Purpose

This study used noninvasive, fluorescence lifetime imaging microscopy (FLIM)-based imaging of NADH and FAD to characterize the metabolic response of mouse embryos to short-term oxygen deprivation. We investigated the response to hypoxia at various preimplantation stages.

Methods

Mouse oocytes and embryos were exposed to transient hypoxia by dropping the oxygen concentration in media from 5–0% over the course of ~1.5 h, then 5% O₂ was restored. During this time, FLIM-based metabolic imaging measurements of oocyte/embryo cohorts were taken every 3 minutes. Experiments were performed in triplicate for oocytes and embryos at the 1- to 8-cell, morula, and blastocyst stages. Maximum hypoxia response for each of eight measured quantitative FLIM parameters was taken from the time points immediately before oxygen restoration.

Results

Metabolic profiles showed significant changes in response to hypoxia for all stages of embryo development. The response of the eight measured FLIM parameters to hypoxia was highly stage-dependent. Of the eight FLIM parameters measured, NADH and FAD intensity showed the most dramatic metabolic responses in early developmental stages. At later stages, however, other parameters, such as NADH fraction engaged and FAD lifetimes, showed greater changes. Metabolic parameter values generally returned to baseline with the restoration of 5% oxygen.

Conclusions

Quantitative FLIM-based metabolic imaging was highly sensitive to metabolic changes induced by hypoxia. Metabolic response profiles to oxygen deprivation were distinct at different stages, reflecting differences in metabolic plasticity as preimplantation embryos develop.

中文翻译：

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响应诱导缺氧的小鼠胚胎代谢的非侵入性成像。

目的

本研究使用基于 NADH 和 FAD 的无创荧光寿命成像显微镜 (FLIM) 成像来表征小鼠胚胎对短期缺氧的代谢反应。我们研究了植入前各个阶段对缺氧的反应。

方法

通过在约 1.5 小时内将培养基中的氧浓度从 5-0% 降低，小鼠卵母细胞和胚胎暴露于短暂缺氧，然后恢复5% O ₂。在此期间，每 3 分钟对卵母细胞/胚胎群体进行基于 FLIM 的代谢成像测量。对 1 至 8 细胞、桑椹胚和胚泡阶段的卵母细胞和胚胎进行了三次重复实验。八个测量的定量 FLIM 参数中的每一个的最大缺氧响应取自氧气恢复之前的时间点。

结果

代谢谱显示胚胎发育所有阶段对缺氧反应的显着变化。八个测量的 FLIM 参数对缺氧的反应高度依赖于阶段。在测量的八个 FLIM 参数中，NADH 和 FAD 强度在早期发育阶段显示出最显着的代谢反应。然而，在后期阶段，其他参数，例如参与的 NADH 分数和 FAD 寿命，显示出更大的变化。代谢参数值通常随着 5% 氧气的恢复而恢复到基线。

结论

基于 FLIM 的定量代谢成像对缺氧引起的代谢变化高度敏感。对缺氧的代谢反应谱在不同阶段是不同的，反映了随着植入前胚胎发育的代谢可塑性的差异。