murine corneal nerve axotomy for the induction of neurotrophic keratitis

用于轴突再生研究的角膜感觉纤维损伤方案

The cornea, which is the transparent surface of the eye, is composed of three distinct layers: the epithelium, the stroma, and the endothelium. This organ has the highest density of innervation in the body and is composed mainly of sensory fibers (types A&#948; and C) originating from the ophthalmic branch of the trigeminal ganglion. Sensory fibers penetrate the periphery of the cornea in the mid-stroma in the form of big bundles that branch out to cover the surface. They then bifurcate to pierce the Bowmann&#39;s membrane and form the subbasal plexus, which is easily recognizable by the formation of a vortex in the center of the cornea. Those fibers terminate as free nerve endings at the external surface of the epithelium. They are able to transduce thermal, mechanical, and chemical stimuli and to release trophic factors that are essential for epithelium homeostasis1,2. Neurotrophic keratitis (NK) is a degenerative disease affecting the corneal sensory innervation. This rare disease stems from a decrease or a loss of corneal sensitivity that results in lower tear production and poor healing properties of the cornea3. NK progresses through three well-described stages, from stage 1 where patients suffer epithelial defects, to stage 3 where stromal melting and/or corneal perforation occur4.
Clinically, the origins of this disease can be diverse. Patients can lose corneal innervation after physical injury to the eye, surgery, or through chronic diseases, such as diabetes5,6. To date, the NK pathogenesis process remains poorly understood, and therapeutical options for this sight-threatening condition are very limited. Therefore, a better understanding of the characteristics of epithelial defects is needed to better understand the mechanisms behind the regeneration of those fibers and potentially promote them. Here, we propose several models of corneal injury that induce NK in mice.
The first model is the abrasion of the epithelial layer of the cornea with an ocular burr. This model has mainly been studied in the context of the regeneration of the epithelium in different animals, such as rodents and fish7,8,9, and to test molecules promoting corneal healing10,11. Physiologically, it takes 2-3 days for the epithelial cells to close the wound. The physiological pattern of the innervation, however, takes more than four weeks to recover from the abrasion12,13. During the surgery, the ocular burr removes the epithelial layer of the cornea that contains the subbasal plexus and the fibers&#39; free nerve endings. This procedure can be clinically compared to patients with photorefractive keratectomy (PRK) to correct eye refractive defects. The procedure consists of removing the epithelium of the cornea and then reshaping the stroma with a laser14. Patients can experience several side effects following such surgery, such as a decrease of corneal nerve density for 2 years and a reduction in sensitivity for a duration of 3 months to one year post-surgery15. Given that the surgery induces a fragility of the corneal microenvironment, this model could help investigate these side effects and develop therapeutical approaches that would promote faster reinnervation, thus reducing the side effects in question.
The second model consists of sectioning the axons at the periphery of the cornea with a biopsy punch, inducing a Wallerian degeneration of the central innervation 16. Clinically, this method could be compared to anterior lamellar keratoplasty, in which the surgeon realizes a partial trephination of the cornea to remove a part of the anterior thickness of the cornea and replace it with a donor transplant 17. Following lamellar keratoplasty, patients may suffer from a number of symptoms including dry eye, loss of corneal innervation and graft rejection18. This axotomy model performed on corneal nerves could provide insight into the mechanisms of fiber degeneration, which occurs after a graft, followed by the axons&#39; regeneration.
The third method damages the corneal nerves with a laser. By using a multiphoton microscope on the cornea of anesthetized animals, degeneration of the nerves localized in the optical field is induced as a result of reactive oxygen species (ROS) formation, which leads to DNA damage and cellular cavitation19. This method recapitulates the corneal photodamage induced by overexposure to natural UV (sunburn), which also triggers ROS formation, leading to DNA damage20. Patients who suffer from corneal sunburn experience great pain, as the deterioration of epithelial cells deprives the corneal fibers&#39; extremities of all.
The three methods described here are designed to enable the investigation of the NK pathogenesis process and axon regeneration. They are easily reproducible and precise. Moreover, they allow quick recovery and easy monitoring of the animals.

