Prolonged Early Exposure to a High-Fat Diet Augments the Adverse Effects on Neurobehavior and Hippocampal Neuroplasticity
2023; Elsevier BV; Volume: 193; Issue: 10 Linguagem: Inglês
10.1016/j.ajpath.2023.06.005
ISSN1525-2191
AutoresXiuting Yao, Jingyi Zhao, Yang Yuan, Conghui Wang, Zhehao Yu, Zhihui Huang, Chen Chen, Chenxi Yang, Jiayi Ren, Yu Ma, Yi Rong, Yi Huang, Yue Ming, Lijie Liu,
Tópico(s)Adipose Tissue and Metabolism
ResumoHigh-fat diet (HFD) consumption may contribute to the high prevalence of cognitive–emotional issues in modern society. Mice fed a HFD for a prolonged period develop more severe neurobehavioral disturbances when first exposed to a HFD in the juvenile period than in adulthood, suggesting an initial age-related difference in the detrimental effects of long-term HFD feeding. However, the mechanism underlying this difference remains unclear. Here, male C57BL/6J mice initially aged 4 (IA4W) or 8 (IA8W) weeks were fed a control diet (CD) or HFD for 6 months and then subjected to metabolic, neurobehavioral, and histomorphological examinations. Although the detrimental effects of long-term HFD feeding on metabolism and neurobehavior were observed in mice of both ages, IA4W-HFD mice showed significant cognitive inflexibility accompanied by significantly greater levels of anxiety-like behavior than age-matched controls. Hippocampal neuroplasticity and microglial phenotype were altered by HFD feeding, whereas significant morphological alterations were more frequently observed in IA4W-HFD mice than in IA8W-HFD mice. Additionally, significantly increased hippocampal microglial engulfment of postsynaptic proteins and elevated phospho-insulin-receptor levels were observed in IA4W-HFD, but not in IA8W-HFD, mice. These findings suggest that aberrant microglia-related histomorphological changes in the hippocampus underlie the exacerbated detrimental neurobehavioral effects of prolonged early HFD exposure and indicate that enhanced insulin signaling might drive microglial dysfunction after prolonged early HFD exposure. High-fat diet (HFD) consumption may contribute to the high prevalence of cognitive–emotional issues in modern society. Mice fed a HFD for a prolonged period develop more severe neurobehavioral disturbances when first exposed to a HFD in the juvenile period than in adulthood, suggesting an initial age-related difference in the detrimental effects of long-term HFD feeding. However, the mechanism underlying this difference remains unclear. Here, male C57BL/6J mice initially aged 4 (IA4W) or 8 (IA8W) weeks were fed a control diet (CD) or HFD for 6 months and then subjected to metabolic, neurobehavioral, and histomorphological examinations. Although the detrimental effects of long-term HFD feeding on metabolism and neurobehavior were observed in mice of both ages, IA4W-HFD mice showed significant cognitive inflexibility accompanied by significantly greater levels of anxiety-like behavior than age-matched controls. Hippocampal neuroplasticity and microglial phenotype were altered by HFD feeding, whereas significant morphological alterations were more frequently observed in IA4W-HFD mice than in IA8W-HFD mice. Additionally, significantly increased hippocampal microglial engulfment of postsynaptic proteins and elevated phospho-insulin-receptor levels were observed in IA4W-HFD, but not in IA8W-HFD, mice. These findings suggest that aberrant microglia-related histomorphological changes in the hippocampus underlie the exacerbated detrimental neurobehavioral effects of prolonged early HFD exposure and indicate that enhanced insulin signaling might drive microglial dysfunction after prolonged early HFD exposure. Excessive high-fat diet (HFD) consumption is associated with a range of cognitive dysfunctions1Holloway C.J. Cochlin L.E. Emmanuel Y. Murray A. Codreanu I. Edwards L.M. Szmigielski C. Tyler D.J. Knight N.S. Saxby B.K. Lambert B. Thompson C. Neubauer S. Clarke K. 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The present study evaluated the behavioral effects of prolonged HFD consumption starting from the juvenile or adult stage in mice and investigated the underlying mechanism through a series of studies focused on metabolic parameters, neuronal morphology, microglial phenotype, and microglial insulin signaling in the hippocampus. All experiments were approved by the University Ethics Committee for Laboratory Animals of Southeast University, China (Approval number 2021030201). The experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals.23Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research CouncilGuide for the Care and Use of Laboratory Animals.Eighth Edition. National Academies Press, Washington, DC2011Crossref Google Scholar Only male C57BL/6 mice purchased from Nanjing Biomedical Research Institute of Nanjing University (SCXK (SU)2015-0001; Nanjing, China) were included for all experiments