Objective
Our objective was to evaluate whether brain-derived neurotrophic factor (BDNF) expression is affected by prenatal alcohol exposure and whether the neuroprotective effects of the vasoactive intestinal peptide (VIP)-related peptides, NAPVSIPQ (NAP) and SALLRSIPA (SAL), are mediated through BDNF.
Study Design
Using a well-characterized fetal alcohol syndrome (FAS) model, timed pregnant C57BL6/J mice were treated on gestational day (E) 8 with alcohol (0.03 mL/g), placebo, or alcohol plus (NAP plus SAL). Embryos were harvested at 6 hours (E8), 24 hours (E9), and 10 days (E18) and pups at postnatal day 40. Calibrator-normalized relative real time polymerase chain reaction was performed to quantify BDNF with hypoxanthine phosphoribosyl transferase-1 standardization.
Results
BDNF expression was lower in the alcohol-exposed embryos than in controls at 6 hours and higher at 24 hours and 10 days (all P < .05). Pretreatment with NAP plus SAL prevented the alcohol-induced rise in BDNF expression ( P < .05) at 24 hours and 10 days after alcohol exposure. We found no difference between alcohol and control in young-adults’ brain ( P > .05).
Conclusion
NAP plus SAL treatment prevented alcohol-induced changes in BDNF expression 24 hours and 10 days after alcohol exposure in mouse embryos. This may explain, at least in part, the peptides’ prevention of neurodevelopmental anomalies in FAS.
Alcohol use during pregnancy results in a spectrum of adverse outcomes known as fetal alcohol spectrum disorders (FASD). The incidence of FASD is estimated to be as high as 1 in 100 births. FASD includes a range of neurobehavioral abnormalities, learning deficits, and structural brain defects as shown by magnetic resonance imaging. Prenatal alcohol exposure is the most common nongenetic cause of mental retardation.
Fetal alcohol syndrome (FAS) represents the severe end of the FASD spectrum. FAS is characterized by specific craniofacial dysmorphology, growth restriction, and neurodevelopmental anomalies. The mechanism by which prenatal alcohol exposure affects brain development is multifactorial with numerous mechanisms contributing to ethanol-related neuropathology.
The neurotrophins promote differentiation and survival of specific neuronal populations. Alterations in neurotrophic activity may be involved in the etiology of alcohol-related neurodevelopmental desorders and ethanol-induced neurodegeneration. There is considerable evidence that some of the teratogenic consequences of developmental ethanol exposure are mediated by alteration of neurotrophin expression and function.
Brain-derived neurotrophic factor (BDNF) is a neurotrophin that is essential in normal neuronal development and survival as well as synaptic plasticity, an important factor in learning and memory. It has been hypothesized that prenatal alcohol exposure alters BDNF function contibuting to the development of alcohol-related disorders. Indeed, the similarities between FAS mouse models and BDNF haplodeficient mice suggest that the 2 phenotypes may be developmentally linked.
Vasoactive intestinal peptide (VIP) is a neuropeptide fundamental in embryonic development. VIP stimulation of high-affinity receptors on astrocytes results in the release of neurotrophic factors including activity-dependent neuroprotective protein (ADNP) and activity-dependent neurotrophic factor (ADNF), which have demonstrated neuroprotective proprieties.
A functional relationship between VIP and the neurotrophins is present in the postimplantation embryo. NAPVSIPQ (NAP), a sequence contained in ADNP, and SALLRSIPA (SAL), derived from ADNF, are both regulated by VIP and are protective against alcohol-induced damage in FAS. Prenatal treatment with NAP and SAL prevents alcohol-induced damage, including fetal death, growth abnormalities, and learning deficits in adult offspring. Moreover, young adult treatment with NAP and SAL reverses the alcohol-induced learning deficit in a model of FAS.
Our objective was to delineate BDNF expression during embryonic development and evaluate whether BDNF expression is affected by prenatal alcohol exposure in mouse embryos and young adult mouse brains. Moreover, we evaluated whether NAP and SAL’s neuroprotective effects are mediated by preventing alcohol-induced changes in BDNF expression during embryonic development.
