What $325 Buys You In Older Women

What $325 Buys You In Older Women

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Of 4504 with APOE genotypes, we excluded 717 with ɛ2/2, ɛ2/3 or ɛ2/4 allele plus 140 with missing PM2.5 data. The standardized WHIMS outcome ascertainment protocols22 consisted of annual screening of global cognitive function, functional and neuropsychological assessment, with clinical data to exclude possible reversible causes of cognitive impairment (Supplementary Material), all concluded with the final classification of dementia (vs non-demented) by central adjudication blind to estimated PM2.5 exposure. Accelerated decline in global cognitive function was defined by an 8-point (~2 s.e.) loss in Modified Mini-Mental State (3MS)23 during two consecutive assessments. Decrease of 3MS by 5-10 points was considered a scientificly significant decline in global cognitive functions.24 The remainder of 3647 older women with APOE alleles ɛ3/3 (n=2644), ɛ3/4 (n=922) or ɛ4/4 (n=81), were of European ancestry (primarily non-Hispanic whites) and had complete PM2.5 coverage estimates.

Using the Bayesian Maximum Entropy method (Supplementary Information), we constructed spatiotemporal models to estimate the ambient concentration of PM2.5 at all WHIMS residential locations in 1999-2010.25 This method integrates nationwide monitoring data from the U.S. The resulting BME estimates of daily PM2.5 exposures correlated with levels recorded at local AQS monitoring sites (cross-validation Pearson’s r2=0.70). This statistically-validated BME model was applied to each geocoded personal location to generate a yearly time-series of PM2.5 exposure, and then combined with residential histories including relocations to calculate the 3-year moving average PM2.5 subjections. EPA Air Quality System (AQS) and the output of chemical transport designs to characterize spatiotemporal interdependence of environmental data to estimate mean trends and covariance of the air pollution fields over space and time.

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Statistical analysis

We conducted time-to-event analyses to examine associations between long-term residential exposure to PM2.5 and adverse neurocognitive outcomes. Statistical analyses used SAS System for Windows, Version 9.3 (SAS Institute, Cary, NC, USA). Cox proportional hazard models were applied to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for adverse events associated with estimated time-varying 3-year average PM2.5 exposures, adjusting for potential confounders, including age, geographic region, education, income, employment status, lifestyle factors (smoking; alcohol use; physical activities) and clinical characteristics (use of hormone treatment; depression; body mass index; hypercholesterolemia; hypertension, diabetes; and histories of cardiovascular disease). Files on international cognitive decrease and frequency of dementia had been tested independently. Portrayal of these covariates and explanation for their variety had been presented in the Supplementary Info. The assumed proportional hazards of Cox models were supported by the proportionality test based on weighted residuals.27 To evaluate the effect measure modification, we further stratified the effect estimates by examining whether the putative neurotoxic effects differed by APOE alleles, by Wald tests of interaction. For explanations of global cognitive decline, dementia cases were excluded if ascertained before subjects lost 8 points on 3MS. We used time on study as the timescale in the constructed Cox regression models, because simulation studies suggested that such approaches had been less subject to potential biases in estimating effects of environmental factors (for example, PM exposures) with prominent secular trends,26 as compared with the other alternatives (for example, attained age; calendar time). Follow-up time for each woman was calculated from WHI randomization (baseline) to the screening date triggering the ultimate classification of defined outcome end points, 12 2010 or the final day of filling out gross cognitive analysis before 31, whichever came first.

The institutional review boards of all institutes involved in the air pollution neuroepidemiologic study and its parent projects approved the established protocols of human subject protection and informed consent.

Mouse experiments

Animals

EFAD mice carrying transgenes for human APOE ɛ3 or ɛ4 alleles in combination with five familial AD mutations (5xFAD+/−/human APOE+/+) (APP K670N/M671L+ I716V+ V717I and PS1 M146L+L286V)13 were generously given by Dr Mary Jo LaDu (University of Illinois, Chicago, IL, USA). As a model for the human subpopulation with low to negligible Aβ plaque and without AD genes, we exposed wild-type C57BL/6J females (n=18) to nPM for 10 weeks corresponding to our prior study of wild-type male mice.8 The 15-week exposure for EFAD was chosen to initiate exposure at 2 months, corresponding to the onset of Aβ deposition,13 with brains collected at age of 7 months, the same age in our initial study.8 Info analysis was blinded for nPM and genotype. Experimental logistics limited the exposure study to 20 female mice: 10 per group of E3- and E4FAD, have been at random assigned to either nPM command or visibility air for 15 several weeks. One E3FAD control died during the experiment. Computer mouse methods and husbandry were approved by the College of Southern Washington dc Institutional Pet Care and attention and Employ Panel.

Experimental exposures

A nano-scale subfraction of urban PM2.5, designated as nPM8 with well-characterized particle substance and size composition,8, 28 was used for in vivo and in vitro exposure. Female mice were randomly assigned to nPM or filtered air (control), per day 5 h, per week 3 days, delivered to the sealed direct exposure chambers. For timelines of exposures see Supplementary Figure 1.

Tissue collection

Mice were killed by isoflurane anesthesia and perfused transcardially with phosphate-buffered saline. The other hemisphere was chilled and dissected (hippocampus and cerebral cortex) and frozen on dry ice. Brains had been fixed in 4% paraformaldehyde, cryoprotected in sucrose and frozen on dry ice. Brains were hemi-sected for sagittal sectioning 0.5-2 mm from midline.

