Neuroprotective peptides (SALLRSIPA [SAL] and NAPVSIPQ [NAP]) can prevent some alcohol-induced damage in fetal alcohol syndrome (FAS). Fractalkine, a chemokine constitutively expressed in the central nervous system reduces neuronal death from activated microglia. Using a model of FAS, we evaluated whether fractalkine is altered and whether NAP + SAL work through fractalkine.
With an FAS model, C57BL6/J-mice were treated on gestational day 8 with alcohol (0.03 mL/g), placebo, or alcohol + peptides. Embryos were harvested after 6 hours and 10 days later. Fractalkine was measured in the protein lysate. Statistical analysis included the Kruskal-Wallis test.
Fractalkine was significantly elevated at 6 hours (median, 341pg/mL; range, 263–424 pg/mL) vs controls (median, 228 pg/mL; range, 146–332 pg/mL; P < .001). NAP + SAL prevented the alcohol-induced increase (median, 137 pg/mL; range, 97–255 pg/mL; P < .001). Ten days later, fractalkine levels were similar in all groups ( P = .7).
Prenatal alcohol exposure acutely elevates fractalkine, perhaps in an effort to counter the alcohol toxicity. Pretreatment with NAP + SAL prevents the acute increase in fractalkine.
Prenatal alcohol exposure may result in fetal alcohol syndrome (FAS), the most prevalent noninheritable cause of mental retardation or the less severe fetal alcohol spectrum disorder (FASD). The latter is characterized by a less severe phenotype and typically includes neurodevelopmental and neurobehavioral deficits that persist through adulthood. Gestational timing, dose, frequency, genetic predisposition, and not clearly elucidated factors impact the confounding effects of alcohol on a fetus. Therefore, although not all children who are exposed to alcohol prenatally will experience FAS, it has been estimated that, in the United States, 1 in 100 children are born with FASD that results in a substantial number of children who are susceptible to cognitive learning impairment. To better understand the complex mechanisms underlying FAS and FASD, we have used well-defined animal models that mimic the human condition, with careful consideration to gestational timing, brain structure vulnerability, dose, and duration of alcohol exposure to induce the dysfunctional regulatory mechanisms of neuronal plasticity, which is a direct measure of cognitive learning. Previously, we demonstrated that treatment with novel peptides, SALLRSIPA (SAL) and NAPVSIPQ (NAP), prevented alcohol-induced fetal growth restriction, microcephaly, oxidative damage, inflammatory cytokine release, and learning dysfunction in the mouse model of FAS. Using the FAS animal model, our laboratory demonstrated that prenatal alcohol increased tumor necrosis factor–α and interleukin-6 levels in the embryos vs control, which was attenuated with NAP + SAL treatment. These proinflammatory cytokines (tumor necrosis factor–α and IL6) affect long-term potentiation, which is a molecular model for learning. Given the importance of oxidative stress and inflammatory-mediated alcohol-induced injury in FAS and FASD, we report here on the role of fractalkine. Fractalkine is a chemokine with neuroprotective properties that acts as an antiinflammatory molecule in vitro by attenuating the secretion of interleukin-6 and tumor necrosis factor–α and the up-regulation of inducible nitric oxide synthase in lipopolysaccharide-activated microglia. Fractalkine is expressed constitutively in neurons throughout the central nervous system and has a role in neuroprotection-reducing neuronal death from activated microglia. Our objective was to evaluate whether fractalkine is altered in FAS and whether the mechanism of action of the neuroprotective peptides NAP + SAL includes fractalkine.
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).