.0 International License; (https:// creat iveco mmons. org/ licen ses/ by/4. 0/). Data are presented as mean + SEM, **p < 0.01 effect of diet, ****p < 0.0001 effect of diet, ^p < 0.05 effect of dementia, ^^^p < 0.001 effect of dementia, ^^^^p < 0.0001 effect of dementia, 2‑ way ANOVA, (n = 13–25/group) Gannon et al. Journal of Neuroinflammation (2022) 19:110 Page 4 of 20 Fig. 1 (See legend on previous page.) G annon et al. Journal of Neuroinflammation (2022) 19:110 Page 5 of 20 objects (rubber ducks). Mice were then returned to a performed using moorFLPI Review V4.0 software (Moor recovery cage for 1 h. For the second trial, mice were Instruments, Wilmington, DE, USA). Average flux values returned to the arena, with the one familiar object were extracted from regions of interest (ROIs) using a replaced with a novel object (saltshaker). Episodic-like published protocol [36]. Measurements are presented as memory was assessed by recognition index [(time with %difference in blood flow between the left (non-ischemic novel object/total time with objects) *100]. Between tests, for MxD mice) and right (ischemic for MxD mice) objects were cleaned with 70% ethanol to mask olfactory hemisphere. cues. Mice that spent less than ≤ 2 s with the objects were excluded (total of 26 mice: 0–4/group, 12 males and 14 Immunofluorescence females). Additionally, 2 statistical outliers were removed Mice were perfused with ice-cold 0.9% saline. Brains from this test (1 WT LF M and 1 WT HF M). were removed and fixed in 4% paraformaldehyde for 24 h, followed by immersion in 30% sucrose for at least MWM 72 h. Brains were then snap frozen in OCT and stored Hippocampus-dependent spatial learning and mem- at − 80 °C until sectioning. Brains were sectioned at 40 ory were assessed using a modified 3-day version of microns on a Leica CM1950 cryostat into 6 series. Sec- the MWM that has been shown to be optimal for older, tions were washed in PBS containing 0.01% sodium azide, cognitively impaired, obese mice [34]. The protocol has permeabilized at room temperature for 1 h (0.3% TPBS), been previously described in detail [19]. On day 1, 5 vis- and blocked for 1 h at room temperature in 4% donkey ible trials were performed in which mice learn to find serum in 0.3% TPBS before being incubated in blocking the platform with a visual cue (flag). The entry point was buffer with primary antibodies at 4 °C overnight. Primary alternated for each trial. On day 2, mice underwent 5 hid- antibodies for one series included rabbit anti-beta Amy- den trials, in which the visual cue was removed from the loid (1:300, Cat# 71-5800, Lot# SH257822; Invitrogen, platform. All trials were 3 min long with a 30-min inter- Waltham, MA). Primary antibodies for another series trial interval. The distance traveled to reach the platform included rat anti-glial fibrillary acidic protein (1:2500, (pathlength) was used as a measurement of non-spatial AB5804, Millipore, Lot # TA265137) or goat anti-Iba1 (visual trials) and spatial (hidden trials) learning. On day (1:1000, PA5-18,039, Lot #TI2638761, SJ2467805; Ther - 3, a single probe trial was performed in which the plat- moFisher, Waltham, MA). Sections were incubated with form was removed from the pool. Spatial memory was secondary antibodies and DAPI (1:1000, Cat# D1306, calculated as the percent of time spent in the target quad- ThermoFisher, Waltham, MA) in blocking buffer for rant of the pool during the first minute of the probe trial. 2 h at room temperature. Secondary antibodies used A total of 2–4 mice were excluded from this test for being included Rhodamine Red-X Donkey Anti-Rabbit (1:100), statistical outliers via Grubb’s outlier test [4 mice in the Alexa Fluor 647 Donkey Anti-Goat (1:300), DyLight hidden trial and 2 mice in the probe trial] leaving group 405 AffiniPure Donkey Anti-Rat (1:300) (Jackson Immu - sizes of 8–13/group. noResearch, West Grove, PA), Alexa Fluor 647 Affin - iPure Donkey Anti-Goat IgG (H + L) (1:1000, Cat# Nest building AB_2340437, Jackson ImmunoResearch, West Grove, Mice were singly housed in Allentown cages with pine PA), and Alexa Fluor 488 Donkey Anti-Rabbit (1:1000, chip bedding and two pre-weighed Nestlets each. After Cat# ab150073, Jackson ImmunoResearch, West Grove, 16 h (overnight), the mice were removed from their test PA). Images for quantification were taken at 10 × using cage and returned to group housing. Nests were rated the Axio Observer fluorescent microscope (Carl Zeiss on a 1–5 scale (with half-point scores allowed) based Microscopy, Jena, Germany). All analyses were per- on published criteria [35] by 3 experimenters who were formed in the right hemisphere of coronal sections unless blinded to treatment group. The 3 ratings were averaged. otherwise noted by an experimenter who was blinded One mouse was excluded from this test for being a statis- to treatment group. For Aβ plaque quantification, 8 tical outlier via Grubb’s outlier test leaving group sizes of 40-micron-thick sections between − 1.46 and − 3.52 mm 8–13/group. from bregma were used to hand count numbers of Aβ plaques in the left and right hemisphere (Fig. 4B). For Cerebral blood flow measurement intracellular Aβ quantification, regions of interest (ROI) Cerebral blood flow was measured via laser speckle were drawn in the cortex and a number of cells positive contrast imaging (moor FLPI full field laser perfusion for Aβ were counted (Additional file 5: Fig. S5A). For imager; Moor Instruments, Wilmington, DE, USA) under neuroinflammation analysis (microglia and astrocytes), isoflurane anesthesia as previously described [19]. Image ROIs were drawn in CA1, CA2, CA3, and dentate gyrus acquisition [5-min scan], processing, and analysis were Gannon et al. Journal of Neuroinflammation (2022) 19:110 Page 6 of 20 of the hippocampus in ~ 3 brain sections per mouse and (n = 4 mice/group) were thawed and homogenized then % area covered was measured using ImageJ. spun at 4C 12,100 g for 30 min. The supernatant (40uL) was retrieved and neutralized with 20 volumes of neutral- izing solution (800uL; 1 M Tris base, 0.5 M Na2HPO4, Western blot 0.05% NaN3). A protease and phosphatase cocktail was Frozen hippocampi (ipsilateral to VCID or sham surgery) added (HALT, ThermoScientific #1861284) that contains from 4 mice per group were thawed and homogenized in the serine protease inhibitor ABESF to prevent degrada- 50µL RNA-Later (45-R0901-100MLsigma). 25µL of that tion of AB peptides. Amyloid peptides were then quanti- homogenate was transferred into 100 µL T-Per buffer fied in this fraction using ELISA kit from ThermoFisher (ThermoScientific #78,510) supplemented with protease Scientific (AB40: KHB3481, and AB42: KHB3441) and phosphatase inhibitor cocktail (HALT, ThermoSci - according to the manufacturer’s instructions. Quantities entific #1861284) and spun at 21,000 g for 20 min. The were normalized to total soluble protein quantities. supernatant was collected, and the protein concentration was determined using a Pierce BCA Protein Assay Kit (Thermo Scientific #23,227). 20 µg of protein was dena - Quantitative reverse transcriptase‑PCR (RT‑qPCR) tured in LDS (4× Bolt LDS Sample Buffer, Invitrogen, Frozen hippocampi (ipsilateral to VCID or sham surgery) B0007) with 0.2 M DTT and boiled for 5 min at 95 °C. from 4 to 5 mice per group were thawed and homog- Proteins were then separated using a 10% Tris Bis gel enized in 50µL RNA-Later (45-R0901-100MLsigma). (ThermoFisher, NW00107BOX) before being transferred RNA was extracted from 25uL of homogenate using the onto a nitrocellulose membrane (Nitrocellulose/Filter RNeasy Plus Mini Kit (Qiagen, Catalog number 74134) Paper Sandwich, 0.2 μm, 8.3 × 7.3 cm, ThermoFisher, and RNA concentrations were measured using Thermo - LC2000). Membranes were blocked using LI-COR Scientific NanoDrop One. RNA was converted to cDNA blocking solution (Intercept (TBS) Blocking Buffer, LI- using a High-Capacity cDNA Reverse Transcription Kit COR, 927–60,001) for one hour at room temperature (Applied Biosystems, Catalog number: 4368814). The then exposed to primary antibodies at 4 °C overnight. qPCR reactions were performed using TaqMan Gene The following day membranes were washed then incu - Expression Master Mix (Applied Biosystems, Catalog bated with the corresponding secondaries before being number 4369016) in the presence of TaqMan Assays washed and scanned using an Odyssey CLx LI-COR with primer/probes for Iba1 (Mm00479862_g1), GFAP scanner. All membranes were scanned simultaneously (Mm01253033_m1), and CD68 (Mm00839636_g1) as to ensure similar exposure and scanning parameters. target genes. RPL13A (Mm05910660_g1) was used as the Membranes were then stripped using LI-COR stripping housekeeping gene. Data were presented as fold change buffer (NewBlot IR Stripping Buffers for NIR Western relative normalized expression compared to WT LF mice Blots, LI-COR, 928–40,028) for 20 min and rescanned to by ΔΔCq method using Bio-Rad CFX Maestro software. ensure loss of signal before being incubated with an anti- GAPDH antibody (Sigma, 45-G9545-100UL) as a loading Statistics control. Western blot band analysis was conducted using Statistical analyses were performed using Prism 8.1 the LI-COR Image Studio and normalized to loading (GraphPad Software, San Diego, CA, USA). All data control. Antibodies used were for total tau ((T46) mouse are presented as mean + SEM, with significance set abCam203179, lot #GR33658561), early phosphorylated at p < 0.05 except for categorical data (nest building is tau (S199-202, rabbit Invitrogen 44-768G lot SG255287), expressed as median + interquartile range). Following a and later phosphorylated tau (PHF1, rabbit Invitrogen Grubbs’ test for statistical outliers, a 2-way ANOVA was PA5-56,621 lot TB2525148). All primary antibodies were performed with Tukey’s correction for multiple compar- used at 1:1000 in Tris-buffered saline supplemented with isons [dementia type (WT vs. AD vs. MxD) X diet (LF 0.1% Tween (TBST). Secondary antibodies were used at vs. HF)] in data segregated by sex. The exception to this 1:15,000 in TBST with 0.02% SDS. Secondary antibodies is the test for spatial learning (Morris water maze hid- included IRDye 800CW Donkey anti-Mouse IgG (CAT# den trial, average pathlength) in which we used Dun- 926–32212, Lot#D00930-09, LI-COR, Lincoln, Nebraska) nett’s multiple comparison test. We used this post hoc and IRDye 680RD Donkey anti-Rabbit IgG (CAT# 926– test because we specifically defined the deficit in average 68,073, Lot#D00421-09, LI-COR, Lincoln, Nebraska). pathlength as greater than the LF WT group. In second- ary analyses to assess sex differences, 3-way ANOVAs Enzyme‑linked immunoassay (ELISA) were performed (sex X dementia type X diet) with- The pellet from the protein extraction (insoluble fraction) out post hoc analysis (except for analysis in which WT was dissolved in 50ul of freshly prepared 70% formic acid groups were not included we used Sidak’s multiple com- and stored at -80C. On the day of the assay, the samples parison test). In measures with large sample sizes (n ≥ 13 G annon et al. Journal of Neuroinflammation (2022) 19:110 Page 7 of 20 for all groups) a ROUT test was performed (metabolic females had greater metabolic impairment (weight gain, data only). One-sample t-tests were performed for meas- visceral fat accumulation, and glucose intolerance) than urements where values are compared to chance (50% in HF-fed WT females (p < 0.0001 for all measures). Con- NORT, 25% in MWM probe trial, and 0% hemispheric versely, in males, LF-fed AD and LF-fed MxD males difference in blood flow). Each correlation matrix was had less visceral fat than LF-fed WT males (p < 0.001 created by computing Pearson correlation coefficients for for AD, p < 0.05 for MxD). Analysis of sex differences via each pair of datasets. a 3-way ANOVA (Additional file 6: Table S1) showed a sex X dementia interaction and sex X diet interaction Results (p < 0.0001 for each interaction) in which both visceral Animal models and timeline fat and glucose intolerance were exacerbated in AD/ In order to create a mouse model of MxD, 3xTg-AD MxD and HF-fed females, but not males. Further, there mice (~ 3 months of age) underwent a unilateral com- was also a sex X diet X dementia interaction for % weight mon carotid artery occlusion surgery to induce chronic gain (p < 0.0001), which was driven by the large % weight cerebral hypoperfusion/vascular pathology. These mice increase in HF-fed AD and HF-fed MxD females. Taken were compared to AD mice (3xTg-AD that underwent a together, the data show that HF diet caused greater met- sham surgery) and WT controls (WT mice that under- abolic impairment in AD or MxD females compared to went a sham surgery). In order to determine the effects WT females or AD or MxD males. of HF diet-induced prediabetes on outcomes in the AD and MxD models, one week following surgery, mice were HF diet caused a wider array of cognitive deficits in females placed on either a HF diet (60% fat) or a control LF diet Our prior work has demonstrated that HF diet causes a (10% fat) from ~ 3 to 7 months of age. A study timeline is wider array of cognitive deficits in middle-aged females, shown in Fig. 1A. compared to males, in a mouse model of VCID [19]. Whether HF diet would differentially impact AD and HF diet caused greater metabolic impairment in AD MxD in each sex was unknown. Preference for the novel and MxD females compared to males. object in the NORT (episodic-like memory; Fig. 2A) We have previously reported [29] that metabolic effects was assessed with a one-sample t-test vs. no preference of a HF diet are more severe in 3xTg-AD females com- (recognition index of 50%). In males, all WT mice dem- pared to males. Here we show that these sex differ - onstrated a preference for the novel object (LF-fed WT ences persist in the MxD model. Within each sex, there p < 0.001, HF-fed WT p < 0.001). In females, LF-fed WT was a main effect of HF diet to increase % weight gain mice show intact memory (p < 0.001), while HF-fed WT (p < 0.0001; Fig. 1B), visceral fat accumulation (p < 0.0001; females only showed a trend toward preference for the Fig. 1C), and glucose intolerance (p < 0.0001; Fig. 1D, E). novel object (p = 0.09). In both sexes, both AD and MxD Monthly weigh gain is shown in Additional file : Figure groups on either diet demonstrated no preference for the S1A. Importantly, as others have reported in previous novel object, indicating an impairment in episodic-like studies [27, 28], the HF diet induced a prediabetic pheno- memory. A 3-way ANOVA showed no sex differences. type with significantly elevated fasting blood glucose lev - Although there were some group differences in explora - els (Fig. 1D, zero time point and Additional file 1: Figure tory behavior and anxiety-like behavior in the open S1B) that are below the 250 mg/dl cut-off that has been field (Additional file 2: Figure S2A, B), neither of these established in the literature for mouse models of diabe- measures correlated with NORT performance (Addi- tes [37]. Of note, the highest fasting blood glucose among tional file 1: Figure S1C). There were no significant dif - any of our HF-fed mice was still only 221 mg/dl. Demen- ferences in performance during the NORT training trial tia X diet interactions were sex dependent. In females, (Additional file 1: Figure S1D). The MWM was used to post hoc tests showed that HF-fed AD and HF-fed MxD examine spatial learning and memory. All groups were (See figure on next page.) Fig. 2 HF diet caused a wider array of cognitive impairment in females compared to males. A Episodic‑like memory was assessed in the novel object recognition test (NORT ). Recognition index (% time spent with the novel object) was calculated. Performance not significantly greater than chance (50%, indicated by the red line) indicates impaired memory. B–D Spatial learning and memory was assessed using the Morris Water Maze (MWM). Five hidden trials (B) assessed spatial learning via pathlength to reach the target platform (shorter pathlength = better per formance). Average pathlength over the 5 hidden trials (C) was longer (more impaired memory) in MxD females and AD females on a HF diet. Spatial memory was assessed in the probe trial (D), as % time spent in the target quadrant vs. chance (25%, indicated by the red line). Performance above 25% indicates intact memory. E The nest building task was used to assess activities of daily living. Nests were graded on 1–5 scale (average of scores by 3 experimenters