Reproduction Abstracts

Introduction: Preterm birth associates with later neurological conditions. Many of the underlying mechanisms are unknown however studies suggest changes in DNA methylation may be important. We have previously identified differential DNA methylation at astrocyte-specific genes, Slc1a2 and Lrg1 in preterm infants. Here we set out to describe the normal developmental patterns of gene expression and DNA methylation of Slc1a2 and Lrg1 in vivo in an animal model and assessed the potential utility of cultured astrocytes to study these changes.

Methods: Astrocytes were isolated from Wistar rat forebrain through neural dissociation and selection of Glast+ cells using Magnetic Activated Cell Sorting, at developmental time points corresponding to human brain development: Embryonic day E20.5 (24 weeks), Postnatal day P1 (28 weeks) and P10 (term). Astrocytes isolated at E20.5 and P1 were cultured for 10 days. Gene expression was analysed using RT-qPCR and DNA methylation using pyrosequencing. Experiments were approved under Home Office project licence (PPL60/7874) after local ethical approval.

Results and discussion: Expression of Slc1a2 increased 58-fold (P<0.01) during development and expression of Lrg1 decreased 9-fold from P1 to P10 (P<0.05) DNA methylation in vivo was low at Slc1a2 and did not change between E20.5/P1 and P10 whereas DNA methylation decreased at Lrg1 over time (P<0.05). In culture, Slc1a2 expression was lost but Lrg1 expression was equivalent to P10 in vivo levels. Cell culture altered DNA methylation: levels increased at Lrg1 and decreased at Slc1a2 (P<0.05). Thus, our study suggests that cultured astrocytes are not suitable for studying changes in vitro alternative options including co-culture systems may provide a suitable alternative. Expression and DNA methylation at Slc1a2 and Lrg1 change during early development during a time period corresponding to early postnatal life in many preterm infants. We hypothesize that these processes may be susceptible to disruption following preterm birth.