The Institutional Animal Care and Use Committee at the University of California Davis
approved all animal procedures, which were performed at the California National Primate
Research Center (CNPRC), University of California, Davis. Time-mated pregnant rhesus
macaques were sedated with ketamine at approximately day 130 of gestation (term is
165 days) for ultrasound exam and IA injection of either LPS (1 mg, E. coli O55:B5, Sigma Aldrich, Saint Louis, MO) diluted in 1 mL of sterile saline solution
or 1 mL of sterile saline as the control injection. After 16 or 48 h, fetuses were
surgically delivered for fetal tissue collection. We collected cerebrospinal fluid
(CSF) by occipital puncture. The left brain was dissected and the cortex, periventricular
white matter (PVWM), thalamus, hippocampus, and cerebellum were snap frozen for molecular
analysis. The right brain was fixed in 10 % formalin. We used four to five animals
for each group.

mRNA quantitation

Total RNA was isolated from frozen brains after homogenization with TRIzol (Invitrogen,
Carlsbad, CA) and column purified with RNeasy Universal MiniKit (Qiagen, Valencia,
CA) according to the manufacturer’s instructions. Reverse transcription was performed
using Verso complementary DNA (cDNA) kit (Thermo Scientific, Waltham, MA) to produce
single-strand cDNA. The genes for IL-1?, CCL2, TNF-?, IL-6, IL-8, IL-10, cyclooxygenase-2
(COX-2), and prostaglandin E synthase 2 (PTGES2) were amplified by RT-PCR using the
cDNA template and rhesus macaque-specific primers along with Taqman probes (Applied
Biosystems, Foster City, CA). The messenger RNA (mRNA) expression for each gene was
normalized to the mRNA for the ribosomal protein 18 s as internal standard. Data are
expressed as fold increase over the mean control value.

Cytokine measurements

We measured cytokine concentrations in the CSF by Luminex using nonhuman primate multiplex
kits (Millipore, Billerica, MA) according to the manufacturer’s protocol. Concentrations
were calculated from standard curves using recombinant proteins and expressed in picograms
per milliliter.


Sections from formalin fixed tissues in paraffin blocks were deparaffinized and rehydrated
before microwave-assisted antigen retrieval in citric acid buffer at pH 6.0. Endogenous
peroxidase activity was reduced with CH
treatment, and the tissue was blocked with 2 % donkey serum in phosphate buffer saline
(PBS). Sections were incubated overnight at 4 °C with the primary antibody diluted
in 2 % serum in PBS. We used the following primary antibodies: goat polyclonal Anti-Iba1
(Abcam, Cambridge, MA, cat. #5076, dilution 1:200), rabbit monoclonal Anti-glial fibrillary
acidic protein (GFAP) (Abcam, Cambridge, MA, cat. #48050, dilution 1:500), rabbitt
monoclonal Anti-Olig2 (Abcam, Cambridge, MA, cat. #109186, dilution 1:500), mouse
monoclonal Anti-MBP (Abcam, Cambridge, MA, cat. #62631, dilution 1:500), rabbit monoclonal
Anti-Tau (Abcam, Cambridge, MA, cat. #32057, dilution 1:500), mouse monoclonal anti-NeuN
(Abcam, Cambridge, MA, cat. #ab104224, dilution 1:200), rabbit polyclonal anti-CD3
(DakoCytomation, Carpinteria, CA, cat. #A0452, dilution 1:100), and mouse monoclonal
anti-CD14 (BD Biosciences, San Diego, CA, cat. #557742, dilution 1:100). Sections
were then washed and incubated with the appropriate species-specific secondary antibody
diluted 1:200 in 2 % serum for 2 h at room temperature. After further washing, antigen/antibody
complexes were visualized using a Vectastain ABC peroxidase kit (Vector Laboratories
Inc., Burlingame, CA). Antigen detection was enhanced with nickel-diaminobenzidine,
followed by incubation with Tris-cobalt. Slides were counterstained with Nuclear Fast
Red for photomicroscopy.

Double-labeling immunofluorescence

To identify proliferating microglia, we performed double-labeling immunofluorescence
for Iba-1 and Ki-67. Antigen retrieval and blocking were conducted as described above,
followed by overnight incubation with goat polyclonal Anti-Iba1 (Abcam, Cambridge,
MA, cat. #5076, dilution 1:100) and rat monoclonal anti-Ki67 (LifeSpan Biosciences,
Seattle, WA, cat. #LS-C175347, dilution 1:50). The following day, sections were washed
and incubated with anti-goat Alexa Fluor 594 (Life technologies, Carlsbad, CA, cat.
#A-11058, dilution 1:200) and anti-rat Alexa Fluor 488 (Life technologies, Carlsbad,
CA, cat. #A-21470, dilution 1:200) for 2 h at room temperature, followed by washing
and incubation with DAPI (Life technologies, Carlsbad, CA, cat. #D1306, dilution 1:2000)
for 15 min at room temperature. Sections were washed and mounted with ProLong Gold
(Life technologies, Carlsbad, CA, cat. #P36930). Stained slides were imaged on confocal
microscopy for co-localization at ×40x magnification with 1024?×?1024 pixel resolution
on a Nikon Eclipse A1RSi inverted microscope (Nikon Instruments Inc, Melville, NY).

Cell counting

Cells immunostained for CD3 (T cells), CD14 (monocytes), and caspase-3 (apoptotic
cells) were counted in six randomly selected fields per animal on photomicrographs
(×40) from the periventricular area and counted by a masked investigator. A similar
strategy was used to assess the number (Iba1+) and proportion of proliferating microglia
(Ki67+/Iba1+) in the white matter. A masked investigator counted the number of cells
positive for Iba1, and the number of cells positive for both Iba1 and Ki67 in each
determined both the number of Iba1+ cells and the percentage of Iba1+/Ki67+ cells.

Statistical analysis

Results of the mRNA quantitation and cell counting were analyzed by ANOVA followed
by post-testing comparison of each experimental group against the control group with
Tukey correction. Statistical analysis was performed on GraphPad Prism version 6.0
for Mac OS X (GraphPad Software, San Diego, CA). Data are presented as means?±?standard
deviation. Values of p??0.05 were considered significant.