作者聚焦：通过创新模型推进角膜神经支配研究 

诱导小鼠角膜神经营养性角膜炎和神经再生的三种策略

Watch this Scientific Journal Video about Murine Corneal Nerve Axotomy for the Induction of Neurotrophic Keratitis at JoVE.com

Murine Corneal Nerve Axotomy for the Induction of Neurotrophic Keratitis  

小鼠角膜神经轴切术诱导神经营养性角膜炎

要开始角膜神经轴索切开术，请将麻醉的小鼠放在桌子上。在接受手术的眼睛上滴一滴人工泪液。在对侧眼上滴一滴眼部凝胶。将鼠标放在双目放大镜下，以评估正在手术的眼睛。将培养皿的底部放在鼠标的头部下方，以保持眼睛水平。用棉签小心地从眼睛上擦去人工泪液，并在不接触眼睛的情况下去除睫毛。将一对光滑的弯曲柱子放在动物的眼睛下方，然后轻轻地将其弹出轨道。轻轻地将活检打孔器垂直放在角膜表面，施加压力，然后将打孔器扭转数次。然后，取下活检打孔器，角膜上应该可以看到一个圆形标记。在应用活检穿孔的部位看到一个薄的外周环，这损坏了基底上皮。周围神经似乎被切开，引发了Wallerian轴突变性。观察到严重的变性，导致角膜中心神经支配完全丧失。这导致上皮层崩解，形成溃疡。

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JoVE Journal

Neuroscience

Murine Corneal Nerve Axotomy for the Induction of Neurotrophic Keratitis

123 次观看. 要开始角膜神经轴索切开术，请将麻醉的小鼠放在桌子上。在接受手术的眼睛上滴一滴人工泪液。在对侧眼上滴一滴眼部凝胶。将鼠标放在双目放大镜下，以评估正在手术的眼睛。将培养皿的底部放在鼠标的头部下方，以保持眼睛水平。用棉签小心地从眼睛上擦去人工泪液，并在不接触眼睛的情况下去除睫毛。将一对光滑的弯曲柱子放在动物的眼睛下方，然后轻轻...

视频: Murine Corneal Nerve Axotomy for the Induction of Neurotrophic Keratitis  

Three Strategies to Induce Neurotrophic Keratitis and Nerve Regeneration in Murine Cornea

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation provides sensation and trophic function to the eye and contributes to preserving corneal integrity. The pathological disruption of this innervation is termed neurotrophic keratitis. This can be triggered by injury to the eye, surgery, or disease. In this study, we propose three different protocols for inflicting damage on the innervation in ways that recapitulate the three types of cases generally encountered in the clinic.
The first method consists in making an abrasion of the epithelium with an ophthalmic burr. This involves the removal of the epithelial layer, the free nerve endings, and the subbasal plexus in a manner similar to the photorefractive keratectomy surgery performed in the clinic. The second method&#160;only targets the innervation by sectioning it at the periphery with a biopsy punch, maintaining the integrity of the epithelium. This method is similar to the first steps of lamellar keratoplasty and leads to a degeneration of the innervation followed by regrowth of the axons in the central cornea. The last method damages the innervation of a transgenic mouse model using a multiphoton microscope, which specifically localizes the site of cauterization of the fluorescent nerve fibers. This method inflicts the same damage as photokeratitis, an overexposure to UV light.
This study describes different options for investigating the physiopathology of corneal innervation, particularly the degeneration and regeneration of the axons. Promoting regeneration is crucial for avoiding such complications as epithelium defects or even perforation of the cornea. The proposed models can help test new pharmacological molecules or gene therapy that enhance nerve regeneration and limit disease progression.

Here, we propose three different methods of damaging the sensory fibres innervating the cornea. These methods facilitate the study of axon regeneration in mice. These three methods, which are adaptable to other animal models, are ideal for the study of corneal innervation physiology and regeneration.

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation ...