Materials and Methods

In this study, a well-defined FAS mouse model that exposed fetal mice to ethanol during a critical period of organogenesis and neurogenesis and renders a high incidence of anomalies and death was used. C57Bl6/J female mice (Jackson Laboratories, Bar Harbor, ME) were kept under a 12-hour light, 12-hour dark regimen, with food and water available at all times. The mice received humane animal care in compliance with the National Institutes of Health guidelines for the care and use of experimental animals. The protocol was approved by the National Institute of Child Health and Human Development Animal Care and Use Committee. Six-week-old females (21–24 g) were mated with C57Bl6/J males for 4 hours. Presence of the vaginal copulation plug was considered day 0 of pregnancy There were 3 treatment groups: alcohol, placebo, and alcohol + NAP + SAL. On gestational day 8, we treated pregnant mice intraperitoneally with 25% ethyl alcohol in saline solution (volume per volume) or vehicle alone at 0.03 mL/g body weight.

The peptides, NAP and SAL (20 mg in 0.2 mL), were administered immediately before the alcohol/placebo treatment. NAP was diluted in 50 mL dimethyl sulfoxide and diluted in filtered Dulbecco’s phosphate-buffered saline solution. SAL was dissolved and diluted in filtered Dulbecco’s phosphate-buffered saline solution. The peptide concentrations reflect previously established protective levels for each peptide in the prevention of alcohol-induced fetal death, growth restriction, and microcephaly. At 6 hours after injection, embryos were explanted with microdissection and homogenized in a buffer that contained 20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 0.05% Tween-20, and a cocktail of protease inhibitors (Roche Diagnostics Corporation, Indianapolis, IN). At 10 days after injection (embryonic day 18), brain tissues were isolated from the embryo and homogenized in a buffer that contained 20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 0.05% Tween-20, and a cocktail of protease inhibitors (Roche Diagnostics Corporation). The homogenates were centrifuged at 10,000 g for 5 minutes. The supernatant was removed and analyzed for protein concentration; a Luminex analysis (Luminex Corp, Austin, TX) was carried out for the measurement of fractalkine. Each gestational time point included at least 4 samples; each sample represented 2–7 litters (a gestation typically includes 8–10 embryos).

Fractalkine levels were normalized to total protein. Fractalkine concentrations were determined with the use of antibodies for the analyte that had been immobilized covalently to a set of microspheres according to protocol that was developed and validated at Linco Research, Inc (St. Louis, MO). The analyte on the surface of microspheres was then detected by a cocktail of biotinylated antibodies. After the binding of streptavidin-phycoerythrin conjugate, the reporter fluorescent signal was measured with a Luminex100 reader (Luminex Corp). Data were calculated with a calibration curve that was obtained in each experiment with the use of the respective recombinant proteins that had been diluted in kit matrix for plasma samples and lysis buffer for tissue samples. Concentrations of fractalkine was calculated with StatLIAs software (Brendan Scientific Corp, Carlsbad, CA) with a 5-parameter logistic curve-fitting method and normalized to the amount of protein in each sample. Statistical analysis included Kruskal-Wallis test for comparison of fractalkine levels; a probability value of < .05 was considered to be significant. Data are reported as median (range).