blinded to treatment). Lower scores are indicative of impairment. Data are presented as mean + SEM except for nest building (median + interquartile range), +++p < 0.001 vs chance, +p < 0.05 vs. chance, **p < 0.01 effect of diet, ^p < 0.05 effect of dementia, ^^p < 0.01 effect of dementia, ^^^^p < 0.0001 effect of dementia, Red line = chance, 2‑ way ANOVA, (n = 5–11/group NOR, n = 8–13/group MWM, nest building) Gannon et al. Journal of Neuroinflammation (2022) 19:110 Page 8 of 20 Fig. 2 (See legend on previous page.) G annon et al. Journal of Neuroinflammation (2022) 19:110 Page 9 of 20 trained to swim to a visible platform (main effect of trial females compared to males, particularly when females p < 0.05, Additional file 2: Figure S2E). When the plat- were fed a HF diet. form was hidden (spatial learning trials; Fig. 2B, C), no group differences were observed in males. In females, Neuropathology was exacerbated in AD/MxD females. there was a dementia x diet interaction (p < 0.05), in We have previously reported sex-dependent effects of HF which HF-fed AD females, LF-fed MxD females, and diet on hypothalamic neuroinflammation in 3xTg-AD HF-fed MxD females all had significantly impaired spa - mice; however, neuroinflammation in brain areas associ - tial memory compared to LF-fed WT females (p < 0.01 ated with cognition and other forms of neuropathology for each group; post hoc tests). A 3-way ANOVA (Addi- had not been assessed. Microgliosis (immunolabeling for tional file 6: Table S1) showed a sex X dementia X diet Iba-1) and astrogliosis (immunolabeling for glial fibril - interaction (p < 0.01) in which HF-fed AD/MxD females lary acid protein; GFAP) were assessed in the hippocam- had the most severe spatial learning deficits. In the probe pal CA1, CA2, CA3, and dentate gyrus regions (Fig. 3A). trial (spatial memory; Fig. 2D), preference for the tar- In males, there was a main effect of dementia on micro - get quadrant was assessed with a one-sample t-test of % gliosis (Iba1% area covered; Fig. 3B, D, Additional file 3: time in target quadrant vs. chance (25%). In males, only Figure S3A–C, and Additional file 4: Figure S4A), in WT mice (on either diet) and LF-fed AD mice showed a which AD and MxD males showed decreased hip- preference for the target quadrant (p < 0.05), indicating pocampal microgliosis in several regions (CA1 p < 0.001, that spatial memory was impaired in HF-fed AD males CA2 p < 0.0001, CA3 p < 0.01). In females, microglio- and MxD males on either diet. In females, only the LF- sis was unaffected by either diet or dementia in any of fed WT mice showed a preference for the target quad- the regions assessed. A 3-way ANOVA showed a sex X rant (p < 0.05), indicating that HF-fed WT females and dementia interaction in several regions (CA1 p < 0.01, AD or MxD females on either diet had impaired spatial CA2 p < 0.01, CA3 p < 0.05), which was driven by higher memory. A 3-way ANOVA (Additional file 6: Table S1) microgliosis in WT males (regardless of diet) compared showed only a slight trend toward worse spatial memory to all other groups. In males, astrogliosis (GFAP % area in females (p = 0.08) with no significant interactions. covered; Fig. 3C, E, Additional file 3: Figure S3D-F, and Due to known decreases in swim speed in obese mice, Additional file 4: Figure S4B) was unaffected by either we used non-speed-based measures (pathlength/% time diet or dementia in any of the regions assessed. In in the target quadrant). Although there were some group females, there was a main effect of dementia, in which differences in swim speed, speed did not correlate with AD and MxD females showed increased hippocampal learning or memory in the MWM (Additional file 2: astrogliosis (CA1 p < 0.05, CA2 p < 0.01, dentate gyrus Figure S2F), further supporting that cognitive measures p < 0.01). A 3-way ANOVA showed a sex X demen- were speed independent. The nest building test (Fig. 2E) tia interaction in which AD/MxD females had greater was used to assess activities of daily living (ADLs). In astrogliosis than males (CA1 p < 0.05, CA2 p < 0.05, CA3 males, all mice performed well although there was a p < 0.01, dentate p < 0.05). In addition to examining % area main effect of diet (p < 0.05) to impair ADLs. In females, covered, we assessed expression of hippocampal genes AD and MxD females showed clear impairments (main associated with microgliosis (Iba-1 and CD68, a lysoso- effect of dementia p < 0.0001), with LF-fed MxD (p < 0.01) mal protein upregulated during microglial activation: and HF-fed MxD (p < 0.05) females building poorer nests Fig. 3F, G) and astrogliosis (GFAP: Fig. 3H). In males, than control LF-fed WT females. HF-fed AD females also GFAP gene expression in hippocampal homogenate was showed a trend (p = 0.056) toward impaired ADLs. A increased overall by dementia (p < 0.05). However, GFAP 3-way ANOVA showed a main effect of sex X dementia gene expression was unaffected in females. CD68 expres - interaction (p < 0.01) in which AD and MxD females were sion was unaffected by diet or dementia in both males more impaired than males. Taken together, these data and females. In females, HF diet decreased Iba-1 expres- show a wider array of cognitive deficits in AD or MxD sion (p < 0.05). To examine AD pathology, we assessed Aβ pathology via immunohistochemistry for Aβ, ELISA for (See figure on next page.) Fig. 3 Astrogliosis is exacerbated in AD/MxD females, while microglia coverage is decreased in AD/MxD males. A Hippocampal regions of interest examined: CA1, CA2, CA3, and the dentate gyrus (image created with BioRender.com). B Microgliosis in the CA1 region of the hippocampus was gauged through Iba1 immunofluorescence (greater % area covered indicating greater microgliosis). C Astrogliosis was gauged through GFAP immunofluorescence (greater % area covered indicating greater astrogliosis). D Representative images of Iba‑1 immunofluorescence in the CA1 regions. E Representative images of GFAP immunofluorescence in the CA1 regions. F–H Hippocampal expression of markers for microgliosis (Iba‑1: F and CD68: G) and astrogliosis (GFAP: H) normalized to RPL13A expression. Data are presented as mean + SEM, effect of dementia: ^p < 0.05 effect of dementia, ^^^^p < 0.0001 effect of dementia, effect, *p < 0.05 effect of diet, 2‑ way ANOVA, (n = 4–5/group) Gannon et al. Journal of Neuroinflammation (2022) 19:110 Page 10 of 20 Fig. 3 (See legend on previous page.) G annon et al. Journal of Neuroinflammation (2022) 19:110 Page 11 of 20 insoluble Aβ40 and 42 in the hippocampus, and Western of all MxD groups (main effect of MxD, p < 0.0001; Addi - blot for phosphorylated tau in the hippocampus. Soluble tional file 5: Figure S5B, C). No sex differences were Aβ was not assessed, as it is not elevated until 18 months detected. Taken together, these data show that although of age in 3xTg-AD mice [38]. A 3-way ANOVA showed some pathology was equivalent between the sexes, hip- that females had higher levels of Aβ plaques than males pocampal astrogliosis and Aβ pathology were more (p < 0.05; Fig. 4A, B) and higher levels of insoluble Aβ40 severe in AD/MxD females, compared to males. (p < 0.01; Fig. 4C) and 42 (p < 0.05; Fig. 4D) compared to males. Cortical intracellular Aβ was also assessed, Sex‑dependent correlations between cognitive in which a 3-way ANOVA showed that AD/MxD performance and metabolic measures females had higher levels of Aβ-positive cells than males To further examine the relationship between cognitive, (p < 0.0001) and that there was a main effect of MxD to metabolic, and neuropathology measures, we performed increase Aβ (p < 0.01; Additional file 5: Figure S5A). No linear regression analyses and correlation analyses in differences in phosphorylated or total tau were detected males and females separately. In Fig. 5A, B, we examined between AD and MxD groups, regardless of sex or diet correlations between behavioral outputs and measure- (Fig. 4E-H). To validate that the unilateral carotid artery ments of metabolic impairment in males and females occlusion surgery modeled VCID by inducing chronic separately. In both males and females, each metabolic cerebral hypoperfusion, cortical blood flow was meas - measure was strongly correlated with the others (visceral ured using laser speckle contrast imaging at ~ 7 months fat, % weight gain, and AUC; r = 0.7–0.9, p < 0.0001). In of age (4 months post-surgery). As expected, blood flow males, the effects of metabolic impairments on cognitive deficits were found in the right (occluded) hemisphere function were mixed. In males, higher % weight gain and Fig. 4 Female AD/MxD mice have greater Aβ, but not tau, pathology. A Representative image of Aβ plaques. B The number of Aβ plaques/brain section (averaged across 8, 40‑μM ‑thick brain sections between ‑1.46 and ‑3.52 mm from bregma). C Hippocampal insoluble Aβ‑40, measured by ELISA. D Hippocampal insoluble Aβ‑42, measured by ELISA. E Representative Western blot images of tau protein in right (ischemic for MxD) hippocampal isolates. Measurements for F total tau, G pTau S199, and H pTauPHF1 were normalized to GAPDH. Data are presented as mean + SEM, #p < 0.05 effect of sex, ##p < 0.01 effect of sex, 3‑ way ANOVA, (n = 4–5/group) Gannon et al. Journal of Neuroinflammation (2022) 19:110 Page 12 of 20 more severe glucose intolerance (GTT AUC) were both is shown in Fig. 5E, F in which we examined the relation- associated with poorer spatial memory (MWM % time ship between microgliosis in the CA1 region (CA1 Iba1% in the target quadrant; p < 0.05 for each). Additionally in area covered) and glucose intolerance (AUC) separately males, % weight gain was associated with reduced ability in males and females via linear regression and presented to perform activities of daily living (nest score; p < 0.05); the r and p value. Taken together, these data show that however, more severe glucose intolerance showed a sur- more severe glucose intolerance was the only metabolic prising association with better episodic-like memory parameter that was associated with worse neuroinflam - (NORT recognition index; p < 0.05). In females, meta- mation, and that this increase in neuroinflammation bolic impairment showed a consistent association with (microgliosis) occurred in females only. worse cognitive function. In females, higher % weight A summary of findings is presented in Fig. 6. gain or more visceral fat was associated with poorer spa- tial memory (p < 0.01 for weight gain, p < 0.05 for visceral Discussion fat), and more severe glucose intolerance was associated This study set out to address gaps in knowledge regard - with reduced ability to perform activities of daily living ing sex differences in mid-life metabolic risk factors (p < 0.01). In males, the degree of metabolic impairment for dementia – obesity and prediabetes. Recent clini- was not associated with alterations in cerebral blood flow, cal evidence suggests that prediabetes is a risk factor and blood flow was not associated with cognitive func - for cognitive decline and dementia in women, but not tion. In females, metabolic impairment was associated men [17]. How sex and prediabetes interact to influ - with reduced cerebral blood flow (% blood flow in the ence cognitive function and neuropathology in the two right hemisphere; p < 0.05 for % weight gain, visceral fat, most common forms of dementia – AD and MxD (co- and glucose intolerance). Additionally in females, greater morbid VCID + AD pathology) – is unknown. To fill reduction in cerebral blood flow was associated with this gap in knowledge, we combined mouse models of more severe deficits in the ability to perform activities VCID (chronic cerebral hypoperfusion) and AD (3xTg- of daily living (nest building). Taken together, these data AD mice) to model MxD. We used a chronic HF diet show that impairment in metabolic measures was more to model obesity and prediabetes. While HF diet ubiq- consistently associated with reductions in cognitive func- uitously caused metabolic impairments, this was aug- tion in females. mented in AD/MxD females, but not males. AD, MxD, In Fig. 5C, D we examined correlations between neu- and HF diet differentially affected cognition in males ropathological and metabolic outputs and presented our and females. While episodic-like memory deficits were findings in the form of a correlation matrix. In males, observed in AD and MxD mice of both sexes, additional greater cortical Aβ burden was associated with more cognitive impairments were observed in females. AD/ CA2 astrogliosis (p < 0.05), while in females it was asso- MxD females also showed impairments in activities of ciated with less CA2 astrogliosis. In males, more severe daily living, but males did not. When fed a HF diet, AD glucose intolerance was associated with less CA2 astro- and MxD females, but not males, also exhibited deficits gliosis (p < 0.02), and there were no significant associa - in spatial learning. Metabolic impairment was also more tions with microgliosis. In females, more severe glucose consistently associated with reductions in cognitive func- intolerance was associated with more microgliosis in tion in females. Moreover, AD and MxD females had both CA1 (p < 0.01) and CA2 (p < 0.05), but not associated more hippocampal astrogliosis and a greater burden of with astrogliosis. An example of a sex-specific association Aβ than males. Finally, more severe glucose intolerance (See figure on next page.) Fig. 5 Cognitive impairments and pathological outcomes are correlated with metabolic deficits in male and female mice. Using a correlation matrix, we compared relationships between cognitive, metabolic, and pathological factors. A, B Correlations between cognitive and metabolic measures. Visc fat: visceral fat pad weight normalized to body weight (n = 55–58/sex); AUC: area under the curve from the glucose tolerance test, high AUC indicates greater glucose intolerance (n = 54–58/sex); %decrease in blood flow: the percent difference in cerebral blood flow in the temporal region of the cortical brain surface (n = 50–51/sex); MWM % target: % of the time spent in the target quadrant of the probe trial of the MWM test, higher percentage indicates better spatial memory (n = 58/sex); NOR% time: % time spent with the novel object in the testing trial of the NOR test, higher percentage indicates better episodic‑like memory (n = 46–49/sex); ADLs: activities of daily living assessed by score in the nest building test, a lower score indicates worse ADLs (n = 57–58/sex). C, D Correlations between metabolic and pathological measures in the subset of mice designated for IHC. AUC: area under the curve from the glucose tolerance test, high AUC indicates greater glucose intolerance (n = 18–21/ sex); amyloid count: the number of cells in a cortical ROI that were positive for beta‑amyloid (n = 19–20/sex); CA1 GFAP: the % area covered by GFAP staining in the CA1 region of the hippocampus, greater coverage indicates greater astrogliosis in that region (n = 18–19/sex); CA2 GFAP, (n = 17–19/ sex); CA1 Iba2: the % area covered by Iba1 staining in the CA1 region of the hippocampus, greater coverage indicates greater astrogliosis (n = 17–18/sex) CA2 Iba1, (n = 18/sex). