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Page 348 vonderEmbse et al. Neuroimmunol Neuroinflammation 2020;7:345-59 I http://dx.doi.org/10.20517/2347-8659.2019.29

neurimmiRs known to regulate microglial quiescence and neuronal maturation and sprouting, thereby
promoting lifelong neuroimmune imbalances associated with increased vulnerability to AD. By including
indo exposure, we aimed to discriminate sex-biased effects derived from sexually dimorphic microglial
signaling during this postnatal period related to PGE2. Here, we confirm the hypothesis that postnatal
Pb exposure in a genetic mouse model of AD significantly alters early epigenetic regulation related to
the neuroimmune interface in a sexually dimorphic manner, highlighting the exacerbating effect of the
GxE model in the formation of a developmental phenotype for later-life female-biased susceptibility to
neurodegeneration.

METHODS
Animal handling
All handling and experimental manipulations were carried out in accordance with procedures approved by
the East Carolina University Institutional Animal Care and Use Committee (IACUC). Pregnant wildtype
(WT) and transgenic dams [3xTgAD; B6; 129Psen1 tm1Mpm Tg (APPSwe, tauP301L) 1Lfa/Mmjax] were
obtained from the seed colony in the ECU Department of Comparative Medicine and kept on a 12:12 hour
light/dark cycle, with access to food and water ad libitum. Litters were culled to eight after birth (postnatal
day, PND 1), if needed, and monitored for overt signs of toxicity.

Dosing and tissue preparation
Exposure to lead acetate was based on our previous findings that an identical exposure concentration
and timing in the 3xTgAD mouse model resulted in significantly altered microglia and exacerbated AD
[4,5]
pathology in adult females . Indomethacin concentration (1 mg/kg/day) was chosen to recapitulate
previous reports of efficacious concentrations for PGE2 inhibition in rodent microglia [35,44,45] . Dosing
solutions were dissolved in sterile water and prepared fresh weekly for lead acetate (100 ppm) and
indomethacin (1 mg/kg/day) (Sigma-Aldrich, Milwaukee, WI, USA). From PND 5-9 neonates were dosed
once per day (10 µL/g body weight/day) with a vehicle, indomethacin (1 mg/kg), lead acetate (100 ppm), or
[46]
indomethacin followed by lead acetate 30 min later using a modified gavage technique . One mouse per
sex, litter, and treatment group were randomly assigned and euthanized at PND 10, 15, or 21, with n = 3
mice/sex/age/treatment/strain for each assay. As per ethical use protocol, animals were euthanized with
inhaled isofluorane followed by immediate decapitation. The brain was then carefully removed and placed
in ice-cold PBS. For histochemical analysis, the left hippocampus was dissected and fixed for 24 h in 10%
neutral buffered formalin followed by 70% ethyl alcohol before paraffin fixation. The right hemisphere sans
cerebellum was flash-frozen whole and stored at -80 °C.

Immunohistochemistry
Formalin-fixed, paraffin-embedded hippocampi were sliced on a rotary microtome at 10 μm and mounted
on Superfrost Plus slides (Azer Scientific, Germany). Briefly, slides were dewaxed in Histo-Clear II
(Electron Microscopy Sciences, Halfield, PA, USA), followed by washes in 100% and 95% ethyl alcohol
and phosphate-buffered saline (PBS). Antigen unmasking was accomplished using a heat-mediated
citrate buffer, followed by incubation in 0.3% hydrogen peroxide for 30 min. All subsequent staining
was performed using Sequenza-Coverplate racks (Thermo Scientific, Waldorf, Germany). Sections were
permeabilized with PBS with Tween-20 and blocked with diluted normal serum (ABC Vectastain; Vector
Laboratories, Burlingame, CA, USA). Slides were then incubated with anti-DAP12 primary antibody
(4 µg/mL; unconjugated rabbit polyclonal IgG, Cat#orb156537, Biorbyt, Cambridge, UK), for 60 min at
room temperature or overnight at 4 °C. Indirect labeling was then performed using biotinylated anti-rabbit
IgG secondary antibodies and reagents from a high-sensitivity avidin-biotin kit with peroxidase-based
detection (Vectastain Elite ABC-HRP kit, Peroxidase (Rabbit IgG), Cat# PK-6101; Vector Laboratories).
Slides were visualized with diaminobenzidine (DAB) (DAB Substrate Kit, Peroxidase (HRP), Cat# SK-4100;
Vector Laboratories), with a consistent DAB development time of 60 s for all slides, and counterstained