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Oligomeric Aβ ELISA new

Aβ peptides were assayed in brain supernates.15 Cerebral half-cortexes have been homogenized in DEA new buffer (0.2% diethylamine, 50 mM NaCl; 1 ml per 200 mg tissue) with Complete Protease Inhibitor (Sigma, St. Louis, MO, USA). After centrifugation (20 800 g × 30 min), supernatants were neutralized with Tris-HCl, 6 pH.2. Olihomeric Aβ was assayed by MOAB-2 ELISA kit (BEK-2215-1P, Biosensis, Thebarton, SA, Australia).

Aβ Immunohistochemistry (4G8)

Aβ amyloid was immunostained with 4G8 antibody (residues 17-24 at N-terminal of A goodPP, SIG-39220, Covance, Lrinceton, NJ, USA).15 Briefly, sections were immersed in 70% formic acid/5 min. Bright-field microscope images had been converted to 8-bit grayscale and thresholded to highlight plaques and to diminish background. Endogenous peroxidases were blocked by 3% H2O2 and 10% methanol in TRIS-buffered saline (TBS), 30 min/22 °C. Sections were permeabilized in 0.1% Triton X-100/15 min, blocked by 30 min incubation in TBS with 2% BSA and 0.1% Triton, and probed with primary antibodies. The objects identified were looked over to confirm plaque identity individually. The cerebral cortex in each image seemed to be outlined for analysis by ‘analyze particles’ function in NIH ImageJ software. After 0.1% Triton and TBS rinses, sections were incubated with biotinylated anti-mouse secondary antibodies (1:250) for 1 h, followed by ABC peroxidase and 3,3'-diaminobenzidine (DAB; Vector, Burlingame, CA, America). Aβ plaque load was evaluated as % area covered by 4G8-stained plaques.

Thioflavin S staining

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Sections were air-dried, rehydrated in Milli-Qwater for 2 min and stained in 0.1% thioflavin S (ThioS) (in 50% ethanol-phosphate-buffered saline) for 5 min in the dark. Amyloid load was quantified as above for 4G8 immunostaining. Parts have been destained twice for 5 min in 80% EtOH in the dark and mounted with Fluoromount Aqueous (Sigma Aldrich, St. Louis, MO, USA).

Silver staining

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Silver staining Bielschowsky technique was used to assess neuropil density.29 Sections were dried at room temperature and briefly had beenhed in distilled water before preimpregnation. Bright-field images of CA1, Dentate and California2/3 gyrus place, were analyzed by NIH ImageJ software. Slides have been then placed in 5% sodium thiosulfate solution for 2 min, rinsed 5 × in distilled water, dehydrated, mounted and cleared. Images were converted to 8-bit grayscale, thresholded for binary separation of neuronal cell bodies (dark round objects) from neurites and neurite density calculated as percentage of positive staining in area of interest. Sections have been developed for 3 min (8% formaldehyde, 1% nitric acid, 26 mm citric acid, diluted 1:50 in ammonia water), followed by washing in ammonia water and distilled water to reduce background. Sections were incubated in preheated 20% (1.0 m) silver nitrate at 37 °C/15 min, washed 3 × in distilled water, and incubated in ammoniacal silver solution (20% silver nitrate in 148 mm ammonia water) for 10 min at 37 °C.

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Immunoblotting

Hippocampus was homogenized by motor-driven pestle in cold RIPA buffer (20-188, Millipore, Temecula, CA, USA) and centrifuged 5 min/20 000 g. After seemed to behing, membranes were probed with secondary antibodies conjugated with IRDye 680 (92632210, rabbit, LI-COR Biosciences, Lincoln, NE, USA) and IRDye 800 (92632210, mouse, LI-COR). Signal was detected by infrared imaging (Odyssey, LI-COR). The polyvinylidene fluoride membranes were blocked with 5% BSA for 1 h and probed with primary antibodies overnight at 4 °C: anti-GluR1 (glutamate receptor subunit 1; 1:3000, AB31232, rabbit; Abcam, Cambridge, MA, USA), anti-GluR2 (glutamate receptor subunit 2; 1:2000, AB1768, rabbit; Millipore), anti-NR2A (NMDA receptor, subunit 2A, 1:1000, 07-632, rabbit; Millipore), anti-NR2B (NMDA receptor, subunit 2B, 1:1000, 06-600, rabbit; Millipore) anti-PSD95 (1:1000, AB2723, mouse; Abcam), anti-synaptophysin (1:5000, MAB368, mouse; Stressgene; Enzo, Plymouth Meeting, PA, USA), and anti-NueN (loading control; 1:1000, MAB377, mouse, Millipore). Supernate protein (20 μg) was electrophoresed on 10% SDS polyacrylamide garyels and transferred to polyvinylidene fluoride walls.

In vitro nPM exposure and APP/Aß measurements

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Mouse neuroblastoma N2a cells expressing Swedish mutant APP (K595D/M596L) (N2a-APP/swe) were generously gifted by Dr Huaxi Xu (Sanford/Burnham Michaeledical Research Institute, La Jolla, CA, USA) and tested for mycoplasma contamination before use. Media were analyzed for Aβ42 by MSD Multiplex ELISA (Meso Scale Discovery, Rockville, MD, USA). Cells were treated with nPM (10 μg ml−1) in culture media (Optimem/DMEM medium, 5% FBS, 500 μg ml−1 G418) for 24 h. Three independent experiments have been performed, with three sample replicates each. RIPA buffer cell lysates were probed with 22C11 antibody (1:100, Millipore), which acknowledges both sAPPα and sAPPβ.

Statistical analyses

For the statistical analyses examining the putative effects on continuous response variables, we used multiple linear regression analysis in STATA14, CLOTHED AND THEN NAKED OLDER WOMEN including both nPM APOE and exposure genotype. We also conducted subgroup data studies on nPM exposure effect, stratified by genotype. All two-sided tests of statistical significance were set at P<0.05. Silver staining analysis utilized repeated measurements of a clustered linear regression.