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, significant correlation; Pearson r values are presented. Yellow: positive correlation, Blue: negative correlation. E, F Linear regression of CA1 Iba1% area covered and glucose tolerance test from the AUC (males: n = 19; females n = 17). r value and p value are presented G annon et al. Journal of Neuroinflammation (2022) 19:110 Page 13 of 20 Fig. 5 (See legend on previous page.) Gannon et al. Journal of Neuroinflammation (2022) 19:110 Page 14 of 20 33]. In line with our prior findings, in the current study, the diet was initiated in young adulthood and thus WT males and females showed similar levels of metabolic impairment. However, AD/MxD females developed greater metabolic impairments, including increased weight gain and more severe glucose intolerance, than males in response to HF diet. In a subset of mice from the current study (WT and AD groups with the sham surgery), we previously reported that 3xTg-AD females did not display increased plasma levels of other diabe- tes markers (e.g., insulin, GIP, GLP-1, PAI-1, resistin) or peripheral inflammation [29]. This enhanced meta - bolic effect observed in AD/MxD females in the cur - rent study is reminiscent of our findings in middle-aged mice in which females are more adversely affected than males. One possibility is that the AD and MxD females might have displayed a form of accelerated aging of their metabolic system and/or brain regulation of metabolic function. We have previously reported this female-spe- cific interaction between the effects of a HF diet and the 3xTg-AD genotype that worsens metabolic impairment and examined potential underlying mechanisms [29]. In a subset of mice from the current study (WT and AD groups with the sham surgery), we reported increased food intake, reduced activity levels, and markedly increased hypothalamic expression of GFAP and IL-1β, as well as GFAP labeling in several hypothalamic nuclei that regulate energy balance in the HF-fed 3xTg-AD females [29]. Thus, neuroinflammation in the hypothala - mus likely contributes to the observed sex difference in Fig. 6 Summary of our major findings. We demonstrate relative accumulation of cognitive impairments and neuropathology of metabolic function in 3xTg-AD mice. A limitation in our female male (left, blue) and (right, pink) mice along a center scale interpretation of these data is in the generalizability of with the top of the scale indicating less impairment and pathology using a diet composed of 60% fat from lard (saturated fat). and the bottom of the scale representing greater impairment and Diets of different fat composition or combinations with pathology sugar (a Western diet) may have different consequences. While we were unable to address changes in blood pres- sure in this study, others have demonstrated that high-fat was associated with worse microgliosis in females only. diet in wild-type mice and in the 5xFAD mouse model of Taken together, the data demonstrate heightened suscep- AD does not increase blood pressure [39]. Overall, our tibility of females to the negative metabolic, cognitive, results show that a chronic 60% fat diet caused greater and neuropathological effects of AD, MxD, and HF diet. metabolic impairment in AD/MxD females compared to These data, along with recent clinical findings [17], sug - males. gest that prediabetes might contribute to multiple forms of dementia in women. Cognitive findings Although HF diet elicited some cognitive deficits in Metabolic findings both sexes, HF-fed females displayed a wider array of Here, we found that AD and MxD females had more cognitive impairments, even in WT mice (summarized adverse metabolic responses to chronic HF diet. We have in Fig. 6). While numerous studies have shown that HF previously shown that sex differences in response to HF diet exacerbates cognitive impairment in AD [20–23, diet are age sensitive. If the diet is initiated in juvenile 25], few have examined sex differences. Here, we report mice, males show greater metabolic impairments [33]. that HF diet caused sex-specific cognitive deficits in This sex difference is eliminated if the diet is initiated in spatial learning. AD males, regardless of diet, did not adulthood [33], and reversed if initiated in middle age – show impairment in spatial learning; however, AD with females showing greater metabolic impairment [19, G annon et al. Journal of Neuroinflammation (2022) 19:110 Page 15 of 20 Neuropathology findings females showed an impairment in spatial learning only In assessing underlying neuropathology, we found that when fed a HF diet. In both males and females, addi- astrogliosis and Aβ accumulation were more severe in tive effects of HF diet were observed in spatial memory. AD/MxD females, compared to males. First, we assessed In males, LF-fed AD mice did not show spatial memory markers of neuroinflammation. Female AD/MxD mice deficits; however, with a combination of HF diet and had greater astrogliosis than males in several regions AD these impairments emerged. In females, even WT of the hippocampus. We, and others, have previously mice showed spatial memory impairment in response reported increased astrogliosis in 3xTg-AD mice [29, to HF diet (as did AD mice). Overall, we found that 40] and in cerebral hypoperfusion mouse models of poorer metabolic measures were also more consistently VCID [32, 52, 53]. In comparison to our 7-month-old associated with poorer cognitive function in females, mice, greater microglia activation has been reported in compared to males. In addition to the sex differences 12-month-old 3xTg-AD females compared to males [54], observed with HF diet, we also observed a wider array and more Iba1 + cells in the subiculum in 18-month- of cognitive impairment in LF-fed AD females com- old 3xTg-AD females compared to males [55]; thus, pared to LF-fed males. In support of the current data, sex differences may have emerged at later time points. prior studies have demonstrated exacerbation of cog- Although there were no sex differences in the extent of nitive deficits in 3xTg-AD mice by a HF diet [20– 25] microgliosis, we did observe sex differences in the rela - and greater cognitive impairments in AD females tionship between microgliosis and metabolic markers. compared to males [40–45]. However, there have been In females, but not males, more severe glucose intoler- some reports of greater deficits in males specifically in ance was associated with more severe microgliosis in the regard to working memory [46] and fear conditioning CA1 region of the hippocampus; mechanistic links driv- [47]. Greater female susceptibility to negative cogni- ing this outcome require further investigation. In addi- tive effects of a HF diet is in line with our prior work tion to neuroinflammation, we assessed other classic AD demonstrating that HF diet also causes a wider range neuropathology, including Aβ and phosphorylated tau. of cognitive deficits in WT middle-aged females [19]. While we observed no group differences in hippocampal Taken together, our data suggest that females may be phosphorylated tau levels, recent work by the LaFerla lab more adversely cognitively affected by metabolic dis - has demonstrated sex differences in hippocampal tau in ease which could contribute to cognitive deficits during the 3xTg-AD mouse[55]. Overall, we found that females normal aging or dementia. had greater levels of amyloid pathology both in terms In both sexes, MxD elicited a wider array of cognitive of Aβ plaque counts as well as in Aβ 40 and 42 levels in deficits beyond what was observed in the AD model, with the hippocampus with no added effects of diet or MxD. females being most impacted. Among LF-fed mice, spa- Others have found increased Aβ levels in female 3xTg- tial learning deficits emerged in MxD females that were AD mice [21, 40, 54–58] as well as increased Aβ-positive not observed in the AD females or MxD males. Among cell counts in the dorsal hippocampus [44]. Others have LF-fed males, spatial memory deficits emerged with MxD found no hypoperfusion-induced elevation in Aβ lev- that were not observed in AD males but were observed els in 3xTg-AD mice [51]; however, this was after only in AD/MxD females. Due to floor effects, we were unable 2 months of hypoperfusion suggesting that changes we to detect effects of MxD in females for some tests or to observed may have emerged at a later time point. While detect the effect of HF diet in the MxD groups in either we did not observe an effect of diet on Aβ pathology, oth - sex for most tests. However, it is important to note that ers have found that HF diet increases Aβ in 3xTg-AD MxD females were impaired in all four cognitive tests, mice [21, 24, 25, 59], with some studies showing that this while MxD males had preserved spatial learning and abil- effect is specific to females [57, 58]. This may be due to ity to perform activities of daily living. Our MxD model increased Aβ production that can occur in metabolic involved a unilateral common carotid artery occlusion disease, which is exacerbated when plaques have formed surgery, which leads to chronic cerebral hypoperfusion [60]. While further sex differences in dementia pathology [31–49] and cognitive deficits even in WT mice [19, 31, may develop with age, increased Aβ and astrogliosis in 50, 32]. While others have examined the pathological females support the heightened sensitivity of females to effects of chronic cerebral hypoperfusion in 3xTg-AD AD/MxD and may underlie the wider array of cognitive mice [51], we are the first to examine sex differences deficits in females. in this model. Our data support that added vascular In addition to AD pathology, we also assessed the pathology in MxD further exacerbates cognitive deficits, degree of chronic cerebral hypoperfusion. We found compared to AD alone, and that females may be more that unilateral carotid artery occlusion surgery (MxD adversely cognitively affected by chronic cerebral hypop - mice) caused deficits in cerebral blood flow in the erfusion in MxD. Gannon et al. Journal of Neuroinflammation (2022) 19:110 Page 16 of 20 cortical surface that persisted 4 months after surgery. female MxD mice are more adversely affected, MxD This is in line with our previous work, and that of oth - may pose a greater cognitive threat to women. ers, documenting prolonged deficits in blood flow in the ipsilateral hemisphere [19, 31–49, 61]. While we Potential mechanisms underlying observed sex differences observed the expected effect of MxD to cause hypop - Sex hormones may influence dementia on many levels, in erfusion, we did not observe any sex or diet differences. both men and women, and their effects must be viewed This is in line with a previous study by Bracko et al. with consideration to age and disease state. Estrogens are showing that HF diet-induced obesity do not cause present in both sexes but are higher in reproductive-age hypoperfusion or capillary stalling in AD[62]. However, women. Estrogen also protects against AD risk factors we did observe that greater metabolic impairment was (diabetes) and VCID risk factors (hypertension, obesity, associated with more severe hypoperfusion in females, diabetes, stroke) in women [15]. It is unknown how estro- but not males. Thus, the degree of metabolic impair - gen would impact AD and VCID when the pathologies ment may be important. Pires et al. have shown that HF overlap in MxD. Generally, estrogen can protect the brain diet causes cerebral vessel remodeling [63]. Blood flow through its vasodilatory, anti-apoptotic, antioxidant, and recovery following unilateral carotid artery occlusion anti-inflammatory actions [15, 64]. Rodent studies have surgery could be affected by the degree of collateral also shown protective effects of estrogen against Aβ [59, blood flow, angiogenesis, and compensation follow - 65, 66]. A limitation of our work is that menopause is not ing surgery. A limitation of the current study is that we taken into consideration. Most women with dementia only examined relative blood flow in the cortical sur - are post-menopausal, and menopause decreases estro- face; there may be differences in blood flow in deeper gen levels. Thus, we are currently assessing the effects brain structures that we were unable to detect. We have of menopause in several mouse models of dementia. previously assessed changes in blood flow using arterial While the evidence for the protective effects of estrogen spin labeling MRI and found that although hippocam- is robust, we have found that AD/MxD female mice are pal blood flow was reduced by the unilateral common more strongly impacted by HF diet. The 3xTg-AD geno - carotid occlusion surgery, it was not further reduced type may diminish the capacity of the brain to produce by diet in this deeper brain structure [61]. Sex differ - estrogen, given that women with AD have lower levels ences in the 3xTg-AD mouse have been noted, includ- of brain aromatase (the enzyme that produces estrogen) ing a greater association between plaques and markers [65, 67] and women with AD have lower brain estrogen of hypoxia [54]. These findings suggest that there could levels [67]. There is evidence that estrogen’s effect may be a stronger connection between vascular dysfunction turn from protective to damaging when acting in the and Aβ pathology in females. Our finding that meta - presence of inflammation or a disease state. For exam - bolic impairment is associated with more severe hypop- ple, estrogen can increase inflammation if administered erfusion in females only prompts further research into in older mice after a long period of estrogen withdrawal the potential of metabolic disease to interfere with [68] or lose effectiveness in ameliorating AD pathology blood flow compensation and recovery in a sex-specific [59]. Additionally, studies examining the impact of hor- manner. mone replacement therapy on dementia risk have flagged There may be additional neuropathologies not diabetic women as a population in which HRT increases addressed in the current study that could have contrib- dementia risk [69]. Diabetes has been shown to dimin- uted to sex differences in cognitive function. For exam - ish and sometimes reverse the protective effect of estro - ple, we have previously shown that HF diet impairs gen in rodent models of ischemia [70, 71]. In this study, adult hippocampal neurogenesis in WT females, but chronic HF diet-induced obesity could have created an not males [18]. This deficit occurred in the dorsal “accelerated aging” effect in females that negated the pro - hippocampus, where neurogenesis supports spatial tective influence of estrogen. learning and memory. Additionally, HF diet can accel- Androgens may also contribute to sex differences erate AD pathology in 3xTg-AD mice through several reported here. In men, age-related androgen decline is mechanisms not assessed in the current study, such as found as a risk factor for developing AD [72]. In male by increasing neuronal cell death, oxidative stress [23], 3xTg-AD mice, gonadectomy (which reduces testoster- and brain atrophy [20]. Future work is needed to find one) increases Aβ and tau pathology but is rescued by mechanistic insights behind the sex differences we and treatment with testosterone [73, 74]. While the effects of others have observed. Given the greater AD pathol- testosterone on MxD specifically are unclear, testosterone ogy in females, the interconnectedness of vascular and has a complicated influence on the cerebral vasculature AD pathology, and our current findings showing that [64, 75] with potentially harmful effects, such as increas - ing vasoconstriction [76–78] and inflammation [79, 80], G annon et al. Journal of Neuroinflammation (2022) 19:110 Page 17 of 20 but decreasing inflammation in disease states [81, 82] in studies assessing the overlap of other midlife risk factors, males. Our lab has previously reviewed the cerebrovas- such as menopause, will be important for the develop- cular actions of androgens and how that relates to meta- ment and utilization of therapeutic strategies for demen- bolic and cardiovascular disease [75]. More research is tia treatment and prevention. needed to determine if testosterone level changes in pre- diabetes affect long-term risk of MxD. Abbreviations AD: Alzheimer’s disease; ADL: Activities of daily living; ANOVA: Analysis of variance; AUC : Area under the curve; Aβ: Amyloid beta; BACE1: Beta‑secretase Clinical relevance 1; CA1–3: Cornu Ammonis 1–3; CD68: Cluster of differentiation 68; GFAP: Glial Given that there are well-documented sex differences fibrillary acidic protein; GTT : Glucose tolerance test; HF: High fat (diet); Hif1‑ in both AD and VCID, sex differences likely play a role alpha: Hypoxia inducible factor 1‑alpha; IBA1: Ionized calcium‑binding adaptor molecule 1; IHC: Immunohistochemistry; IL‑1β: Interleukin‑1beta; LF: Low fat in MxD. Prediabetes research is clinically important (diet); MWM: Morris water maze; MxD: Mixed dementia; NORT: Novel object because the prediabetic population is growing, there is recognition test; SEM: Standard error of the mean; VCID: Vascular contributions accelerating awareness of its connection to dementia, to cognitive impairment and dementia; Visc: Visceral (Fat); WT: Wild type. and, importantly, it is a potentially modifiable dementia risk factor. To incorporate this risk factor into clinical Supplementary Information prevention and treatment of dementia, more research is The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s12974‑ 022‑ 02466‑2. needed particularly in its effects on MxD, which is inad - equately represented in research. Women with diabetes Additional file 1: Figure S1. HF diet increased weight gain and fasting have greater risk of developing cognitive impairments, blood glucose in males