to avoid sex-dependent differences. Mice at the initial age of 4 weeks (IA4W, juvenile) and 8 weeks (IA8W, adult) were randomly assigned to either the control diet (CD, 3.57 kcal/g energy with 10% fat, TP23403; Trophic Animal Feed High-Tech Co., Ltd. Nantong, China) or a HFD (5.0 kcal/g energy with 60% fat, TP23400; Trophic Animal Feed High-Tech Co., Ltd.) group (termed IA4W-CD, IA4W-HFD, IA8W-CD, and IA8W-HFD, respectively) and were fed for 6 months (Figure 1). The age-matched mice were housed in cages (two to four per cage) under constant temperature (22°C ± 2°C) and humidity (55% ± 5%) on a 12-hour light/dark cycle (lights on 07: 00 to 19:00), with food and water available ad libitum. The dietary treatment periods covered the period from the juvenile stage to young adulthood (IA4W cohort) or most of young adulthood (IA8W cohort).24Dutta S. Sengupta P. Men and mice: relating their ages.Life Sci. 2016; 152: 244-248Crossref PubMed Scopus (883) Google Scholar,25Brust V. Schindler P.M. Lewejohann L. Lifetime development of behavioural phenotype in the house mouse (Mus musculus).Front Zool. 2015; 12: S17Crossref PubMed Scopus (133) Google Scholar The Morris water maze (MWM) test was conducted in a circular pool (120 cm in diameter and 50 cm in depth) filled with opaque white water at 21°C to 23°C, with various prominent visual cues surrounding it. The test included four periods: initial spatial training, the first probe test, spatial reversal training, and the second probe test. Initial spatial training and the first probe test: After 2 days of cued learning, mice were subjected to 3-day spatial acquisition training (four trials with a 5-minute interval per day) in which a circular hidden platform (10 cm in diameter) was submerged 1 cm under the surface of the water, followed by a probe test 24 hours later for mice with no platform. During the spatial acquisition phase, mice were placed at one of four different starting positions (north, south, east, and west) at the start of each trial and allowed to swim to search for the platform in 60 seconds. If the mice failed to reach the platform in this time, they were gently guided to the platform and left there for 20 seconds. The escape latency and time spent in the target or other quadrants were recorded to evaluate learning ability and spatial memory, respectively. Spatial reversal training and the second probe test: During the reversal learning phase, the hidden platform was relocated to the quadrant opposite the initial target location. Reversal learning entailed 3 additional days with four training trials per day, as in the initial spatial training phase. Finally, a reversal probe trial was performed 24 hours later. The open field test (OFT) is now one of the most popular procedures to evaluate anxiety-like behaviors.26Prut L. Belzung C. The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review.Eur J Pharmacol. 2003; 463: 3-33Crossref PubMed Scopus (2263) Google Scholar The test was performed in a nontransparent white box (40 × 40 × 40 cm), with mice gently placed individually in the corner of the box and allowed to freely explore for 5 minutes. Test animal movement was recorded by a video camera coupled with an automated tracking system (Visutrack 3.0; Xinruan Information Technology Company, Shanghai, China), which allowed analysis of the total distance traveled, and duration in and the number of entries into the center zone. The elevated zero maze (EZM) is also widely employed to assess anxious-like behavior.27Tucker L.B. McCabe J.T. Behavior of male and female C57BL/6J mice is more consistent with repeated trials in the elevated zero maze than in the elevated plus maze.Front Behav Neurosci. 2017; 11: 13Crossref PubMed Scopus (78) Google Scholar The instrument, 100 cm above the ground, consists of a white elevated zero maze (45 cm in diameter and 6 cm in width) with two open arms and two closed arms. The mice were placed individually at a random boundary between an open and closed arm, and allowed to explore for 5 minutes while a video tracking system recorded their behavior. Several variables were assessed, including the duration the mouse spent in the open arms. After each experiment, the bottom of the apparatus was cleaned with 70% ethanol and then water to prevent the residual odor of the previous mouse from interfering with this experiment. The sucrose preference test (SPT) is a method used to assess depression-like behavior by evaluating the response to rewards.28Liu M.-Y. Yin C.-Y. Zhu L.-J. Zhu X.-H. Xu C. Luo C.-X. Chen H. Zhu D.-Y. Zhou Q.-G. Sucrose preference test for measurement of stress-induced anhedonia in mice.Nat Protoc. 2018; 13: 1686-1698Crossref PubMed Scopus (399) Google Scholar Two identical drinking bottles, one containing 1% sucrose and the other containing tap water, were placed in all mouse cages for 24 hours. The positions of the two bottles were interchanged every 12 hours to avoid the effects of position preference. At the end of the test, the weight of the two bottles was measured, and the sucrose preference rate was calculated, which was positively correlated with the pleasure of mice. The forced swim test (FST) is a highly reliable experiment for evaluating depression-like behavior. During the test, an animal was placed individually in a transparent glass container (13 cm in diameter) filled with 13-cm–deep water (24°C ± 1°C) for 6 minutes, and its behavior was automatically recorded with a video camera. The immobility time, which reflects the degree of depression, was defined as the time spent floating with the absence of any movement or only small limb movements to keep their head above water. Tissue collections were performed between 09:00 and 11:00 AM to avoid potential effects of the circadian rhythm. On the day after the behavioral test, the mice were deeply anesthetized with pentobarbital sodium (100 mg/kg, i.p.). The epididymal fat pads, which are masses of white adipose tissue, were then carefully collected and weighed. After collecting blood by cardiac puncture, the animals used in immunohistochemical experiments were transcardially perfused with 20 mL of 0.1 mol/L phosphate-buffered saline (PBS) followed by 50 mL of ice-cold 4% paraformaldehyde in PBS. The brains were quickly excised and then fixed in 4% paraformaldehyde for 6 to 8 hours at 4°C, followed by cryoprotection in 30% sucrose in PBS until they sank. The whole brains were embedded in an OCT compound and stored at −80°C. For Golgi staining, the harvested brains were immersed in a mixture of solutions provided by the FD Rapid GolgiStain Kit (Cat. #: PK401; FD NeuroTechnologies, Inc., Columbia, MD) and processed according to the manufacturer's protocol. For fasting blood glucose (FBG) measurement, blood was collected from the tail vein of 4-hour-fasted mice. Blood glucose was measured using a glucometer (Bayer HealthCare LLC, Whippany, NJ). For fasting blood insulin (FBI) measurement, blood of fasted mice was allowed to coagulate at room temperature for 1 hour. Serum samples were obtained after centrifugation and frozen at −80°C until later processing. Circulating levels of insulin were measured using a commercial enzyme-linked immunoassay kit (EZRMI-13 K; Millipore, Billerica, MA) following the manufacturer's protocol. Homeostatic model assessment of insulin resistance (HOMA-IR) was calculated with fasting blood glucose and blood insulin. HOMA-IR = FBI (μU/mL) × FBG (mmol/L)/22.5. Whole brains taken from rapidly decapitated mice were immersed in a Golgi-Cox impregnation solution (FD Rapid GolgiStain kit, PK401; FD NeuroTechnologies) and kept away from light for 3 weeks. Sagittal sections (150 μm) of the brain were cut on a vibratome and mounted on gelatin-coated slides. After drying in a humid chamber, the sections were placed in RapidGolgi Staining solution for 10 minutes, dehydrated with graded ethanol, cleared with xylene, and then coverslipped. Images of both the dorsal and ventral hippocampus were captured by light microscopy (Olympus BX53, Tokyo, Japan). All microscopic analyses were blindly conducted using ImageJ software version 1.52a (NIH, Bethesda, MD; http://imagej.nih.gov/ij). For analysis of neuron complexity, neurons were selected randomly and neuronal processes were reconstructed by semimanual tracing using the plugin NeuronJ version 1.4.3 (https://imagescience.org/meijering/software/neuronj). The total dendritic length and number of dendritic intersections with concentric circles positioned at radial intervals of 10 μm were calculated to analyze the complexity of neurons using Sholl analysis. The average dendritic spine density was expressed as the number of spines/10 μm of dendrite. For every cell, dendritic segments selected for analysis met the following criteria: on secondary or tertiary dendrites and ≥20 μm away from the beginning of the branches. The neurons were randomly selected based on the numbers of neurons randomly assigned by using ImageJ. Briefly, the authors created selections of somas of all granule or pyramidal neurons in the studied area by using the “Selection Brush Tool” in ImageJ and then made a particle analysis to obtain the number of neurons. The random number function (RAND) in Microsoft Excel (Richmond, WA) was used to generate the number of neurons to be analyzed except where the processes or dendrite spines of that neuron were hard to discern; in that case, the next randomly picked cell was assessed. Up to four cells within each target region were analyzed for each section. In total, 10 mice were used for the analysis of neuron complexity and dendrite spine density (two to three mice per group). Every fifth sagittal section (40-μm thick) of each hemisphere (2.40 to 2.88 mm lateral to the midline) containing the hippocampus was chosen for immunofluorescence staining. As described in previous reports,11Yao X. Yang C. Wang C. Li H. Zhao J. Kang X. Liu Z. Chen L. Chen X. Pu T. Li Q. Liu L. High-fat diet consumption in adolescence induces emotional behavior alterations and hippocampal neurogenesis deficits accompanied by excessive microglial activation.Int J Mol Sci. 2022; 23: 8316Crossref PubMed Scopus (9) Google Scholar,12Zhuang H. Yao X. Li H. Li Q. Yang C. Wang C. Xu D. Xiao Y. Gao Y. Gao J. Bi M. Liu R. Teng G. Liu L. Long-term high-fat diet consumption by mice throughout adulthood induces neurobehavioral alterations and hippocampal neuronal remodeling accompanied by augmented microglial lipid accumulation.Brain Behav Immun. 