Introduction

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).

Objectives

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.

Methods

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.

Results

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.

Conclusion

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.

中文翻译：

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安乐死和摘除术对小鼠角膜上皮轴突密度和神经末梢形态的影响。

介绍

在这里，我们研究了使用 CO2 气体或颈椎脱位 (CD) 进行安乐死以及使用不同技术摘除眼睛对保持上皮内角膜神经 (ICN) 的轴突密度和形态的影响。

目标

确定是否使用 CO2 气体或 CD 进行安乐死并通过切割或拉出眼睛去核会影响小鼠角膜中的轴突密度和神经末梢形态。

方法

小鼠用CO2气体或CD安乐死；还评估了安乐死后延迟固定 5 分钟的影响。我们测试了两种不同的摘除眼睛的技术：用弯曲的剪刀剪断视神经或将眼睛拉出。每个变量至少使用 5 只雄性和雌性 BALB/c 小鼠的 10 个角膜。轴突和上皮内角膜神经末梢 (ICNT) 使用 βIII 微管蛋白和 L1CAM 进行可视化，并使用共聚焦显微镜进行量化。

结果

个体小鼠之间轴突密度的变化与性别或安乐死无关。在通过拉出视神经而摘除的眼睛中观察到轴突密度显着降低和 ICNT 形态的丧失。在雄性和雌性小鼠中获得了类似的结果。

结论

虽然在安乐死中测试的变异不影响雄性和雌性小鼠角膜的轴突密度，但与将眼睛拉出并保持视神经附着相比，通过突出和轻轻切掉眼睛进行摘除会增加轴突密度并改善 ICNT 形态。