and females. A) Body weight was measured and in certain types of dementia experience greater cog- monthly starting just prior to the surgery and onset of diet (“Pre”) and ending at tissue collection (“End”), (n = 15–28/group). B) Fasting blood nitive impairments than men with diabetes [3, 83, 84]. glucose levels were measured at the beginning of the glucose tolerance Metabolic disease in general appears to be a greater test. Data are presented as mean + SEM, ^p < 0.05 effect of dementia, **** dementia risk factor for women [15, 64]. This also extends p < 0.0001 effect of diet, 2‑ way ANOVA, (n = 17–26/group). to prediabetes, which is an all-cause dementia risk factor Additional file 2: Figure S2. HF diet decreased locomotor activity in females and increased center time in MxD males and females. A) General [85, 86]. A 2021 study found that prediabetes is associ- locomotor activity was measured by tracking the distance traveled (in ated with accelerated dementia onset and declining cog- meters) during the open field test. B) Anxiety‑like behavior and disorienta‑ nitive function only in women [17]. Diabetes is more tion were measured using the %time that the mice spend in the center of the testing arena during the open field test. C) Correlation matrix for open aggressively treated than prediabetes; this difference field measures (distance traveled and % time in the center of the arena) may manifest in long-term consequences. For example, a and episodic‑like memory as measured by the NOR recognition index (RI) recent study found a relationship between Aβ burden and for males and females. Pearson r values and p values are presented. Yellow: positive correlation, Blue: negative correlation. (n = 45–58/sex). D) Explora‑ prediabetes but not diabetes [87]. The authors hypothe - tion time during the training trial of the NOR test. The red line marks 2 s of sized that this is due to beneficial effects of diabetes treat - exploration, which was used as the cut‑ off for minimum object explora‑ ment. It is clear that further research is needed to grasp tion to be included in the test. E) MWM visible trial (day 1) pathlength by trial. F) Correlation matrix for average swim speed in the visible trials how prediabetes fits into the overall picture of sex differ - of the MWM (avg. speed visible) and spatial learning (hidden trial) and ences in dementia. spatial memory (probe trial). Pearson r values and p values are presented. Yellow: positive correlation, Blue: negative correlation, (n = 6–13 /group). A‑B and D ‑E) Data are presented as mean + SEM, *p < 0.05 effect of diet, Conclusion ****p < 0.0001 effect of diet, ^p < 0.05 effect of dementia, ^^p < 0.01 effect In summary, using a mouse model of mixed (AD + VCID) of dementia, 2‑ way ANOVA. dementia, AD and MxD females showed a wider array of Additional file 3: Figure S3. Microglia coverage is decreased in AD/MxD cognitive deficits, compared to males. Astrogliosis and males, while astrogliosis is exacerbated in AD/MxD females. Microglia coverage in the CA1 region of the hippocampus was gauged through Aβ pathology were also more severe in AD/MxD females, Iba1 immunofluorescence (larger % area covered indicating greater compared to males. When challenged with a HF diet, AD microgliosis). Iba1 immunoreactivity was used to calculated microglia or MxD females also had increased metabolic impair- coverage as the percent area covered by Iba1. Hippocampal regions of interest examined: CA2 (A), CA3 (B), and the dentate gyrus (C). Astrogliosis ment compared to males. Metabolic impairment was also in multiple regions of the hippocampus was gauged through GFAP immu‑ more consistently associated with reductions in cogni- nofluorescence (greater % area covered indicating greater astrogliosis). tive function in females. More severe glucose intolerance Hippocampal regions of interest examined:CA2 (D), CA3 (E), and the dentate gyrus (F). Data are presented as mean + SEM, ^ p < 0.05 effect was associated with worse microgliosis in females only. of dementia, ^^p < 0.01 effect of dementia, ^^^^p < 0.0001 effect of Here, we demonstrate the importance of considering dementia, 2‑ way ANOVA, (n = 4–5/group). how sex modulates the relationship between risk factors Additional file 4: Figure S4. Hippocampal Iba1 and GFAP immunofluo ‑ and dementia. This work supports the importance of pre - rescence intensity. Immunofluorescence intensity of Iba1 (A) and GFAP (B) was measured. 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Midlife The authors would like to thank Nathan Albert and David Riccio for their assis‑ overweight and obesity increase late‑life dementia risk: a population‑ tance with tissue collection and imaging, Avi Sura for assisting with ELISAs, based twin study. Neurology. 2011;76(18):1568–74. and Melissa Thomas for feeding and weighing mice. 10. 2020 Alzheimer’s disease facts and figures. Alzheimer’s & Dementia. 2020;16(3):391–460. Authors contributions 11. Attems J, Jellinger KA. The overlap between vascular disease and Alzhei‑ KLZ obtained funding for the experiments. LSR, OJG, and KLZ designed the mer’s disease–lessons from pathology. BMC Med. 2014;12:206. experiments. OJG and AES performed the animal work. OJG, LSR, AES, and 12. Brun A, Englund E. A white matter disorder in dementia of the Alzheimer CAG performed the experiments. LSR, OJG, CAG, FM, RDK, AT, RB, and JO type: a pathoanatomical study. Ann Neurol. 1986;19(3):253–62. analyzed the data. OJG prepared the figures. OJG prepared the manuscript. 13. Fernando MS, Ince PG, Function MRCC. Vascular pathologies and LSR and KLZ edited the manuscript. All authors read and approved the final cognition in a population‑based cohort of elderly people. J Neurol Sci. manuscript. 2004;226(12):13–7. 14. Ruitenberg A, Ott A, van Swieten JC, Hofman A, Breteler MM. Inci‑ Funding dence of dementia: does gender make a difference? Neurobiol Aging. This work was funded by the American Heart Association 16SDG27190001 2001;22(4):575–80. (KLZ), American Heart Association 20PRE35080166 (OJG), NINDS/NIA 15. Gannon OJ, Robison LS, Custozzo AJ, Zuloaga KL. Sex differences in risk R01NS110749 (KLZ), NIA U01AG072464 (KLZ), Albany Medical College startup factors for vascular contributions to cognitive impairment & dementia. funds, and NINDS F31 NS115290 (OJG). Neurochem Int. 2019;127:38–55. 16. Liu CL, Lin MY, Hwang SJ, Liu CK, Lee HL, Wu MT. Factors associated with Availability of data and materials type 2 diabetes in patients with vascular dementia: a population‑based The datasets used and/or analyzed during the current study are available from cross‑sectional study. BMC Endocr Disord. 2018;18(1):45. the corresponding author on reasonable request. 17. Sundermann EE, Thomas KR, Bangen KJ, Weigand AJ, Eppig JS, Edmonds EC, et al. Prediabetes is associated with brain hypometabolism and cognitive decline in a sex‑ dependent manner: a longitudinal study of Declarations nondemented older adults. Front Neurol. 2021;12: 551975. 18. Robison LS, Albert NM, Camargo LA, Anderson BM, Salinero AE, Riccio DA, Ethics approval and consent to participate et al. High‑Fat Diet ‑Induced Obesity Causes Sex ‑Specific Deficits in Adult Human data or tissue was not used in this study. All work involving animals Hippocampal Neurogenesis in Mice. Neuro. 2020;7(1):19. were approved by IACUC. 19. 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Estrogen receptor beta Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : dependent attenuation of cytokine‑induced cyclooxygenase ‑2 by andro ‑ gens in human brain vascular smooth muscle cells and rat mesenteric fast, convenient online submission arteries. Steroids. 2012;77(8–9):835–44. thorough peer review by experienced researchers in your ﬁeld 83. McCollum M, Hansen LS, Lu L, Sullivan PW. Gender differences in diabetes mellitus and effects on self‑ care activity. Gend Med. 2005;2(4):246–54. rapid publication on acceptance 84. Dufouil C, Seshadri S, Chene G. Cardiovascular risk profile in women and support for research data, including large and complex data types dementia. J Alzheimers Dis. 2014;42(Suppl 4):S353–63. • gold Open Access which fosters wider collaboration and increased citations 85. Xu W, Qiu C, Winblad B, Fratiglioni L. The effect of borderline diabetes on the risk of dementia and Alzheimer’s disease. Diabetes. 2007;56(1):211–6. maximum visibility for your research: over 100M website views per year 86. Xue M, Xu W, Ou YN, Cao XP, Tan MS, Tan L, et al. Diabetes mellitus and risks of cognitive impairment and dementia: A systematic review and At BMC, research is always in progress. meta‑analysis of 144 prospective studies. Ageing Res Rev. 2019;55: Learn more biomedcentral.com/submissions http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Neuroinflammation Springer Journals http://www.deepdyve.com/lp/springer-journals/high-fat-diet-exacerbates-cognitive-decline-in-mouse-models-of-6lh9CGtom0

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Publisher: Springer Journals
Copyright: Copyright © The Author(s) 2022
eISSN: 1742-2094
DOI: 10.1186/s12974-022-02466-2
Publisher site: See Article on Publisher Site

Abstract

Background: Approximately 70% of Alzheimer’s disease (AD) patients have co‑morbid vascular contributions to cognitive impairment and dementia ( VCID); this highly prevalent overlap of dementia subtypes is known as mixed dementia (MxD). AD is more prevalent in women, while VCID is slightly more prevalent in men. Sex differences in risk factors may contribute to sex differences in dementia subtypes. Unlike metabolically healthy women, diabetic women are more likely to develop VCID than diabetic men. Prediabetes is 3× more prevalent than diabetes and is linked to earlier onset of dementia in women, but not men. How prediabetes influences underlying pathology and cognitive outcomes across different dementia subtypes is unknown. To fill this gap in knowledge, we investigated the impact of diet‑induced prediabetes and biological sex on cognitive function and neuropathology in mouse models of AD and MxD. Methods: Male and female 3xTg‑AD mice received a sham (AD model) or unilateral common carotid artery occlusion surgery to induce chronic cerebral hypoperfusion (MxD model). Mice were fed a control or high fat (HF; 60% fat) diet from 3 to 7 months of age. In both sexes, HF diet elicited a prediabetic phenotype (impaired glucose tolerance) and weight gain. Results: In females, but not males, metabolic consequences of a HF diet were more severe in AD or MxD mice compared to WT. In both sexes, HF‑fed AD or MxD mice displayed deficits in spatial memory in the Morris water maze (MWM). In females, but not males, HF‑fed AD and MxD mice also displayed impaired spatial learning in the MWM. In females, but not males, AD or MxD caused deficits in activities of daily living, regardless of diet. Astrogliosis was more severe in AD and MxD females compared to males. Further, AD/MxD females had more amyloid beta plaques and hippocampal levels of insoluble amyloid beta 40 and 42 than AD/MxD males. In females, but not males, more severe glucose intolerance (prediabetes) was correlated with increased hippocampal microgliosis. Olivia J. Gannon and Lisa S. Robison contributed equally to this work *Correspondence: zuloagk@amc.edu Department of Neuroscience & Experimental Therapeutics, Albany Medical College, 47 New Scotland Avenue; MC‑136, Albany, NY 12208, USA Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Gannon et al. Journal of Neuroinflammation (2022) 19:110 Page 2 of 20 Conclusions: High‑fat diet had a wider array of metabolic, cognitive, and neuropathological consequences in AD and MxD females compared to males. These findings shed light on potential underlying mechanisms by which pre ‑ diabetes may lead to earlier dementia onset in women. Keywords: Sex, Vascular, Dementia, Alzheimer’s disease, High‑fat diet, Inflammation, Obesity, Cerebral hypoperfusion, Metabolic, Glucose intolerance, Diabetes Background have shown that HF diet impairs adult hippocampal neu- Diabetes increases the risk of developing dementia by rogenesis in female, but not male mice [18]. Further, we twofold [1–5]. While diabetes is increasingly common, recently found that HF diet in middle-aged mice causes prediabetes is estimated to affect 1 out of every 3 Ameri - a wider array of cognitive deficits in females compared cans [6], and most people are unaware of their status. to males [19]. Others have found that a HF diet in the Like diabetes, prediabetes is characterized by impaired 3xTg-AD model of AD exacerbates cognitive impairment glucose tolerance; however, those with prediabetes show [20–25] and AD pathology, such as brain atrophy [20], slight elevations in insulin and fasting blood glucose, inflammation [22, 26], and Aβ load [26–28]. Examina- rather than hyperglycemia. Diabetes and prediabetes tion of sex differences in the cognitive effects of HF diet are shared risk factors and common co-morbidities for in the 3xTg-AD mouse have been mixed, with some stud- the two most common forms of dementia: Alzheimer’s ies finding greater cognitive impairment in females [20], disease (AD) and vascular contributions to cognitive while others have found no sex differences [21]. Previ - impairment and dementia (VCID) [1–5]. Obesity, which ously, we reported that HF diet results in greater meta- is often co-morbid with prediabetes or type 2 diabetes, bolic impairment (weight gain, visceral fat, and glucose increases AD risk threefold and VCID fivefold [7–9]. AD intolerance) in female compared to male 3xTg-AD mice is characterized by amyloid plaques, tau tangles, and neu- [29]. HF-fed females also showed increased astrogliosis in rodegeneration culminating in brain atrophy and cogni- the hypothalamus, a brain region that controls metabolic tive impairment. VCID is caused by deficits in cerebral function. Whether these greater metabolic disturbances blood flow and/or damage to cerebral vessels. In reality, in females would also lead to more severe cognitive defi - the distinction between AD and VCID is less clear-cut. cits and neuropathology in brain regions associated with Dementia pathologies often overlap, with more than half learning and memory had not yet been tested. In the cur- of dementia patients having multiple pathologies [10], a rent study, using mouse models of both AD and MxD, we condition known as mixed dementia (MxD). The most found that diet-induced obesity with prediabetes led to a common form of MxD is a mix of AD and VCID, as this wider array of cognitive deficits and neuropathology in occurs in ~ 70% of AD patients [11–13]. MxD is under- females compared to males. represented in animal research despite its high clinical prevalence. Understanding the interaction between AD Methods and VCID risk factors will provide insight into MxD. Animals and experimental design Dementia risk and prevalence vary by sex: women are This study was conducted in accordance with the more likely to develop AD[10], while men are slightly National Institutes of Health Guidelines for the Care and more likely to develop VCID [14, 15]. Discrepancies in Use of Laboratory Animals, and protocols were approved risk factors may drive these sex differences. In patients by the Institutional Animal Care and Use Committee at with diabetes, the sex difference observed in the non- Albany Medical College (Albany, NY, USA). Temperature diabetic population is reversed: diabetic women are at a and humidity were set at 72 °F, 30–70% humidity, with a 19% greater risk of VCID than diabetic men [3]. Among 12-h light/dark cycle (7 a.m. on/7 p.m. off ). Mice were those who have VCID, women are also more likely to fed a standard chow diet (Purina Lab Diet 5P76) until the have diabetes [16]. Prediabetes is associated with cogni- start of this study. They were housed in Allentown cages tive impairment and earlier onset of dementia in women, at a density of 2–5 mice. Mice were provided with envi- but not men, suggesting it may be a sex-specific risk fac - ronmental enrichment (Nestlets and Shepherd Shacks) tor [17]. However, it is unknown how prediabetes affects and were group housed at all times, except during the MxD, and whether these effects differ by sex. nest building test. Male and female wild-type (WT) High-fat (HF) diet is commonly used to induce meta- B6129SF2/J mice (#101045) and 3xTg-AD (#34830-JAX) bolic disease in rodents, as it causes both obesity and breeding pairs were purchased from Jackson Labora- prediabetes. HF diet can have profound effects on the tories (Bar