Methylation in HT22 cells and primary hippocampal neurons with and without isoflurane exposure

HT22 is an immortalized mouse hippocampal cell line, which is a sub-line derived from parent HT4 cells that were originally immortalized from cultures of primary mouse hippocampal neurons [1]. The HT22 cells were a generous gift from the Salk Institute (La Jolla, CA, USA). HT22 cells were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, and in a 5% CO2 atmosphere at 37 °C.

Primary mouse hippocampal neurons were purchased from Lonza (M-HI-401, LOT numbers: 030807, 301,006, and 240,807, Basel, Switzerland). Mouse hippocampal neurons were isolated from E16–17, CD-1 mice which had been killed by decapitation, and then cryopreserved at primary passage. They stain positive for PGP and Tuj-1 and test negative for mycoplasma. Preparation of medium, coating of cell culture plates, thawing of cells / initiation of culture process, and maintenance of the cell culture was performed according to the manufacturer’s instructions. On day 1 of each experiment, thawing of cells and initiation of the culture process was started. Cells were seeded for experiments on day 4, 5, or 6 and underwent isoflurane or control exposures on the following day (day 5,6, or 7). The morphology of cultured cells was assessed once daily by microscopy.

For isoflurane exposure, HT22 cells or primary hippocampal neurons were transferred into a Billups-Rothenburg chamber (MIC-101, Billups-Rothenburg, Del Mar, CA) [11]. An in-line calibrated anesthetic agent Vaporizer (Dräger, Lübeck, Germany) was used to deliver isoflurane to the gas phase of the culture wells. Control air (5% CO2, 21% O2, balance nitrogen) (Air Liquide, Düsseldorf, Germany) and 3% isoflurane were delivered into the chamber with 5 l/min over 20 min. Effluent isoflurane, and carbon dioxide concentrations were monitored during flushing the chamber via a sampling port connected to an anesthetic agent analyzer (DrägerVamos, Lübeck, Germany). The chamber was then placed immediately into an incubator and remained there for 4 h. Isoflurane concentrations (3%) were again checked after 30, 90, and 180 min after exposure while flushing the chamber for 5 min. Control experiments were performed in the same manner, except that no isoflurane was added.

Three hundred thousand HT22 cells were seeded in 6-well plates and underwent isoflurane exposure or control experiments without isoflurane. The experiments were repeated 6 times.

Primary hippocampal neurons were cultivated according to the manufacturer’s instructions. On culture days 4, 5, and 6 200.000 cells were seeded on a 24-well plate and underwent isoflurane or control exposures the following day (day 5, 6 or 7). For methylation analysis, this experiment was performed twice, resulting in 6 single experiments. Therefore, in the methylation analysis of primary hippocampal neurons, 2 experiments performed on day 5, 2 experiments performed on day 6, and 2 experiments performed on day 7 were included. For analysis of mRNA expression in primary hippocampal neurons, 3 independent experiments, in which cells underwent isoflurane or control exposures at day 5, 6 or 7 were included.

Immediately after 4 h of isoflurane exposure, DNA and RNA were extracted from the respective cells.

DNA was extracted with the DNeasy Blood & Tissue Kits (Qiagen, Hilden, Germany) and DNA concentration was measured by a spectrometer (Eppendorf, Hamburg, Germany).

Methylation of genes was analyzed with EpiTect Methyl II PCR Array Systems (Qiagen, Hilden, Germany), which examines the methylation status across a CpG-rich sequence in gene promoters. The method is based on the detection of remaining input DNA after cleavage with a methylation-sensitive and/or a methylation-dependent restriction enzyme (EpiTect Methyl II DNA Restriction Kit, Qiagen, Hilden, Germany). These enzymes digest unmethylated and methylated DNA, respectively. Following digestion, the remaining DNA is quantified by real-time PCR (RT² SYBR® Green qPCR Mastermix, Qiagen, Hilden, Germany) in each individual enzyme reaction using primers that flank a promoter (gene) region of interest. We analyzed the DNA methylation status of 22 genes of the pathways focused EpiTect Methyl II PCR Array Systems (Qiagen, Hilden, Germany), i.e., apoptosis (EAMM-121Z, Table 1), cytokine production (EAMM-541Z, Table 2), and inflammatory response and autoimmunity (EAMM-521Z, Table 3). The relative fractions of methylated and unmethylated DNA are subsequently determined by comparing the amount in each digest with that of a mock (no enzymes added) digest using the ΔCt method as recommended by the manufactor (Qiagen, Hilden, Germany).