2022; 100: 155-171Crossref PubMed Scopus (21) Google Scholar free-floating sections were rinsed three times for 10 minutes in 0.1 mol/L PBS solution. After washing, the sections were blocked with blocking solution for 2 hours and then incubated with primary antibodies diluted in blocking serum overnight at 4°C. Subsequently, the sections were washed six times for 10 minutes in 0.1 mol/L PBS solution followed by incubation for 2 hours with secondary antibodies diluted in blocking serum in a wet chamber away from light. The following antibodies were used: rabbit anti-ionized calcium binding adaptor molecule 1 (Iba1, for microglia, 1:1000, 019-19741; Wako, Osaka, Japan), rat anti-CD68 (1:1500, MCA1957; Bio-Rad, Oxford, UK), rabbit anti-postsynaptic density 95 (PSD95, 1:1000, ab18258; Abcam, Cambridge, UK), goat anti-Iba1 (for microglia, 1:600, 011-27991; Wako), rabbit anti-IRβ pT1150/1151 (1:400, #3024; Cell Signaling Technology Inc., Beverly, MA), Alexa Fluor 568–conjugated goat anti-rabbit IgG (1:1000, ab175471; Abcam), Alexa Fluor 647–conjugated donkey anti-rabbit IgG (1:1000, A31573; Invitrogen, Waltham, MA), Alexa Fluor 488–conjugated goat anti-rat IgG (1:1000, A11006; Invitrogen), Alexa Fluor 488–conjugated donkey anti-goat IgG (1:1000, ab150129; Abcam), Alexa Fluor 594–conjugated donkey anti-rat IgG (1:1000, ab150156; Abcam), Alexa Fluor 647–conjugated donkey anti-goat IgG (1:1000, ab150135; Abcam), and Alexa Fluor 594–conjugated donkey anti-rabbit IgG (1:1000, ab150064; Abcam). All slides were counterstained with DAPI (1:600, C1027; Beyotime, Shanghai, China) in PBS solution at room temperature for 15 minutes to visualize cell nuclei. Images were captured using a fluorescence microscope (Olympus BX53, Olympus, Tokyo, Japan) or confocal laser scanning microscope (FV1000, FV3000, Olympus) under 20 × and 40 × objectives. All confocal stacks were acquired at a resolution of 1024 × 1024 pixels with a Z-step of 1 μm. Two-dimensional projections of the image stacks were created and analyzed by researchers (J.R., Y.Ma., Y.R., Y.H., and Y.Mi.) blinded to the experimental design using ImageJ software. For the quantitative analysis of microglia, all the cells with complete and clearly visible cellular morphology were selected. The microglial density, average microglial soma area, average territory area of microglia (defined as the area outlined by the outermost points of the dendritic processes of an Iba1+ cell), and percentage of CD68+ microglia (defined as the proportion of CD68+Iba1+ cells among all Iba1+ cells) were analyzed as previously reported.29Verdonk F. Roux P. Flamant P. Fiette L. Bozza F.A. Simard S. Lemaire M. Plaud B. Shorte S.L. Sharshar T. Chrétien F. Danckaert A. Phenotypic clustering: a novel method for microglial morphology analysis.J Neuroinfamm. 2016; 13: 153Crossref PubMed Scopus (72) Google Scholar,30Zhuang H. Yang J. Huang Z. Liu H. Li X. Zhang H. Wang J. Yu S. Liu K. Liu R. Bi M. Wang J. Salvi R.J. Hu B. Teng G. Liu L. 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For quantification of PSD95 engulfment by microglia, only PSD95+ puncta within CD68+Iba1+ structures were considered to ensure that only puncta entirely phagocytosed by microglia were included in the analysis.31Filipello F. Morini R. Corradini I. Zerbi V. Canzi A. Michalski B. Erreni M. Markicevic M. Starvaggi-Cucuzza C. Otero K. Piccio L. Cignarella F. Perrucci F. Tamborini M. Genua M. Rajendran L. Menna E. Vetrano S. Fahnestock M. Paolicelli R.C. Matteoli M. The microglial innate immune receptor TREM2 is required for synapse elimination and normal brain connectivity.Immunity. 2018; 48: 979-991.e8Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar Therefore, a new channel for engulfed PSD95 within CD68+ structures of microglia was created by using the mask function. Quantification of the number and volumes of engulfed PSD95 was performed, and the parameters were exported as separate files for data analysis. For quantitative evaluation of phospho-insulin receptor (p-IR) within hippocampal microglia, Iba1+ microglia and p-IR+ puncta within microglia were reconstructed three-dimensionally using the surface creation wiza
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