Harbor, ME) and used to maintain a colony brain, some of which are sex dependent. For example, we at Albany Medical Center’s Animal Resource Facility. G annon et al. Journal of Neuroinflammation (2022) 19:110 Page 3 of 20 The 3xTg-AD mice, which are on a C57BL/6;129X1/ sites were closed with Vetbond, and the mice were given SvJ;129S1/Sv background, have three mutations that are 100µL 0.03 mg/mL buprenorphine via subcutaneous associated with AD in humans: APPSwe, tauP301L, and injection twice per day for 3 days as an analgesic. tm1Mpm Psen1 [30]. A timeline of the experiment is shown in Fig. 1A. At ~ 3 months of age, 3xTg-AD mice under- Glucose tolerance test went a sham surgery (AD group) or a unilateral com- As previously described [19, 29, 33], mice were given a mon carotid artery occlusion surgery (MxD group). WT glucose tolerance test (GTT) to assess diabetic status controls also received a sham surgery (WT group). One at ~ 5.75 months of age. The mice were fasted overnight, week following surgery, mice were placed on either a HF and their fasting blood glucose levels were measured diet (60% fat, 5.24 kcal/g; D12492, Research Diets, New (t = 0) using a glucometer (Verio IQ, OneTouch, Sun- Brunswick, NJ) or a low-fat (LF) control diet (10% fat, nyvale CA, USA) from their tail vein. Following an i.p. 3.82 kcal/g; D12450B, Research Diets) for the duration injection of 2 g/kg of glucose, blood glucose levels were of the study. At ~ 5.75 months of age, mice underwent a measured at 15, 30, 60, 90, and 120 min post-injection to glucose tolerance test (GTT) and a 2-week rest period, assess glucose tolerance. behavioral testing, followed by blood flow imaging, euthanasia, and tissue collection (including brains, fat, Behavior testing and reproductive organs) at ~ 7 months of age. A total of Following a 2-week recovery post-GTT, mice were tested 251 mice were used in this study. Experiments were con- for exploratory activity and anxiety-like behavior in the ducted in cohorts of up to 20 animals. A subset of mice open field (day 1), episodic-like memory in the novel (n = 118) was designated for collection of metabolic data, object recognition test (NORT; day 2), spatial learn- behavior testing, plasma assays [reported in [29]], and ing and memory in the Morris water maze (MWM; blood flow imaging, and brains were microdissected for days 8–10 or 10–12), and activities of daily living using use in Western blots. The remaining mice were used for a nest building task (days 15–16). Videos were recorded collecting metabolic data and blood flow imaging, and of behavioral performance for open field, NORT, and brains were used for immunofluorescence. In total, 23 MWM and analyzed using automated tracking software mice were excluded due to premature death or the pres- (ANY-maze 5.1, Stoelting, Wood Dale, IL). For each test, ence of other major health exclusions (hydrocephaly, mice were placed into the procedure room under dim large fighting wounds, tumors). Final group sizes ranged light and allowed to acclimate for 1 h. Each test appara- from 13 to 25 per group for metabolic measures, 8 to 13 tus was cleaned with 70% ethanol between each mouse to for behavioral testing, and 4 to 6 for immunohistochem- remove olfactory cues. istry (IHC). During tests, experimenters were blinded to surgical group. Blinding to diet and sex was not possible Open field due to mouse appearance. During analysis, experiment- The mice were placed in the test apparatus (495 × 495 mm ers were blinded to sex, diet, and dementia group. box) for 10 min. Distance traveled was used to determine the general activity levels of the animal. The percent of Surgical model of MxD time spent in center of the arena was used to determine To model MxD, 3xTg-AD mice underwent a right com- anxiety-like behavior. One mouse was excluded from this mon carotid artery occlusion surgery, as previously test for being a statistical outlier via Grubb’s outlier test described, to elicit chronic cerebral hypoperfusion and leaving group sizes of 8–13/group. model VCID [19, 31, 32]. Briefly, under isoflurane anes - thesia, the right common carotid artery was ligated NORT with two 6–0 silk sutures and cauterized (MxD group). NORT consisted of two, five-minute trials performed The sham surgery (WT and AD groups only) consisted in the same open field arena. In the first trial, mice were of exposing the carotid artery without ligation. Incision placed in the box and allowed to explore two identical (See figure on next page.) Fig. 1 HF diet caused greater metabolic impairment in AD and MxD females compared to males. A Experimental timeline. GTT (glucose tolerance test). B Weight gain was assessed by the % change in body weight from the start of the study to the end of the study. C Visceral adiposity was determined by isolating and weighing the visceral fat pads and normalizing to body weight. D, E Glucose intolerance was assessed with a GTT following a 16 h fast. D Glucose clearance was gauged by concentrations of glucose in the blood measured over time (time 0 = fasting blood glucose). E Blood glucose concentration over time was used to calculate area under the curve. We previously reported metabolic data for the Sham WT and Sham AD, but not the MxD, groups in Robison et al. (2020) in the Journal of Neuroinflammation [29]; licensed under a Creative Commons Attribution 4.0 International License; (https:// creat iveco mmons. org/ licen ses/ by/4. 0/). Data are presented as mean + SEM, **p < 0.01 effect of diet, ****p < 0.0001 effect of diet, ^p < 0.05 effect of dementia, ^^^p < 0.001 effect of dementia, ^^^^p < 0.0001 effect of dementia, 2‑ way ANOVA, (n = 13–25/group) Gannon et al. Journal of Neuroinflammation (2022) 19:110 Page 4 of 20 Fig. 1 (See legend on previous page.) G annon et al. Journal of Neuroinflammation (2022) 19:110 Page 5 of 20 objects (rubber ducks). Mice were then returned to a performed using moorFLPI Review V4.0 software (Moor recovery cage for 1 h. For the second trial, mice were Instruments, Wilmington, DE, USA). Average flux values returned to the arena, with the one familiar object were extracted from regions of interest (ROIs) using a replaced with a novel object (saltshaker). Episodic-like published protocol [36]. Measurements are presented as memory was assessed by recognition index [(time with %difference in blood flow between the left (non-ischemic novel object/total time with objects) *100]. Between tests, for MxD mice) and right (ischemic for MxD mice) objects were cleaned with 70% ethanol to mask olfactory hemisphere. cues. Mice that spent less than ≤ 2 s with the objects were excluded (total of 26 mice: 0–4/group, 12 males and 14 Immunofluorescence females). Additionally, 2 statistical outliers were removed Mice were perfused with ice-cold 0.9% saline. Brains from this test (1 WT LF M and 1 WT HF M). were removed and fixed in 4% paraformaldehyde for 24 h, followed by immersion in 30% sucrose for at least MWM 72 h. Brains were then snap frozen in OCT and stored Hippocampus-dependent spatial learning and mem- at − 80 °C until sectioning. Brains were sectioned at 40 ory were assessed using a modified 3-day version of microns on a Leica CM1950 cryostat into 6 series. Sec- the MWM that has been shown to be optimal for older, tions were washed in PBS containing 0.01% sodium azide, cognitively impaired, obese mice [34]. The protocol has permeabilized at room temperature for 1 h (0.3% TPBS), been previously described in detail [19]. On day 1, 5 vis- and blocked for 1 h at room temperature in 4% donkey ible trials were performed in which mice learn to find serum in 0.3% TPBS before being incubated in blocking the platform with a visual cue (flag). The entry point was buffer with primary antibodies at 4 °C overnight. Primary alternated for each trial. On day 2, mice underwent 5 hid- antibodies for one series included rabbit anti-beta Amy- den trials, in which the visual cue was removed from the loid (1:300, Cat# 71-5800, Lot# SH257822; Invitrogen, platform. All trials were 3 min long with a 30-min inter- Waltham, MA). Primary antibodies for another series trial interval. The distance traveled to reach the platform included rat anti-glial fibrillary acidic protein (1:2500, (pathlength) was used as a measurement of non-spatial AB5804, Millipore, Lot # TA265137) or goat anti-Iba1 (visual trials) and spatial (hidden trials) learning. On day (1:1000, PA5-18,039, Lot #TI2638761, SJ2467805; Ther - 3, a single probe trial was performed in which the plat- moFisher, Waltham, MA). Sections were incubated with form was removed from the pool. Spatial memory was secondary antibodies and DAPI (1:1000, Cat# D1306, calculated as the percent of time spent in the target quad- ThermoFisher, Waltham, MA) in blocking buffer for rant of the pool during the first minute of the probe trial. 2 h at room temperature. Secondary antibodies used A total of 2–4 mice were excluded from this test for being included Rhodamine Red-X Donkey Anti-Rabbit (1:100), statistical outliers via Grubb’s outlier test [4 mice in the Alexa Fluor 647 Donkey Anti-Goat (1:300), DyLight hidden trial and 2 mice in the probe trial] leaving group 405 AffiniPure Donkey Anti-Rat (1:300) (Jackson Immu - sizes of 8–13/group. noResearch, West Grove, PA), Alexa Fluor 647 Affin - iPure Donkey Anti-Goat IgG (H + L) (1:1000, Cat# Nest building AB_2340437, Jackson ImmunoResearch, West Grove, Mice were singly housed in Allentown cages with pine PA), and Alexa Fluor 488 Donkey Anti-Rabbit (1:1000, chip bedding and two pre-weighed Nestlets each. After Cat# ab150073, Jackson ImmunoResearch, West Grove, 16 h (overnight), the mice were removed from their test PA). Images for quantification were taken at 10 × using cage and returned to group housing. Nests were rated the Axio Observer fluorescent microscope (Carl Zeiss on a 1–5 scale (with half-point scores allowed) based Microscopy, Jena, Germany). All analyses were per- on published criteria [35] by 3 experimenters who were formed in the right hemisphere of coronal sections unless blinded to treatment group. The 3 ratings were averaged. otherwise noted by an experimenter who was blinded One mouse was excluded from this test for being a statis- to treatment group. For Aβ plaque quantification, 8 tical outlier via Grubb’s outlier test leaving group sizes of 40-micron-thick sections between − 1.46 and − 3.52 mm 8–13/group. from bregma were used to hand count numbers of Aβ plaques in the left and right hemisphere (Fig. 4B). For Cerebral blood flow measurement intracellular Aβ quantification, regions of interest (ROI) Cerebral blood flow was measured via laser speckle were drawn in the cortex and a number of cells positive contrast imaging (moor FLPI full field laser perfusion for Aβ were counted (Additional file 5: Fig. S5A). For imager; Moor Instruments, Wilmington, DE, USA) under neuroinflammation analysis (microglia and astrocytes), isoflurane anesthesia as previously described [19]. Image ROIs were drawn in CA1, CA2, CA3, and dentate gyrus acquisition [5-min scan], processing, and analysis were Gannon et al. Journal of Neuroinflammation (2022) 19:110 Page 6 of 20 of the hippocampus in ~ 3 brain sections per mouse and (n = 4 mice/group) were thawed and homogenized then % area covered was measured using ImageJ. spun at 4C 12,100 g for 30 min. The supernatant (40uL) was retrieved and neutralized with 20 volumes of neutral- izing solution (800uL; 1 M Tris base, 0.5 M Na2HPO4, Western blot 0.05% NaN3). A protease and phosphatase cocktail was Frozen hippocampi (ipsilateral to VCID or sham surgery) added (HALT, ThermoScientific #1861284) that contains from 4 mice per group were thawed and homogenized in the serine protease inhibitor ABESF to prevent degrada- 50µL RNA-Later (45-R0901-100MLsigma). 25µL of that tion of AB peptides. Amyloid peptides were then quanti- homogenate was transferred into 100 µL T-Per buffer fied in this fraction using ELISA kit from ThermoFisher (ThermoScientific #78,510) supplemented with protease Scientific (AB40: KHB3481, and AB42: KHB3441) and phosphatase inhibitor cocktail (HALT, ThermoSci - according to the manufacturer’s instructions. Quantities entific #1861284) and spun at 21,000 g for 20 min. The were normalized to total soluble protein quantities. supernatant was collected, and the protein concentration was determined using a Pierce BCA Protein Assay Kit (Thermo Scientific #23,227). 20 µg of protein was dena - Quantitative reverse transcriptase‑PCR (RT‑qPCR) tured in LDS (4× Bolt LDS Sample Buffer, Invitrogen, Frozen hippocampi (ipsilateral to VCID or sham surgery) B0007) with 0.2 M DTT and boiled for 5 min at 95 °C. from 4 to 5 mice per group were thawed and homog- Proteins were then separated using a 10% Tris Bis gel enized in 50µL RNA-Later (45-R0901-100MLsigma). (ThermoFisher, NW00107BOX) before being transferred RNA was extracted from 25uL of homogenate using the onto a nitrocellulose membrane (Nitrocellulose/Filter RNeasy Plus Mini Kit (Qiagen, Catalog number 74134) Paper Sandwich, 0.2 μm, 8.3 × 7.3 cm, ThermoFisher, and RNA concentrations were measured using Thermo - LC2000). Membranes were blocked using LI-COR Scientific NanoDrop One. RNA was converted to cDNA blocking solution (Intercept (TBS) Blocking Buffer, LI- using a High-Capacity cDNA Reverse Transcription Kit COR, 927–60,001) for one hour at room temperature (Applied Biosystems, Catalog number: 4368814). The then exposed to primary antibodies at 4 °C overnight. qPCR reactions were performed using TaqMan Gene The following day membranes were washed then incu - Expression Master Mix (Applied Biosystems, Catalog bated with the corresponding secondaries before being number 4369016) in the presence of TaqMan Assays washed and scanned using an Odyssey CLx LI-COR with primer/probes for Iba1 (Mm00479862_g1), GFAP scanner. All membranes were scanned simultaneously (Mm01253033_m1), and CD68 (Mm00839636_g1) as to ensure similar exposure and scanning parameters. target genes. RPL13A (Mm05910660_g1) was used as the Membranes were then stripped using LI-COR stripping housekeeping gene. Data were presented as fold change buffer (NewBlot IR Stripping Buffers for NIR Western relative normalized expression compared to WT LF mice Blots, LI-COR, 928–40,028) for 20 min and rescanned to by ΔΔCq method using Bio-Rad CFX Maestro software. ensure loss of signal before being incubated with an anti- GAPDH antibody (Sigma, 45-G9545-100UL) as a loading Statistics control. Western blot band analysis was conducted using Statistical analyses were performed using Prism 8.1 the LI-COR Image Studio and normalized to loading (GraphPad Software, San Diego, CA, USA). All data control. Antibodies used were for total tau ((T46) mouse are presented as mean + SEM, with significance set abCam203179, lot #GR33658561), early phosphorylated at p < 0.05 except for categorical data (nest building is tau (S199-202, rabbit Invitrogen 44-768G lot SG255287), expressed as median + interquartile range). Following a and later phosphorylated tau (PHF1, rabbit Invitrogen Grubbs’ test for statistical outliers, a 2-way ANOVA was PA5-56,621 lot TB2525148). All primary antibodies were performed with Tukey’s correction for multiple compar- used at 1:1000 in Tris-buffered saline supplemented with isons [dementia type (WT vs. AD vs. MxD) X diet (LF 0.1% Tween (TBST). Secondary antibodies were used at vs. HF)] in data segregated by sex. The exception to this 1:15,000 in TBST with 0.02% SDS. Secondary antibodies is the test for spatial learning (Morris water maze hid- included IRDye 800CW Donkey anti-Mouse IgG (CAT# den trial, average pathlength) in which we used Dun- 926–32212, Lot#D00930-09, LI-COR, Lincoln, Nebraska) nett’s multiple comparison test. We used this post hoc and IRDye 680RD Donkey anti-Rabbit IgG (CAT# 926– test because we specifically defined the deficit in average 68,073, Lot#D00421-09, LI-COR, Lincoln, Nebraska). pathlength as greater than the LF WT group. In second- ary analyses to assess sex differences, 3-way ANOVAs Enzyme‑linked immunoassay (ELISA) were performed (sex X dementia type X diet) with- The pellet from the protein extraction (insoluble fraction) out post hoc analysis (except for analysis in which WT was dissolved in 50ul of freshly prepared 70% formic acid groups were not included we used Sidak’s multiple com- and stored at -80C. On the day of the assay, the samples parison test). In measures with large sample sizes (n ≥ 13 G annon et al. Journal of Neuroinflammation (2022) 19:110 Page 7 of 20 for all groups) a ROUT