RNA was extracted with peqGOLD MircoSpin Total RNA Kits (Peqlab Biotechnology, Erlangen, Germany) at the indicated time point. RNA concentration was measured by a spectrometer (Eppendorf, Hamburg, Germany). First-strand cDNA was synthesized from 1 μg of total RNA with oligo-dT primers (Roche, Mannheim, Germany) by using Superscript II reverse transcriptase, as recommended by the supplier (Invitrogen, Carlsbad, CA). Relative quantification of mRNA expression was carried out by using primers for those genes whose methylation level showed a difference greater than 20% among in the primary hippocampal neurons with and without isoflurane exposure (Cxcl12, Cxcl14, Gata3, Il11, Il13, and Il4ra,; for primer details see supplementary Table 1, for gene details see Table 3), taking several considerations of reference gene selection into account [11]. Contamination with genomic DNA was circumvented by DNase treatment of RNA and by using RNA as template. The PCR reaction mix was prepared using the Quantitect SYBR Green Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. PCR was performed in the Step One Plus (Applied Biosystems, Foster City, USA). Real-Time PCR experiments were performed three times.

Data are presented as means ± standard deviation (SD). Data were analyzed using Graph Pad Prism 6 (Graph Pad Software, La Jolla, CA). Methylation analysis was done according to the manufacturer’s instructions. A change in methylation of > 20% was considered as significant, according to the instructions of the manufacturer and comparable to other studies using the EpiTect Methyl II PCR Array System [12, 13]. Here, DNA methylation was compared between apoptosis-related genes in pleomorphic adenomas of the salivary glands [13], respectively in genes involved in inflammation and autoimmunity in inflammatory bowel disease [12]. mRNA expression was done in genes with significant methylation changes, according to references [12, 13], and analyzed with an unpaired t-test. This An a priori alpha error p of less than 0.05 was considered to indicate statistical significance.

In a first step, we measured methylation of genes in HT22 and in primary hippocampal neurons in control experiments without exposure to isoflurane (Fig. 1a-c). Interestingly, the methylation status between the immortalized cell line HT22 and the primary hippocampal neuron cultures was markedly different. In general, methylation of genes in HT22 cells was greater than in the primary cells. Eight of 22 genes of the apoptosis array (Bid, Bik, Cideb, Dapk1, Hrk, Ltbr, Tnfrsf21, and Tnfrsf25) were significantly greater (> 20%) methylated in HT22 cells compared to primary hippocampal neurons (Fig. 1a). For example, methylation of Bik was 99.4% in HT22 cells, whereas it was 0.2% in primary hippocampal neurons (Fig. 1A). In the cytokine array, genes Bcl10, Bcl3, Cd114, Furin, Gata3, Irf1, Ltb, Myd88, and Stat5a showed > 20% methylation changes in the HT22 cells compared to the primary hippocampal neurons (e.g. Ltb: 99.6% vs. 0.4%) (Fig. 1b). Most differences were found in genes of the inflammatory array. Here, 12 of 22 genes (Cx3cl1, Cxcl12, Cxcl14, Gata3, Il10ra, Il11, Il13, Il13ra1, Il17ra, Il7, Ltb, and Tgfb1) were significantly greater (> 20%) methylated in HT22 cells compared to primary hippocampal neurons (Fig. 1c). Methylation of Cxcl12 was 99.7% in HT22 cells, whereas it was 0.6% in primary hippocampal neurons (Fig. 1c). Only one gene (Il18) was nearly completely methylated in the primary neurons (> 99.8%).

We measured whether isoflurane exposure alters methylation of genes in primary hippocampal neurons (Fig. 2). However, no changes were seen for genes of the apoptosis array (Fig. 2a) and the cytokine array (Fig. 2b). There was a greater than 20% methylation change for several genes in the inflammatory array after isoflurane exposure: Cxcl12, Cxcl14, Gata3, Il11, Il13, and Il4ra (Fig. 2c). Methylation of Cxcl12 was 0.6% without, but 27.5% with isoflurane exposure. For all genes, variation between the single experiments was seen (Fig. 2c). In HT22 cells, neither methylation of the genes in the apoptosis array (A), nor those in the cytokine (B) or in the inflammatory arrays (C) was altered by isoflurane (Supplementary Fig. 1).

To elucidate whether greater methylation of several genes in the inflammatory array with isoflurane exposure altered mRNA expression of these genes, RT primer were designed for genes, in which methylation was > 20% different after isoflurane exposure (Cxcl12, Cxcl14, Gata3, Il11, Il13, and Il4ra, for primer sequences see supplementary Table 1). mRNA expression was then measured in cDNA samples of cells exposed to isoflurane and in cells under control conditions. Expression of Cxcl12 was significantly lower under isoflurane exposure (p = 0.023, Fig. 2d), whereas no differences were found for the genes Cxcl14, and Gata3 (data not shown). No RT product could be obtained for genes Il11, Il13, and Il14ra.