Materials and Methods
C57Bl6/J female mice (The Jackson Laboratory, Bar Harbor, ME) were kept under a 12 hours light/12 hours 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 Care and Use of Experimental Animals. The protocol was approved by the Eunice Kennedy Shriver National Institute of Child Health and Human Development Animal and Care Use Committee. Six to 8 week old females were mated with C57BL/6J males for 4-6 hours to minimize time variability of conception. The presence of a vaginal plug was considered day 0 of pregnancy.
A well-described model for FAS was used, based on an acute high exposure to alcohol, resulting in many of the characteristic features including growth restriction, teratogenicity, and embryo lethality. On gestational day 8 (embryonic day [E] 8), pregnant mice were randomly injected intraperitoneally with 25% ethyl alcohol in saline solution at 0.03 mL/g of body weight (alcohol group, n = 15) or saline solution alone (control group, n = 16).
Pretreatment (intraperitoneal) with ADNP/ADNF peptides (NAP plus SAL) was given 30 minutes before alcohol (alcohol plus peptides group, n = 15). Dosages of the peptides were NAP (20 μg) and SAL (20 μg). NAP was diluted in 50 μL of dimethyl sulfoxide and diluted in filtered Dulbeccos’s phosphate-buffered saline solution (DPBS). SAL was diluted in DPBS.
Because the animals receiving alcohol were incapacitated for approximately 6 hours after injection, food and water were withheld from all groups for 6 hours. At 6 hours and 24 hours (E9) after injection, embryo tissues were explanted using microdissection from the uterus and placed in phosphate-buffered saline solution (PBS). At 10 days after injection (E18), brain tissuses were isolated from the embryo and placed in PBS. Each gestational time point included at least 4 samples with each sample representing 3-7 litters (a gestation typically includes 8-10 embryos).
To evaluate long-term alterations in BDNF expression on adult brain, at least 3 litters per treatment were allowed to deliver naturally. Male adult brains were harvested on postnatal day 40; hippocampus and cortex were collected separately and frozen in liquid nitrogen.
For ribonucleic acid (RNA) extraction, samples were homogenized by using disposable micropestles and a sonicator (Janke & Kunkel, Wilmington, NC). The samples were processed with Nucleospin RNA/protein kit (Machery-Nagel, Duren, Germany). One microliter was taken for spectrophotometric determination of RNA content. The remaining sample was stored at −80°C. Using 5 μg of total RNA, the reverse transcriptase (RT) reaction was performed (Applied Biosystem, Foster City, CA) in a final volume of 150 μL. Each RNA sample was run in duplicate. Negative controls included RT reactions omitting RNA or reverse transcriptase.
For real-time polymerase chain reaction (PCR), hypoxanthine phosphoribosyl transferase (HPRT)-1 was used as the internal standard. The HPRT1 primer pair was synthesized by Eurogentec (San Diego, CA), the primer sequences were 5′-CCA GCG TCG TGA TTA GCG ATG A -3′ (sense) and 5′-TGA GCA CAC AGA GGG CCA CAA T-3′ (antisense). BDNF primer pair was synthesized by Superarray (Frederick, MD; the sequences are owned by the company).
With the use of the FastStart DNA Master SYBR Green 1 dye-base detection (Roche, Indianapolis, IN), BDNF and HPRT1 expression was measured by real-time PCR using the LightCycler with relative quantification software (Roche) and melting point analysis to assess the specificity of the amplified genes.
To further eliminate the risk of cross contaminations LightCycler Uracil-DNA Glycosylase (Roche Diagnostic Corp) was added to the master mix in all PCR experiments. The presence and purity of target gene sequence expression in the real time PCR reaction were confirmed by gel electrophoresis. Samples were run in duplicate and relative quantification was performed with calibrator-normalized data with efficiency correction. Results are presented as the normalized ratio of BDNF to HPRT1 expression.
Analysis of variance was performed for overall comparisons at each time point among the treatment groups with post hoc Fisher protected least significant difference analysis performed to compare between pairs, with P < .05 considered significant (StatView, version 5.0.1; SAS Institute, Cary, NC).
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