test was performed (metabolic females had greater metabolic impairment (weight gain, data only). One-sample t-tests were performed for meas- visceral fat accumulation, and glucose intolerance) than urements where values are compared to chance (50% in HF-fed WT females (p < 0.0001 for all measures). Con- NORT, 25% in MWM probe trial, and 0% hemispheric versely, in males, LF-fed AD and LF-fed MxD males difference in blood flow). Each correlation matrix was had less visceral fat than LF-fed WT males (p < 0.001 created by computing Pearson correlation coefficients for for AD, p < 0.05 for MxD). Analysis of sex differences via each pair of datasets. a 3-way ANOVA (Additional file 6: Table S1) showed a sex X dementia interaction and sex X diet interaction Results (p < 0.0001 for each interaction) in which both visceral Animal models and timeline fat and glucose intolerance were exacerbated in AD/ In order to create a mouse model of MxD, 3xTg-AD MxD and HF-fed females, but not males. Further, there mice (~ 3 months of age) underwent a unilateral com- was also a sex X diet X dementia interaction for % weight mon carotid artery occlusion surgery to induce chronic gain (p < 0.0001), which was driven by the large % weight cerebral hypoperfusion/vascular pathology. These mice increase in HF-fed AD and HF-fed MxD females. Taken were compared to AD mice (3xTg-AD that underwent a together, the data show that HF diet caused greater met- sham surgery) and WT controls (WT mice that under- abolic impairment in AD or MxD females compared to went a sham surgery). In order to determine the effects WT females or AD or MxD males. of HF diet-induced prediabetes on outcomes in the AD and MxD models, one week following surgery, mice were HF diet caused a wider array of cognitive deficits in females placed on either a HF diet (60% fat) or a control LF diet Our prior work has demonstrated that HF diet causes a (10% fat) from ~ 3 to 7 months of age. A study timeline is wider array of cognitive deficits in middle-aged females, shown in Fig. 1A. compared to males, in a mouse model of VCID [19]. Whether HF diet would differentially impact AD and HF diet caused greater metabolic impairment in AD MxD in each sex was unknown. Preference for the novel and MxD females compared to males. object in the NORT (episodic-like memory; Fig. 2A) We have previously reported [29] that metabolic effects was assessed with a one-sample t-test vs. no preference of a HF diet are more severe in 3xTg-AD females com- (recognition index of 50%). In males, all WT mice dem- pared to males. Here we show that these sex differ - onstrated a preference for the novel object (LF-fed WT ences persist in the MxD model. Within each sex, there p < 0.001, HF-fed WT p < 0.001). In females, LF-fed WT was a main effect of HF diet to increase % weight gain mice show intact memory (p < 0.001), while HF-fed WT (p < 0.0001; Fig. 1B), visceral fat accumulation (p < 0.0001; females only showed a trend toward preference for the Fig. 1C), and glucose intolerance (p < 0.0001; Fig. 1D, E). novel object (p = 0.09). In both sexes, both AD and MxD Monthly weigh gain is shown in Additional file : Figure groups on either diet demonstrated no preference for the S1A. Importantly, as others have reported in previous novel object, indicating an impairment in episodic-like studies [27, 28], the HF diet induced a prediabetic pheno- memory. A 3-way ANOVA showed no sex differences. type with significantly elevated fasting blood glucose lev - Although there were some group differences in explora - els (Fig. 1D, zero time point and Additional file 1: Figure tory behavior and anxiety-like behavior in the open S1B) that are below the 250 mg/dl cut-off that has been field (Additional file 2: Figure S2A, B), neither of these established in the literature for mouse models of diabe- measures correlated with NORT performance (Addi- tes [37]. Of note, the highest fasting blood glucose among tional file 1: Figure S1C). There were no significant dif - any of our HF-fed mice was still only 221 mg/dl. Demen- ferences in performance during the NORT training trial tia X diet interactions were sex dependent. In females, (Additional file 1: Figure S1D). The MWM was used to post hoc tests showed that HF-fed AD and HF-fed MxD examine spatial learning and memory. All groups were (See figure on next page.) Fig. 2 HF diet caused a wider array of cognitive impairment in females compared to males. A Episodic‑like memory was assessed in the novel object recognition test (NORT ). Recognition index (% time spent with the novel object) was calculated. Performance not significantly greater than chance (50%, indicated by the red line) indicates impaired memory. B–D Spatial learning and memory was assessed using the Morris Water Maze (MWM). Five hidden trials (B) assessed spatial learning via pathlength to reach the target platform (shorter pathlength = better per formance). Average pathlength over the 5 hidden trials (C) was longer (more impaired memory) in MxD females and AD females on a HF diet. Spatial memory was assessed in the probe trial (D), as % time spent in the target quadrant vs. chance (25%, indicated by the red line). Performance above 25% indicates intact memory. E The nest building task was used to assess activities of daily living. Nests were graded on 1–5 scale (average of scores by 3 experimenters blinded to treatment). Lower scores are indicative of impairment. Data are presented as mean + SEM except for nest building (median + interquartile range), +++p < 0.001 vs chance, +p < 0.05 vs. chance, **p < 0.01 effect of diet, ^p < 0.05 effect of dementia, ^^p < 0.01 effect of dementia, ^^^^p < 0.0001 effect of dementia, Red line = chance, 2‑ way ANOVA, (n = 5–11/group NOR, n = 8–13/group MWM, nest building) Gannon et al. Journal of Neuroinflammation (2022) 19:110 Page 8 of 20 Fig. 2 (See legend on previous page.) G annon et al. Journal of Neuroinflammation (2022) 19:110 Page 9 of 20 trained to swim to a visible platform (main effect of trial females compared to males, particularly when females p < 0.05, Additional file 2: Figure S2E). When the plat- were fed a HF diet. form was hidden (spatial learning trials; Fig. 2B, C), no group differences were observed in males. In females, Neuropathology was exacerbated in AD/MxD females. there was a dementia x diet interaction (p < 0.05), in We have previously reported sex-dependent effects of HF which HF-fed AD females, LF-fed MxD females, and diet on hypothalamic neuroinflammation in 3xTg-AD HF-fed MxD females all had significantly impaired spa - mice; however, neuroinflammation in brain areas associ - tial memory compared to LF-fed WT females (p < 0.01 ated with cognition and other forms of neuropathology for each group; post hoc tests). A 3-way ANOVA (Addi- had not been assessed. Microgliosis (immunolabeling for tional file 6: Table S1) showed a sex X dementia X diet Iba-1) and astrogliosis (immunolabeling for glial fibril - interaction (p < 0.01) in which HF-fed AD/MxD females lary acid protein; GFAP) were assessed in the hippocam- had the most severe spatial learning deficits. In the probe pal CA1, CA2, CA3, and dentate gyrus regions (Fig. 3A). trial (spatial memory; Fig. 2D), preference for the tar- In males, there was a main effect of dementia on micro - get quadrant was assessed with a one-sample t-test of % gliosis (Iba1% area covered; Fig. 3B, D, Additional file 3: time in target quadrant vs. chance (25%). In males, only Figure S3A–C, and Additional file 4: Figure S4A), in WT mice (on either diet) and LF-fed AD mice showed a which AD and MxD males showed decreased hip- preference for the target quadrant (p < 0.05), indicating pocampal microgliosis in several regions (CA1 p < 0.001, that spatial memory was impaired in HF-fed AD males CA2 p < 0.0001, CA3 p < 0.01). In females, microglio- and MxD males on either diet. In females, only the LF- sis was unaffected by either diet or dementia in any of fed WT mice showed a preference for the target quad- the regions assessed. A 3-way ANOVA showed a sex X rant (p < 0.05), indicating that HF-fed WT females and dementia interaction in several regions (CA1 p < 0.01, AD or MxD females on either diet had impaired spatial CA2 p < 0.01, CA3 p < 0.05), which was driven by higher memory. A 3-way ANOVA (Additional file 6: Table S1) microgliosis in WT males (regardless of diet) compared showed only a slight trend toward worse spatial memory to all other groups. In males, astrogliosis (GFAP % area in females (p = 0.08) with no significant interactions. covered; Fig. 3C, E, Additional file 3: Figure S3D-F, and Due to known decreases in swim speed in obese mice, Additional file 4: Figure S4B) was unaffected by either we used non-speed-based measures (pathlength/% time diet or dementia in any of the regions assessed. In in the target quadrant). Although there were some group females, there was a main effect of dementia, in which differences in swim speed, speed did not correlate with AD and MxD females showed increased hippocampal learning or memory in the MWM (Additional file 2: astrogliosis (CA1 p < 0.05, CA2 p < 0.01, dentate gyrus Figure S2F), further supporting that cognitive measures p < 0.01). A 3-way ANOVA showed a sex X demen- were speed independent. The nest building test (Fig. 2E) tia interaction in which AD/MxD females had greater was used to assess activities of daily living (ADLs). In astrogliosis than males (CA1 p < 0.05, CA2 p < 0.05, CA3 males, all mice performed well although there was a p < 0.01, dentate p < 0.05). In addition to examining % area main effect of diet (p < 0.05) to impair ADLs. In females, covered, we assessed expression of hippocampal genes AD and MxD females showed clear impairments (main associated with microgliosis (Iba-1 and CD68, a lysoso- effect of dementia p < 0.0001), with LF-fed MxD (p < 0.01) mal protein upregulated during microglial activation: and HF-fed MxD (p < 0.05) females building poorer nests Fig. 3F, G) and astrogliosis (GFAP: Fig. 3H). In males, than control LF-fed WT females. HF-fed AD females also GFAP gene expression in hippocampal homogenate was showed a trend (p = 0.056) toward impaired ADLs. A increased overall by dementia (p < 0.05). However, GFAP 3-way ANOVA showed a main effect of sex X dementia gene expression was unaffected in females. CD68 expres - interaction (p < 0.01) in which AD and MxD females were sion was unaffected by diet or dementia in both males more impaired than males. Taken together, these data and females. In females, HF diet decreased Iba-1 expres- show a wider array of cognitive deficits in AD or MxD sion (p < 0.05). To examine AD pathology, we assessed Aβ pathology via immunohistochemistry for Aβ, ELISA for (See figure on next page.) Fig. 3 Astrogliosis is exacerbated in AD/MxD females, while microglia coverage is decreased in AD/MxD males. A Hippocampal regions of interest examined: CA1, CA2, CA3, and the dentate gyrus (image created with BioRender.com). B Microgliosis in the CA1 region of the hippocampus was gauged through Iba1 immunofluorescence (greater % area covered indicating greater microgliosis). C Astrogliosis was gauged through GFAP immunofluorescence (greater % area covered indicating greater astrogliosis). D Representative images of Iba‑1 immunofluorescence in the CA1 regions. E Representative images of GFAP immunofluorescence in the CA1 regions. F–H Hippocampal expression of markers for microgliosis (Iba‑1: F and CD68: G) and astrogliosis (GFAP: H) normalized to RPL13A expression. Data are presented as mean + SEM, effect of dementia: ^p < 0.05 effect of dementia, ^^^^p < 0.0001 effect of dementia, effect, *p < 0.05 effect of diet, 2‑ way ANOVA, (n = 4–5/group) Gannon et al. Journal of Neuroinflammation (2022) 19:110 Page 10 of 20 Fig. 3 (See legend on previous page.) G annon et al. Journal of Neuroinflammation (2022) 19:110 Page 11 of 20 insoluble Aβ40 and 42 in the hippocampus, and Western of all MxD groups (main effect of MxD, p < 0.0001; Addi - blot for phosphorylated tau in the hippocampus. Soluble tional file 5: Figure S5B, C). No sex differences were Aβ was not assessed, as it is not elevated until 18 months detected. Taken together, these data show that although of age in 3xTg-AD mice [38]. A 3-way ANOVA showed some pathology was equivalent between the sexes, hip- that females had higher levels of Aβ plaques than males pocampal astrogliosis and Aβ pathology were more (p < 0.05; Fig. 4A, B) and higher levels of insoluble Aβ40 severe in AD/MxD females, compared to males. (p < 0.01; Fig. 4C) and 42 (p < 0.05; Fig. 4D) compared to males. Cortical intracellular Aβ was also assessed, Sex‑dependent correlations between cognitive in which a 3-way ANOVA showed that AD/MxD performance and metabolic measures females had higher levels of Aβ-positive cells than males To further examine the relationship between cognitive, (p < 0.0001) and that there was a main effect of MxD to metabolic, and neuropathology measures, we performed increase Aβ (p < 0.01; Additional file 5: Figure S5A). No linear regression analyses and correlation analyses in differences in phosphorylated or total tau were detected males and females separately. In Fig. 5A, B, we examined between AD and MxD groups, regardless of sex or diet correlations between behavioral outputs and measure- (Fig. 4E-H). To validate that the unilateral carotid artery ments of metabolic impairment in males and females occlusion surgery modeled VCID by inducing chronic separately. In both males and females, each metabolic cerebral hypoperfusion, cortical blood flow was meas - measure was strongly correlated with the others (visceral ured using laser speckle contrast imaging at ~ 7 months fat, % weight gain, and AUC; r = 0.7–0.9, p < 0.0001). In of age (4 months post-surgery). As expected, blood flow males, the effects of metabolic impairments on cognitive deficits were found in the right (occluded) hemisphere function were mixed. In males, higher % weight gain and Fig. 4 Female AD/MxD mice have greater Aβ, but not tau, pathology. A Representative image of Aβ plaques. B The number of Aβ plaques/brain section (averaged across 8, 40‑μM ‑thick brain sections between ‑1.46 and ‑3.52 mm from bregma). C Hippocampal insoluble Aβ‑40, measured by ELISA. D Hippocampal insoluble Aβ‑42, measured by ELISA. E Representative Western blot images of tau protein in right (ischemic for MxD) hippocampal isolates. Measurements for F total tau, G pTau S199, and H pTauPHF1 were normalized to GAPDH. Data are presented as mean + SEM, #p < 0.05 effect of sex, ##p < 0.01 effect of sex, 3‑ way ANOVA, (n = 4–5/group) Gannon et al. Journal of Neuroinflammation (2022) 19:110 Page 12 of 20 more severe glucose intolerance (GTT AUC) were both is shown in Fig. 5E, F in which we examined the relation- associated with poorer spatial memory (MWM % time ship between microgliosis in the CA1 region (CA1 Iba1% in the target quadrant; p < 0.05 for each). Additionally in area covered) and glucose intolerance (AUC) separately males, % weight gain was associated with reduced ability in males and females via linear regression and presented to perform activities of daily living (nest score; p < 0.05); the r and p value. Taken together, these data show that however, more severe glucose intolerance showed a sur- more severe glucose intolerance was the only metabolic prising association with better episodic-like memory parameter that was associated with worse neuroinflam - (NORT recognition index; p < 0.05). In females, meta- mation, and that this increase in neuroinflammation bolic impairment showed a consistent association with (microgliosis) occurred in females only. worse cognitive function. In females, higher % weight A summary of findings is presented in Fig. 6. gain or more visceral fat was associated with poorer spa- tial memory (p < 0.01 for weight gain, p < 0.05 for visceral Discussion fat), and more severe glucose intolerance was associated This study set out to address gaps in knowledge regard - with reduced ability to perform activities of daily living ing sex differences in mid-life metabolic risk factors (p < 0.01). In males, the degree of metabolic impairment for dementia – obesity and prediabetes. Recent clini- was not associated with alterations in cerebral blood flow, cal evidence suggests that prediabetes is a risk factor and blood flow was not associated with cognitive func - for cognitive decline and dementia in women, but not tion. In females, metabolic impairment was associated men [17]. How sex and prediabetes interact to influ - with reduced cerebral blood flow (% blood flow in the ence cognitive function and neuropathology in the two right hemisphere; p < 0.05 for % weight gain, visceral fat, most common forms of dementia – AD and MxD (co- and glucose intolerance). Additionally in females, greater morbid VCID + AD pathology) – is unknown. To fill reduction in cerebral blood flow was associated with this gap in knowledge, we combined mouse models of more severe deficits in the ability to perform activities VCID (chronic cerebral hypoperfusion) and AD (3xTg- of daily living (nest building). Taken together, these data AD mice) to model MxD. We used a chronic HF diet show that impairment in metabolic measures was more to model obesity and prediabetes. While HF diet ubiq- consistently associated with reductions in cognitive func- uitously caused metabolic impairments, this was aug- tion in females. mented in AD/MxD females, but not males. AD, MxD, In Fig. 5C, D we examined correlations between neu- and HF diet differentially affected cognition in males ropathological and metabolic outputs and presented our and females. While episodic-like memory deficits were findings in the form of a correlation matrix. In males, observed in AD and MxD mice of both sexes, additional greater cortical Aβ burden was associated with more cognitive impairments were observed in females. AD/ CA2 astrogliosis (p < 0.05), while in females it was asso- MxD females also showed impairments in activities of ciated with less CA2 astrogliosis. In males, more severe daily living, but males did not. When fed a HF diet, AD glucose intolerance was associated with less CA2 astro- and MxD females, but not males, also exhibited deficits gliosis (p < 0.02), and there were no significant associa - in spatial learning. Metabolic impairment was also more tions with microgliosis. In females, more severe glucose consistently associated with reductions in cognitive func- intolerance was associated with more microgliosis in tion in females. Moreover, AD and MxD females had both CA1 (p < 0.01) and CA2 (p < 0.05), but not associated more hippocampal astrogliosis and a greater burden of with astrogliosis. An example of a sex-specific association Aβ than males. Finally, more severe glucose intolerance (See figure on next page.) Fig. 5 Cognitive impairments and pathological outcomes are correlated with metabolic deficits in male and female mice. Using a correlation matrix, we compared relationships between cognitive, metabolic, and pathological factors. A, B Correlations between cognitive and metabolic measures. Visc fat: visceral fat pad weight normalized to body weight (n = 55–58/sex); AUC: area under the curve from the glucose tolerance test, high AUC indicates greater glucose intolerance (n = 54–58/sex); %decrease in blood flow: the percent difference in cerebral blood flow in the temporal region of the cortical brain surface (n = 50–51/sex); MWM % target: % of the time spent in the target quadrant of the probe trial of the MWM test, higher percentage indicates better spatial memory (n = 58/sex); NOR% time: % time spent with the novel object in the testing trial of the NOR test, higher percentage indicates better episodic‑like memory (n = 46–49/sex); ADLs: activities of daily living assessed by score in the nest building test, a lower score indicates worse ADLs (n = 57–58/sex). C, D Correlations between metabolic and pathological measures in the subset of mice designated for IHC. AUC: area under the curve from the glucose tolerance test, high AUC indicates greater glucose intolerance (n = 18–21/ sex); amyloid count: the number of cells in a cortical ROI that were positive for beta‑amyloid (n = 19–20/sex); CA1 GFAP: the % area covered by GFAP staining in the CA1 region of the hippocampus, greater coverage indicates greater astrogliosis in that region (n = 18–19/sex); CA2 GFAP, (n = 17–19/ sex); CA1 Iba2: the % area covered by Iba1 staining in the CA1 region of the hippocampus, greater coverage indicates greater astrogliosis (n = 17–18/sex) CA2 Iba1, (n = 18/sex). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, significant correlation; Pearson r values are presented. Yellow: positive correlation, Blue: negative correlation. E, F Linear regression of CA1 Iba1% area covered and glucose tolerance test from the AUC (males: n = 19; females n = 17). r value and p value are presented G annon et al. Journal of Neuroinflammation (2022) 19:110 Page 13 of 20 Fig. 5 (See legend on previous page.) Gannon et al. Journal of Neuroinflammation (2022) 19:110 Page 14 of 20 33]. In line with our prior findings, in the current study, the diet was initiated in young adulthood and thus WT males and females showed similar levels of metabolic impairment. However, AD/MxD females developed greater metabolic impairments, including increased weight gain and more severe glucose intolerance, than males in response to HF diet. In a subset of mice from the current study (WT and AD groups with the sham surgery), we previously reported that 3xTg-AD females did not display increased plasma levels of other diabe- tes markers (e.g., insulin, GIP, GLP-1, PAI-1, resistin) or peripheral inflammation [29]. This enhanced meta - bolic effect observed in AD/MxD females in the cur - rent study is reminiscent of our findings in middle-aged mice in which females are more adversely affected than males. One possibility is that the AD and MxD females might have displayed a form of accelerated aging of their metabolic system and/or brain regulation of metabolic function. We have previously reported this female-spe- cific interaction between the effects of a HF diet and the 3xTg-AD genotype that worsens metabolic impairment and examined potential underlying mechanisms [29]. In a subset of mice from the current study (WT and AD groups with the sham surgery), we reported increased food intake, reduced activity levels, and markedly increased hypothalamic expression of GFAP and IL-1β, as well as GFAP labeling in several hypothalamic nuclei that regulate energy balance in the HF-fed 3xTg-AD females [29]. Thus, neuroinflammation in the hypothala - mus likely contributes to the observed sex difference in Fig. 6 Summary of our major findings. We demonstrate relative accumulation of cognitive impairments and neuropathology of metabolic function in 3xTg-AD mice. A limitation in our female male (left, blue) and (right, pink) mice along a center scale interpretation of these data is in the generalizability of with the top of the scale indicating less impairment and pathology using a diet composed of 60% fat from lard (saturated fat). and the bottom of the scale representing greater impairment and Diets of different fat composition or combinations with pathology sugar (a Western diet) may have different consequences. While we were unable to address changes in blood pres- sure in this study, others have demonstrated that high-fat was associated with worse microgliosis in females only. diet in wild-type mice and in the 5xFAD mouse model of Taken together, the data demonstrate heightened suscep- AD does not increase blood pressure [39]. Overall, our tibility of females to the negative metabolic, cognitive, results show that a chronic 60% fat diet caused greater and neuropathological effects of AD, MxD, and HF diet. metabolic impairment in AD/MxD females compared to These data, along with recent clinical findings [17], sug - males. gest that prediabetes might contribute to multiple forms of dementia in women. Cognitive findings Although HF diet elicited some cognitive deficits in Metabolic findings both sexes, HF-fed females displayed a wider array of Here, we found that AD and MxD females had more cognitive impairments, even in WT mice (summarized adverse metabolic responses to chronic HF diet. We have in Fig. 6). While numerous studies have shown that HF previously shown that sex differences in response to HF diet exacerbates cognitive impairment in AD [20–23, diet are age sensitive. If the diet is initiated in juvenile 25], few have examined sex differences. Here, we report mice, males show greater metabolic impairments [33]. that HF diet caused sex-specific cognitive deficits in This sex difference is eliminated if the diet is initiated in spatial learning. AD males, regardless of diet, did not adulthood [33], and reversed if initiated in middle age – show impairment in spatial learning; however, AD with females showing greater metabolic impairment [19, G annon et al. Journal of Neuroinflammation (2022) 19:110 Page 15 of 20 Neuropathology findings females showed an impairment in spatial learning only In assessing underlying neuropathology, we found that when fed a HF diet. In both males and females, addi- astrogliosis and Aβ accumulation were more severe in tive effects of HF diet were observed in spatial memory. AD/MxD females, compared to males. First, we assessed In males, LF-fed AD mice did not show spatial memory markers of neuroinflammation. Female AD/MxD mice deficits; however, with a combination of HF diet and had greater astrogliosis than males in several regions AD these impairments emerged. In females, even WT of the hippocampus. We, and others, have previously mice showed spatial memory impairment in response reported increased astrogliosis in 3xTg-AD mice [29, to HF diet (as did AD mice). Overall, we found that 40] and in cerebral hypoperfusion mouse models of poorer metabolic measures were also more consistently VCID [32, 52, 53]. In comparison to our 7-month-old associated with poorer cognitive function in females, mice, greater microglia activation has been reported in compared to males. In addition to the sex differences 12-month-old 3xTg-AD females compared to males [54], observed with HF diet, we also observed a wider array and more Iba1 + cells in the subiculum in 18-month- of cognitive impairment in LF-fed AD females com- old 3xTg-AD females compared to males [55]; thus, pared to LF-fed males. In support of the current data, sex differences may have emerged at later time points. prior studies have demonstrated exacerbation of cog- Although there were no sex differences in the extent of nitive deficits in 3xTg-AD mice by a HF diet [20– 25] microgliosis, we did observe sex differences in the rela - and greater cognitive impairments in AD females tionship between microgliosis and metabolic markers. compared to males [40–45]. However, there have been In females, but not males, more severe glucose intoler- some reports of greater deficits in males specifically in ance was associated with more severe microgliosis in the regard to working memory [46] and fear conditioning CA1 region of the hippocampus; mechanistic links driv- [47]. Greater female susceptibility to negative cogni- ing this outcome require further investigation. In addi- tive effects of a HF diet is in line with our prior work tion to neuroinflammation, we assessed other classic AD demonstrating that HF diet also causes a wider range neuropathology, including Aβ and phosphorylated tau. of cognitive deficits in WT middle-aged females [19]. While we observed no group differences in hippocampal Taken together, our data suggest that females may be phosphorylated tau levels, recent work by the LaFerla lab more adversely cognitively affected by metabolic dis - has demonstrated sex differences in hippocampal tau in ease which could contribute to cognitive deficits during the 3xTg-AD mouse[55]. Overall, we found that females normal aging or dementia. had greater levels of amyloid pathology both in terms In both sexes, MxD elicited a wider array of cognitive of Aβ plaque counts as well as in Aβ 40 and 42 levels in deficits beyond what was observed in the AD model, with the hippocampus with no added effects of diet or MxD. females being most impacted. Among LF-fed mice, spa- Others have found increased Aβ levels in female 3xTg- tial learning deficits emerged in MxD females that were AD mice [21, 40, 54–58] as well as increased Aβ-positive not observed in the AD females or MxD males. Among cell counts in the dorsal hippocampus [44]. Others have LF-fed males, spatial memory deficits emerged with MxD found no hypoperfusion-induced elevation in Aβ lev- that were not observed in AD males but were observed els in 3xTg-AD mice [51]; however, this was after only in AD/MxD females. Due to floor effects, we were unable 2 months of hypoperfusion suggesting that changes we to detect effects of MxD in females for some tests or to observed may have emerged at a later time point. While detect the effect of HF diet in the MxD groups in either we did not observe an effect of diet on Aβ pathology, oth - sex for most tests. However, it is important to note that ers have found that HF diet increases Aβ in 3xTg-AD MxD females were impaired in all four cognitive tests, mice [21, 24, 25, 59], with some studies showing that this while MxD males had preserved spatial learning and abil- effect is specific to females [57, 58]. This may be due to ity to perform activities of daily living. Our MxD model increased Aβ production that can occur in metabolic involved a unilateral common carotid artery occlusion disease, which is exacerbated when plaques have formed surgery, which leads to chronic cerebral hypoperfusion [60]. While further sex differences in dementia pathology [31–49] and cognitive deficits even in WT mice [19, 31, may develop with age, increased Aβ and astrogliosis in 50, 32]. While others have examined the pathological females support the heightened sensitivity of females to effects of chronic cerebral hypoperfusion in 3xTg-AD AD/MxD and may underlie the wider array of cognitive mice [51], we are the first to examine sex differences deficits in females. in this model. Our data support that added vascular In addition to AD pathology, we also assessed the pathology in MxD further exacerbates cognitive deficits, degree of chronic cerebral hypoperfusion. We found compared to AD alone, and that females may be more that unilateral carotid artery occlusion surgery (MxD adversely cognitively affected by chronic cerebral hypop - mice) caused deficits in cerebral blood flow in the erfusion in MxD. Gannon et al. Journal of Neuroinflammation (2022) 19:110 Page 16 of 20 cortical surface that persisted 4 months after surgery. female MxD mice are more adversely affected, MxD This is in line with our previous work, and that of oth - may pose a greater cognitive threat to women. ers, documenting prolonged deficits in blood flow in the ipsilateral hemisphere [19, 31–49, 61]. While we Potential mechanisms underlying observed sex differences observed the expected effect of MxD to cause hypop - Sex hormones may influence dementia on many levels, in erfusion, we did not observe any sex or diet differences. both men and women, and their effects must be viewed This is in line with a previous study by Bracko et al. with consideration to age and disease state. Estrogens are showing that HF diet-induced obesity do not cause present in both sexes but are higher in reproductive-age hypoperfusion or capillary stalling in AD[62]. However, women. Estrogen also protects against AD risk factors we did observe that greater metabolic impairment was (diabetes) and VCID risk factors (hypertension, obesity, associated with more severe hypoperfusion in females, diabetes, stroke) in women [15]. It is unknown how estro- but not males. Thus, the degree of metabolic impair - gen would impact AD and VCID when the pathologies ment may be important. Pires et al. have shown that HF overlap in MxD. Generally, estrogen can protect the brain diet causes cerebral vessel remodeling [63]. Blood flow through its vasodilatory, anti-apoptotic, antioxidant, and recovery following unilateral carotid artery occlusion anti-inflammatory actions [15, 64]. Rodent studies have surgery could be affected by the degree of collateral also shown protective effects of estrogen against Aβ [59, blood flow, angiogenesis, and compensation follow - 65, 66]. A limitation of our work is that menopause is not ing surgery. A limitation of the current study is that we taken into consideration. Most women with dementia only examined relative blood flow in the cortical sur - are post-menopausal, and menopause decreases estro- face; there may be differences in blood flow in deeper gen levels. Thus, we are currently assessing the effects brain structures that we were unable to detect. We have of menopause in several mouse models of dementia. previously assessed changes in blood flow using arterial While the evidence for the protective effects of estrogen spin