The Schepens Eye Research Institute of Massachusetts Eye and Ear and Department of Ophthalmology, Harvard Medical School, Boston, MA, USA; Clinical Eye Research of Boston, Winchester and Boston, MA, USA; Ocular Research of Boston, Boston, MA, USA.
Magnetic Resonance Laboratory, Midwestern University, Downers Grove, IL, USA.
Exp Eye Res. 2020 Mar;192:107932. doi: 10.1016/j.exer.2020.107932. Epub 2020 Jan 18.
This study documents the absorption of glycerylphosphorylcholine (GPC) into corneas ex vivo. Corneas in quadruplicate were incubated in preservation medium containing 30 mM GPC, which is used as a reference marker. The GPC reference marker is used to calibrate P nuclear magnetic resonance (NMR) spectral chemical-shift positions for identification of phosphatic metabolites and to calculate intracorneal pH in intact tissues ex vivo. Following baseline NMR ex vivo analysis, corneas were stored in eye bank chambers in preservation medium containing 30 mM GPC at 4 °C overnight for 8 h. After returning to room temperature, NMR analysis was repeated on the same corneas in fresh GPC-free preservation medium. NMR analysis also was performed on the 30 mM GPC preservation medium alone from the eye bank chambers for detection of the GPC signal. The elevated GPC signal unexpectedly persisted in corneas incubated at 4 °C overnight even though GPC was not present in the fresh GPC-free preservation medium. In fact, the concentration of GPC in the intact cornea was many times higher than that found in the cornea endogenously. The levels of phosphatic metabolites and the energy modulus, after subtracting the spectral contribution of the 30 mM exogenous GPC, as well as the intracorneal pH remained unchanged from pre-refrigeration analyses. Corneas also retained transparency through the time-course of this study irrespective of temperature or change in temperature. The GPC signal in the NMR analysis of the preservation medium from the eye bank chambers was nearly undetectable. GPC was unexpectedly absorbed into the corneal tissue without detectable metabolic or physical toxicity. The intracorneal uptake of GPC at reduced temperatures parallels the increase in GPC that occurs naturally in muscle tissue in animals during wintering periods and the very high concentration of GPC in sperm, a cryogenically compatible cell, suggestive of a potential role for GPC in cryopreservation.
本研究记录了甘油磷酸胆碱（GPC）在离体角膜中的吸收情况。将角膜一式四份置于含有 30mM GPC 的保存液中孵育，GPC 作为参比标记物。GPC 参比标记物用于校准磷核磁共振（NMR）光谱化学位移位置，以鉴定磷酸代谢物，并计算离体完整组织中的角膜内 pH 值。在基线 NMR 离体分析后，角膜在含有 30mM GPC 的眼库腔室中于 4°C 下保存过夜 8 小时。返回室温后，在新鲜不含 GPC 的保存液中对相同角膜重复进行 NMR 分析。还对眼库腔室中的 30mM GPC 保存液单独进行 NMR 分析，以检测 GPC 信号。令人意外的是，即使在新鲜不含 GPC 的保存液中不含 GPC，在 4°C 孵育过夜的角膜中 GPC 信号仍持续升高。实际上，完整角膜中 GPC 的浓度比内源性角膜中的浓度高得多。在用 30mM 外源性 GPC 减去光谱贡献后，磷酸代谢物的水平和能量模量，以及角膜内 pH 值，与冷藏前分析相比保持不变。在这项研究中，无论温度如何变化，角膜都保持透明。眼库腔室保存液中 NMR 分析的 GPC 信号几乎无法检测到。GPC 被意外吸收到角膜组织中，没有检测到代谢或物理毒性。低温下角膜内 GPC 的摄取与动物在冬季肌肉组织中自然发生的 GPC 增加以及在冷冻相容细胞精子中 GPC 的非常高浓度平行，提示 GPC 在冷冻保存中可能具有潜在作用。
