Older Women - Does Dimension Matter?

Older Women - Does Dimension Matter?

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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 bb 5-10 points was considered a significant decline in global cognitive functions clinically.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 visibility estimates.

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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 home 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 direct exposures. EPA Air Quality System (AQS) and the output of chemical transport units 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. 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. Portrayal of these covariates and basis for their collection have been granted in the Supplementary Facts. Cox proportional hazard models were used to estimate danger 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 global cognitive incidence and refuse of dementia were analyzed separately. Statistical analyses used SAS System for Windows, Version 9.3 (SAS Institute, Cary, NC, USA). 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, december 2010 or the last date of completing annual cognitive assessment before 31, whichever came first. For explanations of global cognitive decline, dementia cases were excluded if ascertained before subjects lost 8 points on 3MS. We employed time on study as the timescale in the constructed Cox regression models, because simulation studies suggested that such approaches have 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).

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

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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 Files analysis was blinded for genotype and nPM. Mouse button husbandry and types of procedures have been authorized by the College of Southeast Ca Institutional Canine Attention and Make use of Committee. One E3FAD control died during the experiment. Yxperimental logistics limited the exposure study to 20 female mice: 10 per group of E3- and E4FAD, have been arbitrarily allocated to either nPM subjection or management environment for 15 2 or 3 weeks.

Experimental exposures

A nano-scale subfraction of urban PM2.5, designated as nPM8 with well-characterized particle chemical 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 coverage chambers. For timelines of exposures see Supplementary Figure 1.

Tissue collection

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

Oligomeric Aβ ELISA good

Aβ peptides were assayed in brain supernates.15 Cerebral half-cortexes had been homogenized in DEA good 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. Oligomeric Aβ was assayed by MOAB-2 ELISA kit (BEK-2215-1P, Biosensis, Thebarton, SA, Australia).

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Aβ Immunohistochemistry (4G8)

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

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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

Silver staining Bielschowsky technique was used to assess neuropil density.29 Sections were dried at room temperature and briefly has beenhed in distilled water before preimpregnation. Sections were 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 have been 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. Bright-field images of CA1, Dentate and Florida2/3 gyrus area, were analyzed by NIH ImageJ software. Images had been 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. Slides were then placed in 5% sodium thiosulfate solution for 2 min, rinsed 5 × in distilled water, dehydrated, mounted and cleared.

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. Signal had been 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, NUDE OLDER WOMEN TAKING OILED UP SELFIE IN MIRROR Millipore). Supernate protein (20 μg) was electrophoresed on 10% SDS polyacrylamide gels and transferred to polyvinylidene fluoride membranes. After washing, membranes have been probed with secondary antibodies conjugated with IRDye 680 (92632210, rabbit, LI-COR Biosciences, Lincoln, NE, USA) and IRDye 800 (92632210, mouse, LI-COR).

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In vitro nPM exposure and APP/Aß measurements

Mouse neuroblastoma N2a cells expressing Swedish mutant APP (K595In/M596L) (N2a-APP/swe) were generously gifted by Dr Huaxi Xu (Sanford/Burnham Metersedical Research Institute, La Jolla, CA, USA) and tested for mycoplasma contamination before use. Three independent experiments were performed, with three sample replicates each. 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. RIPA buffer cell lysates were probed with 22C11 antibody (1:100, Millipore), which recognizes both sAPPβ and sAPPα. Media were analyzed for Aβ42 by MSD Multiplex ELISA (Meso Scale Discovery, Rockville, MD, USA).

Statistical analyses

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