Department of Anatomy and Cell Biology, The George Washington School of Medicine and Health Sciences, Washington DC, 20037, USA.
Department of Anatomy and Cell Biology, The George Washington School of Medicine and Health Sciences, Washington DC, 20037, USA; Department of Ophthalmology, The George Washington School of Medicine and Health Sciences, Washington DC, 20037, USA.
Ocul Surf. 2020 Oct;18(4):821-828. doi: 10.1016/j.jtos.2020.07.021. Epub 2020 Aug 13.
Here we study the impact of using either CO2 gas or cervical dislocation (CD) for euthanasia and using different techniques to enucleate the eye on preserving axonal density and morphology of the intraepithelial corneal nerves (ICNs).
To determine whether using CO2 gas or CD for euthanasia and enucleating by cutting or pulling eyes out impacts axon density and nerve terminal morphology in the mouse cornea.
Mice were euthanized by CO2 gas or CD; the impact of delaying fixation for 5 min post-euthanasia was also assessed. We tested two different techniques to enucleate the eyes: cutting the optic nerve by curved scissors or pulling the eye out. A minimum of 10 corneas from 5 male and female BALB/c mice were used for each variable. Axons and intraepithelial corneal nerve terminals (ICNTs) were visualized utilizing βIII tubulin and L1CAM and quantified using confocal microscopy.
The variations seen in axon density between individual mice are not gender- or euthanasia-dependent. A significant reduction in axon density and loss of ICNT morphology are observed in eyes enucleated by pulling the optic nerve out. Similar results are obtained in male and female mice.
While the variations tested in euthanasia do not affect axon density in male and female mouse corneas, enucleation by proptosing and gently cutting out the eyes yields increased axon density and improved ICNT morphology compared to pulling eyes out and leaving the optic nerve attached.
本研究旨在探讨使用二氧化碳（CO2）气体或颈椎脱位（CD）进行安乐死，以及使用不同的眼球摘除技术对角膜上皮内神经（ICN）轴突密度和形态的影响。
确定使用 CO2 气体或 CD 进行安乐死，以及通过切割或拔出眼睛来进行眼球摘除，是否会影响小鼠角膜中的轴突密度和神经末梢形态。
使用 CO2 气体或 CD 对小鼠进行安乐死；还评估了安乐死后延迟 5 分钟固定的影响。我们测试了两种不同的眼球摘除技术：用弯剪切割视神经或拔出眼睛。每个变量至少使用 5 只雄性和雌性 BALB/c 小鼠的 10 只角膜。使用共聚焦显微镜可视化βIII 微管蛋白和 L1CAM 来观察轴突和角膜上皮内神经末梢（ICNT）并进行定量。
个体小鼠之间的轴突密度变化与性别或安乐死无关。通过拔出视神经来摘除眼球会导致轴突密度显著降低和 ICNT 形态丧失。在雄性和雌性小鼠中均观察到类似的结果。
虽然在安乐死中测试的变化不会影响雄性和雌性小鼠角膜中的轴突密度，但与拔出眼睛并保留视神经相比，通过推眼球并轻轻取出眼睛进行眼球摘除可获得更高的轴突密度和改善的 ICNT 形态。
