Methylation in HT22 cells and primary hippocampal neurons with and without isoflurane exposurewhether isoflurane causes

Abstract

Background

Epigenetic modulation may play a role in anesthesia related phenotypes, such as cognitive impairment or memory loss, especially with exposure to anesthetics in the vulnerable phase of brain development. While isoflurane anesthesia can evoke neuroinflammation and neuroapoptosis in young animals, we investigated in a permanent hippocampal cell line (HT22) and in primary hippocampal neurons in an a priori in vitro analysis, whether isoflurane exposure 1) evokes DNA methylation changes in genes involved in apoptosis and inflammation, and 2) results observed in a permanent hippocampal cell line are comparable to primary hippocampal neurons. In case of methylation changes in specific genes, (3) mRNA analysis was performed to assess possible effects on gene expression.

Methods

HT22 cells and primary mouse hippocampal neurons were exposed to 3% isoflurane for 4 h and DNA (each 6 single experiments) and RNA (3 single independent experiments) were extracted. Methylation analysis (EpiTect Methyl II PCR Array Systems, Qiagen) included the methylation status of 66 genes involved in apoptosis, cytokine production, inflammatory response, and autoimmunity. Quantitative Real-Time PCR was performed using the Quantitect SYBR Green Kit on a Step One Plus.

Results

Methylation status was markedly different between immortalized HT22 cells and cultured primary hippocampal neurons without isoflurane exposure. Of 66 genes investigated, 29 were methylated to a significantly greater degree in HT22 cells compared to primary hippocampal neurons. In cultured primary hippocampal neurons, in contrast, there was a greater methylation in several genes involved in inflammation, accompanied with significant downregulation of C-X-C motif chemokine 12 with isoflurane exposure (p = 0.023).

Conclusions

We demonstrate marked differences in gene methylation between HT22 cells and cultured primary hippocampal neurons without isoflurane exposure, with a greater methylation of several genes involved in inflammation upon isoflurane exposure and significant downregulation of Cxcl12 mRNA expression in primary hippocampal neurons. Accordingly, further investigations of anesthesia related DNA methylation should be performed with special consideration being given to the choice of cells targeted for such investigations.