labeling MRI and found that although hippocam- is robust, we have found that AD/MxD female mice are pal blood flow was reduced by the unilateral common more strongly impacted by HF diet. The 3xTg-AD geno - carotid occlusion surgery, it was not further reduced type may diminish the capacity of the brain to produce by diet in this deeper brain structure [61]. Sex differ - estrogen, given that women with AD have lower levels ences in the 3xTg-AD mouse have been noted, includ- of brain aromatase (the enzyme that produces estrogen) ing a greater association between plaques and markers [65, 67] and women with AD have lower brain estrogen of hypoxia [54]. These findings suggest that there could levels [67]. There is evidence that estrogen’s effect may be a stronger connection between vascular dysfunction turn from protective to damaging when acting in the and Aβ pathology in females. Our finding that meta - presence of inflammation or a disease state. For exam - bolic impairment is associated with more severe hypop- ple, estrogen can increase inflammation if administered erfusion in females only prompts further research into in older mice after a long period of estrogen withdrawal the potential of metabolic disease to interfere with [68] or lose effectiveness in ameliorating AD pathology blood flow compensation and recovery in a sex-specific [59]. Additionally, studies examining the impact of hor- manner. mone replacement therapy on dementia risk have flagged There may be additional neuropathologies not diabetic women as a population in which HRT increases addressed in the current study that could have contrib- dementia risk [69]. Diabetes has been shown to dimin- uted to sex differences in cognitive function. For exam - ish and sometimes reverse the protective effect of estro - ple, we have previously shown that HF diet impairs gen in rodent models of ischemia [70, 71]. In this study, adult hippocampal neurogenesis in WT females, but chronic HF diet-induced obesity could have created an not males [18]. This deficit occurred in the dorsal “accelerated aging” effect in females that negated the pro - hippocampus, where neurogenesis supports spatial tective influence of estrogen. learning and memory. Additionally, HF diet can accel- Androgens may also contribute to sex differences erate AD pathology in 3xTg-AD mice through several reported here. In men, age-related androgen decline is mechanisms not assessed in the current study, such as found as a risk factor for developing AD [72]. In male by increasing neuronal cell death, oxidative stress [23], 3xTg-AD mice, gonadectomy (which reduces testoster- and brain atrophy [20]. Future work is needed to find one) increases Aβ and tau pathology but is rescued by mechanistic insights behind the sex differences we and treatment with testosterone [73, 74]. While the effects of others have observed. Given the greater AD pathol- testosterone on MxD specifically are unclear, testosterone ogy in females, the interconnectedness of vascular and has a complicated influence on the cerebral vasculature AD pathology, and our current findings showing that [64, 75] with potentially harmful effects, such as increas - ing vasoconstriction [76–78] and inflammation [79, 80], G annon et al. Journal of Neuroinflammation (2022) 19:110 Page 17 of 20 but decreasing inflammation in disease states [81, 82] in studies assessing the overlap of other midlife risk factors, males. Our lab has previously reviewed the cerebrovas- such as menopause, will be important for the develop- cular actions of androgens and how that relates to meta- ment and utilization of therapeutic strategies for demen- bolic and cardiovascular disease [75]. More research is tia treatment and prevention. needed to determine if testosterone level changes in pre- diabetes affect long-term risk of MxD. Abbreviations AD: Alzheimer’s disease; ADL: Activities of daily living; ANOVA: Analysis of variance; AUC : Area under the curve; Aβ: Amyloid beta; BACE1: Beta‑secretase Clinical relevance 1; CA1–3: Cornu Ammonis 1–3; CD68: Cluster of differentiation 68; GFAP: Glial Given that there are well-documented sex differences fibrillary acidic protein; GTT : Glucose tolerance test; HF: High fat (diet); Hif1‑ in both AD and VCID, sex differences likely play a role alpha: Hypoxia inducible factor 1‑alpha; IBA1: Ionized calcium‑binding adaptor molecule 1; IHC: Immunohistochemistry; IL‑1β: Interleukin‑1beta; LF: Low fat in MxD. Prediabetes research is clinically important (diet); MWM: Morris water maze; MxD: Mixed dementia; NORT: Novel object because the prediabetic population is growing, there is recognition test; SEM: Standard error of the mean; VCID: Vascular contributions accelerating awareness of its connection to dementia, to cognitive impairment and dementia; Visc: Visceral (Fat); WT: Wild type. and, importantly, it is a potentially modifiable dementia risk factor. To incorporate this risk factor into clinical Supplementary Information prevention and treatment of dementia, more research is The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s12974‑ 022‑ 02466‑2. needed particularly in its effects on MxD, which is inad - equately represented in research. Women with diabetes Additional file 1: Figure S1. HF diet increased weight gain and fasting have greater risk of developing cognitive impairments, blood glucose in males and females. A) Body weight was measured and in certain types of dementia experience greater cog- monthly starting just prior to the surgery and onset of diet (“Pre”) and ending at tissue collection (“End”), (n = 15–28/group). B) Fasting blood nitive impairments than men with diabetes [3, 83, 84]. glucose levels were measured at the beginning of the glucose tolerance Metabolic disease in general appears to be a greater test. Data are presented as mean + SEM, ^p < 0.05 effect of dementia, **** dementia risk factor for women [15, 64]. This also extends p < 0.0001 effect of diet, 2‑ way ANOVA, (n = 17–26/group). to prediabetes, which is an all-cause dementia risk factor Additional file 2: Figure S2. HF diet decreased locomotor activity in females and increased center time in MxD males and females. A) General [85, 86]. A 2021 study found that prediabetes is associ- locomotor activity was measured by tracking the distance traveled (in ated with accelerated dementia onset and declining cog- meters) during the open field test. B) Anxiety‑like behavior and disorienta‑ nitive function only in women [17]. Diabetes is more tion were measured using the %time that the mice spend in the center of the testing arena during the open field test. C) Correlation matrix for open aggressively treated than prediabetes; this difference field measures (distance traveled and % time in the center of the arena) may manifest in long-term consequences. For example, a and episodic‑like memory as measured by the NOR recognition index (RI) recent study found a relationship between Aβ burden and for males and females. Pearson r values and p values are presented. Yellow: positive correlation, Blue: negative correlation. (n = 45–58/sex). D) Explora‑ prediabetes but not diabetes [87]. The authors hypothe - tion time during the training trial of the NOR test. The red line marks 2 s of sized that this is due to beneficial effects of diabetes treat - exploration, which was used as the cut‑ off for minimum object explora‑ ment. It is clear that further research is needed to grasp tion to be included in the test. E) MWM visible trial (day 1) pathlength by trial. F) Correlation matrix for average swim speed in the visible trials how prediabetes fits into the overall picture of sex differ - of the MWM (avg. speed visible) and spatial learning (hidden trial) and ences in dementia. spatial memory (probe trial). Pearson r values and p values are presented. Yellow: positive correlation, Blue: negative correlation, (n = 6–13 /group). A‑B and D ‑E) Data are presented as mean + SEM, *p < 0.05 effect of diet, Conclusion ****p < 0.0001 effect of diet, ^p < 0.05 effect of dementia, ^^p < 0.01 effect In summary, using a mouse model of mixed (AD + VCID) of dementia, 2‑ way ANOVA. dementia, AD and MxD females showed a wider array of Additional file 3: Figure S3. Microglia coverage is decreased in AD/MxD cognitive deficits, compared to males. Astrogliosis and males, while astrogliosis is exacerbated in AD/MxD females. Microglia coverage in the CA1 region of the hippocampus was gauged through Aβ pathology were also more severe in AD/MxD females, Iba1 immunofluorescence (larger % area covered indicating greater compared to males. When challenged with a HF diet, AD microgliosis). Iba1 immunoreactivity was used to calculated microglia or MxD females also had increased metabolic impair- coverage as the percent area covered by Iba1. Hippocampal regions of interest examined: CA2 (A), CA3 (B), and the dentate gyrus (C). Astrogliosis ment compared to males. Metabolic impairment was also in multiple regions of the hippocampus was gauged through GFAP immu‑ more consistently associated with reductions in cogni- nofluorescence (greater % area covered indicating greater astrogliosis). tive function in females. More severe glucose intolerance Hippocampal regions of interest examined:CA2 (D), CA3 (E), and the dentate gyrus (F). Data are presented as mean + SEM, ^ p < 0.05 effect was associated with worse microgliosis in females only. of dementia, ^^p < 0.01 effect of dementia, ^^^^p < 0.0001 effect of Here, we demonstrate the importance of considering dementia, 2‑ way ANOVA, (n = 4–5/group). how sex modulates the relationship between risk factors Additional file 4: Figure S4. Hippocampal Iba1 and GFAP immunofluo ‑ and dementia. This work supports the importance of pre - rescence intensity. Immunofluorescence intensity of Iba1 (A) and GFAP (B) was measured. Hippocampal regions of interest examined: CA1, CA2, CA3, diabetes as a risk factor for multiple forms of dementia, and the dentate gyrus. There was a main effect of dementia in Iba1 inten‑ particularly for women, and emphasizes increased cogni- sity in males, but there were no differences in hippocampal GFAP intensity. tive and pathological sensitivity to high-fat diet. Future Gannon et al. Journal of Neuroinflammation (2022) 19:110 Page 18 of 20 References Data are presented as mean + SEM (effect of dementia: ^ p < 0.05 effect of 1. Janson J, Laedtke T, Parisi JE, O’Brien P, Petersen RC, Butler PC. Increased dementia, ^^p < 0.01 effect of dementia, ^^^p < 0.001 effect of dementia, risk of type 2 diabetes in Alzheimer disease. Diabetes. 2004;53(2):474–81. ^^^^p < 0.0001 effect of dementia 2‑ way ANOVA, (n = 4–5/group). 2. Zhang J, Chen C, Hua S, Liao H, Wang M, Xiong Y, et al. An updated meta‑analysis of cohort studies: diabetes and risk of Alzheimer’s disease. 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National Diabetes Statistics Report website. 2020. https:// www. cdc. ^^^^p < 0.0001; 3‑ way ANOVA with Sidak’s multiple comparison test. gov/ diabe tes/ data/ stati stics‑ report/ index. htm. Additional file 6: Table S1. Analysis of sex differences. Results (p values) 7. Whitmer RA, Gunderson EP, Quesenberry CP Jr, Zhou J, Yaffe K. Body mass of 3‑ way ANOVAs examining main effects of sex, diet, dementia, and index in midlife and risk of Alzheimer disease and vascular dementia. Curr interaction effects. Significant results are shown in white, values trending Alzheimer Res. 2007;4(2):103–9. toward significance in light gray, and non‑significant results in dark gray. 8. Pedditizi E, Peters R, Beckett N. The risk of overweight/obesity in mid‑life and late life for the development of dementia: a systematic review and meta‑analysis of longitudinal studies. Age Ageing. 2016;45(1):14–21. Acknowledgements 9. Xu WL, Atti AR, Gatz M, Pedersen NL, Johansson B, Fratiglioni L. Midlife The authors would like to thank Nathan Albert and David Riccio for their assis‑ overweight and obesity increase late‑life dementia risk: a population‑ tance with tissue collection and imaging, Avi Sura for assisting with ELISAs, based twin study. Neurology. 2011;76(18):1568–74. and Melissa Thomas for feeding and weighing mice. 10. 2020 Alzheimer’s disease facts and figures. Alzheimer’s & Dementia. 2020;16(3):391–460. Authors contributions 11. Attems J, Jellinger KA. The overlap between vascular disease and Alzhei‑ KLZ obtained funding for the experiments. LSR, OJG, and KLZ designed the mer’s disease–lessons from pathology. BMC Med. 2014;12:206. experiments. OJG and AES performed the animal work. OJG, LSR, AES, and 12. Brun A, Englund E. A white matter disorder in dementia of the Alzheimer CAG performed the experiments. LSR, OJG, CAG, FM, RDK, AT, RB, and JO type: a pathoanatomical study. Ann Neurol. 1986;19(3):253–62. analyzed the data. OJG prepared the figures. OJG prepared the manuscript. 13. Fernando MS, Ince PG, Function MRCC. Vascular pathologies and LSR and KLZ edited the manuscript. All authors read and approved the final cognition in a population‑based cohort of elderly people. J Neurol Sci. manuscript. 2004;226(12):13–7. 14. Ruitenberg A, Ott A, van Swieten JC, Hofman A, Breteler MM. Inci‑ Funding dence of dementia: does gender make a difference? Neurobiol Aging. This work was funded by the American Heart Association 16SDG27190001 2001;22(4):575–80. (KLZ), American Heart Association 20PRE35080166 (OJG), NINDS/NIA 15. Gannon OJ, Robison LS, Custozzo AJ, Zuloaga KL. Sex differences in risk R01NS110749 (KLZ), NIA U01AG072464 (KLZ), Albany Medical College startup factors for vascular contributions to cognitive impairment & dementia. funds, and NINDS F31 NS115290 (OJG). Neurochem Int. 2019;127:38–55. 16. Liu CL, Lin MY, Hwang SJ, Liu CK, Lee HL, Wu MT. Factors associated with Availability of data and materials type 2 diabetes in patients with vascular dementia: a population‑based The datasets used and/or analyzed during the current study are available from cross‑sectional study. BMC Endocr Disord. 2018;18(1):45. the corresponding author on reasonable request. 17. Sundermann EE, Thomas KR, Bangen KJ, Weigand AJ, Eppig JS, Edmonds EC, et al. Prediabetes is associated with brain hypometabolism and cognitive decline in a sex‑ dependent manner: a longitudinal study of Declarations nondemented older adults. Front Neurol. 2021;12: 551975. 18. Robison LS, Albert NM, Camargo LA, Anderson BM, Salinero AE, Riccio DA, Ethics approval and consent to participate et al. High‑Fat Diet ‑Induced Obesity Causes Sex ‑Specific Deficits in Adult Human data or tissue was not used in this study. All work involving animals Hippocampal Neurogenesis in Mice. Neuro. 2020;7(1):19. were approved by IACUC. 19. Salinero AE, Robison LS, Gannon OJ, Riccio D, Mansour F, Abi‑ Ghanem C, et al. Sex‑specific effects of high‑fat diet on cognitive impairment in a Consent for publication mouse model of VCID. FASEB J. 2020;34(11):15108–22. Not applicable. 20. Rollins CPE, Gallino D, Kong V, Ayranci G, Devenyi GA, Germann J, et al. Contributions of a high‑fat diet to Alzheimer’s disease ‑related decline: A Competing interests longitudinal behavioural and structural neuroimaging study in mouse The authors declare that they have no conflicts of interest. models. Neuroimage Clin. 2019;21: 101606. 21. Barron AM, Rosario ER, Elteriefi R, Pike CJ. Sex ‑specific effects of high fat Author details diet on indices of metabolic syndrome in 3xTg‑AD mice: implications for Department of Neuroscience & Experimental Therapeutics, Albany Medical Alzheimer’s disease. PLoS ONE. 2013;8(10): e78554. College, 47 New Scotland Avenue; MC‑136, Albany, NY 12208, USA. Depar t‑ 22. 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Journal of Neuroinflammation – Springer Journals

Published: May 14, 2022

Keywords: Sex; Vascular; Dementia; Alzheimer’s disease; High-fat diet; Inflammation; Obesity; Cerebral hypoperfusion; Metabolic; Glucose intolerance; Diabetes

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High-fat diet exacerbates cognitive decline in mouse models of Alzheimer's disease and mixed dementia in a sex-dependent manner

High-fat diet exacerbates cognitive decline in mouse models of Alzheimer's disease and mixed dementia in a sex-dependent manner

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High-fat diet exacerbates cognitive decline in mouse models of Alzheimer's disease and mixed dementia in a sex-dependent manner

High-fat diet exacerbates cognitive decline in mouse models of Alzheimer's disease and mixed dementia in a sex-dependent manner

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