Modeling Fetal Alcohol Spectrum Disorder: Validating an Ex Vivo Primary Hippocampal Cell Culture System

Abstract

Background Fetal Alcohol Spectrum Disorder (FASD) is the leading non-genetic cause of mental retardation. There are no treatments for FASD to date. Preclinical in vivo and in vitro studies could help in identifying novel drug targets as for other diseases. Here, we describe an ex vivo model that combines the physiological advantages of prenatal ethanol (E) exposure in vivo with the uniformity of primary fetal hippocampal culture to characterize the effects of prenatal E. The insulin signaling pathways are known to be involved in hippocampal functions. Therefore, we compared the expression of insulin signaling pathway genes between fetal hippocampi (in vivo) and primary hippocampal culture (ex vivo). The similarity of prenatal E effects in these two paradigms would deem the ex vivo culture acceptable to screen possible treatments for FASD. Methods Pregnant Sprague-Dawley rats received one of three diets: ad libitum standard lab chow (control-C), isocaloric pair-fed (PF, nutritional control), and E containing liquid diets from gestational day (GD) 8. Fetal male and female hippocampi were collected either on GD21 (in vivo) or on GD18 for primary culture (ex vivo). Transcript levels of Igf2, Igf2r, Insr, Grb10, Rasgrf1 and Zac1 were measured by RT-qPCR. Results Hippocampal transcript levels differed by prenatal treatment in both males and females with sex differences observed in the expression of Igf2 and Insr. The effect of prenatal E on the hippocampal expression of the insulin pathway genes was parallel in the in vivo and the ex vivo conditions. Conclusions The similarity of gene expression changes in response to prenatal E between the in vivo and the ex vivo conditions ascertain that these effects are already set in the fetal hippocampus at GD18. This strengthens the feasibility of the ex vivo primary hippocampal culture as a tool to test and screen candidate drug targets for FASD.

Alcohol‐Related Alterations in Placental Imprinted Gene Expression in Humans Mediate Effects of Prenatal Alcohol Exposure on Postnatal Growth

“…Given the well-defined role of IGF2 in growth, the association between IGF2 expression and reduced length and weight likely reflects adaptive changes upstream or downstream from IGF2 that are ultimately growth restricting, rather than a direct effect of IGF2 expression. Of note, our finding that alcohol exposure was associated with increased IGF2 expression in this sample is consistent with previous reports of alcohol-related increases in expression in human placenta trophoblast cultures (Joya et al, 2015), rat placental tissue (G ardebjer et al, 2014), hippocampus (Tunc-Ozcan et al, 2016), and human whole blood through age 5 years (Aros et al, 2011). Two studies have shown decreased placental IGF2 expression (1 human [Marjonen et al, 2017], 1 mouse [Downing et al, 2011]).…”

“…Yes (Babak et al, 2015) Yes (Babak et al, 2015) Growth promoting, with roles in insulin signaling (Bergman et al, 2013;Kappil et al, 2015b) Plays roles in working memory and hippocampal memory consolidation Prenatal alcohol exposure has been associated with increased expression in human placenta trophoblast cultures (Joya et al, 2015), rat placental tissue (G ardebjer et al, 2014), hippocampus (Tunc-Ozcan et al, 2016), and human child whole blood through age 5 years (Aros et al, 2011). Two studies have shown decreased placental expression…”

Hippocampus-dependent memory and allele-specific gene expression in adult offspring of alcohol-consuming dams after neonatal treatment with thyroxin or metformin

“…For the ex vivo and in vivo treatment studies, maternal diets and animal procedures were performed as described previously. 58 Briefly, adult female Sprague-Dawley (S) rats (Harlan, Indianapolis, IN, USA) were mated with adult Brown Norway (B) males (Charles River, Wilmington, MA, USA). We chose to study the S by B (SB) offspring because of their vulnerability to FAE.…”

“…Primary hippocampal cultures were prepared from embryonic day 18 fetuses of dams on C, PF, or E diets (n=3 dams/diet) as described previously. 58 Hippocampal neurons were plated at a density of 8×10 5 cells/60mm dish and cultured for 10 days. Metformin (final concentration of 0.4mM in sterile water) was administered every 48h.…”

“…RNA was isolated by using Direct-zol™ RNA MiniPrep (Zymo Research, Orange, CA, USA) and reverse transcription was performed using SuperScript ® VILO™ Master Mix (Invitrogen, Carlsbad, CA, USA). qPCR was conducted as described previously 58 with 5ng cDNA, specific primer pairs ( Supplemental Table 1 ) and SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) using the ABI 7900HT cycler.…”