PDF(Pubmed)
Abstract：
Dramatic postmortem prostanoid (PG) enzymatic synthesis in the brain causes a significant artifact during PG analysis. Thus, enzyme deactivation is required for an accurate in situ endogenous PG quantification. To date, the only method for preventing postmortem brain PG increase with tissue structure preservation is fixation by head-focused microwave irradiation (MW), which is considered the gold standard method, allowing for rapid in situ heat-denaturation of enzymes. However, MW requires costly equipment that suffers in reproducibility, causing tissue loss and metabolite degradation if overheated. Our recent study indicates that PGs are not synthesized in the ischemic brain unless metabolically active tissue is exposed to atmospheric O2. Based on this finding, we proposed a simple and reproducible alternative method to prevent postmortem PG increase by slow enzyme denaturation before craniotomy. To test this approach, mice were decapitated directly into boiling saline. Brain temperature reached 100°C after ∼140 s during boiling, though 3 min boiling was required to completely prevent postmortem PG synthesis, but not free arachidonic acid release. To validate this fixation method, brain basal and lipopolysaccharide (LPS)-induced PG were analyzed in unfixed, MW, and boiled tissues. Basal and LPS-induced PG levels were not different between MW and boiled brains. However, unfixed tissue showed a significant postmortem increase in PG at basal conditions, with lesser differences upon LPS treatment compared to fixed tissue. These data indicate for the first time that boiling effectively prevents postmortem PG alterations, allowing for a reproducible, inexpensive, and conventionally accessible tissue fixation method for PG analysis.
摘要：
大脑中巨大的死后前列腺素（PG）酶促合成在PG分析过程中会引起明显的伪影。因此，酶失活是准确的原位内源性PG定量所必需的。迄今为止,通过头部聚焦微波辐射（MW）来防止死后脑PG增加并保留组织结构的唯一方法是固定，这被认为是黄金标准方法，允许酶的快速原位热变性。然而,MW需要昂贵的设备，这些设备在再现性方面受到影响，如果过热会导致组织损失和代谢物降解。我们最近的研究表明，除非代谢活跃的组织暴露于大气O2，否则缺血脑中不会合成PG。基于这一发现,我们提出了一种简单且可重复的替代方法，通过开颅手术前缓慢的酶变性来防止死后PG增加。为了测试这种方法，将小鼠直接断头放入沸腾的盐水中。脑温度达到100°C后，〜140秒在沸腾，尽管需要3分钟沸腾才能完全防止死后PG合成，但不是游离的花生四烯酸释放。要验证此固定方法，脑基底和脂多糖（LPS）诱导的PG在未固定的分析，MW,和煮的纸巾。基础和LPS诱导的PG水平在MW和煮沸的大脑之间没有差异。然而,未固定的组织在基础条件下显示出PG的死后显着增加，与固定组织相比，LPS处理后差异较小。这些数据首次表明沸腾有效地防止了死后PG的改变，允许可重现的，便宜,以及用于PG分析的常规可接近的组织固定方法。
