Klenke Stefanie, Specking Christian, Stegen Maike, Engler Andrea, Peters Jürgen
Klinik für Anästhesiologie & Intensivmedizin, Universität Duisburg-Essen and Universitätsklinikum Essen, Hufelandstr. 55, D-45122, Essen, Germany.
BMC Anesthesiol. 2020 Mar 14;20(1):66. doi: 10.1186/s12871-020-00981-4.
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.
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.
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).
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.
表观遗传调控可能在与麻醉相关的表型中发挥作用，如认知障碍或记忆丧失，尤其是在大脑发育的脆弱阶段接触麻醉剂时。虽然异氟烷麻醉可在幼龄动物中引发神经炎症和神经细胞凋亡，但我们在先验的体外分析中，在永久性海马细胞系（HT22）和原代海马神经元中研究了异氟烷暴露是否：1）引发参与细胞凋亡和炎症的基因的DNA甲基化变化；2）在永久性海马细胞系中观察到的结果是否与原代海马神经元相当。如果特定基因发生甲基化变化，（3）进行mRNA分析以评估对基因表达的可能影响。
将HT22细胞和原代小鼠海马神经元暴露于3%异氟烷4小时，然后提取DNA（每组6个独立实验）和RNA（3个独立实验）。甲基化分析（EpiTect Methyl II PCR Array Systems，Qiagen）包括66个参与细胞凋亡、细胞因子产生、炎症反应和自身免疫的基因的甲基化状态。使用Quantitect SYBR Green试剂盒在Step One Plus上进行定量实时PCR。
在未暴露于异氟烷的永生化HT22细胞和培养的原代海马神经元之间，甲基化状态明显不同。在所研究的66个基因中，与原代海马神经元相比，HT22细胞中有29个基因的甲基化程度明显更高。相比之下，在培养的原代海马神经元中，几个参与炎症的基因存在更高的甲基化，同时异氟烷暴露导致C-X-C基序趋化因子12显著下调（p = 0.023）。
我们证明了在未暴露于异氟烷的HT22细胞和培养的原代海马神经元之间基因甲基化存在显著差异，异氟烷暴露后几个参与炎症的基因甲基化程度更高，且原代海马神经元中Cxcl12 mRNA表达显著下调。因此，应进一步开展与麻醉相关的DNA甲基化研究，并特别考虑此类研究的靶向细胞选择。
