localized laser ablation of murine corneal nerves for the induction of neurotrophic keratitis

Corneal Sensory Fiber Damage Protocols for Axon Regeneration Studies

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.

Author Spotlight: Advancing Corneal Innervation Research Through Innovative Models 

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

The cornea, which is the transparent tissue covering the eye, requires a balanced microenvironment to maintain its integrity. The dense innervation of the cornea participates to this microenvironment. If there is a loss, which could be partial or total, of this innervation, then a specific condition arises, which is called neurotrophic keratitis.To find new cures and efficient cures for this disease, we need to have specific models. The expansion of the use of transgenic animal bearing fluorescent reporters and microscopic advances have opened new possibilities in visualizing axonary generation. In vivo imagine has become more accessible to any research lab willing to follow the neurogeneration process.The various causes inducing neurotrophic keratitis underlying the difficulties of having only one model to test nerve regeneration therapies. These three models are reproducing various situations seen in clinics, and sharing those methods will allow other labs to uncover new aspects of corneal innervation from which innovative treatments will be designed.

Watch this Scientific Journal Video about Localized Laser Ablation of Murine Corneal Nerves for the Induction of Neurotrophic Keratitis at JoVE.com

Localized Laser Ablation of Murine Corneal Nerves for the Induction of Neurotrophic Keratitis  

To begin, place an anesthetized mouse on a table. Apply a drop of ocular gel on both eyes. Place the animal in a head holder to secure its position.Rotate the animal by 90 degrees so that the eye being operated on faces the ceiling. Secure the animal's vibrancy to clear the area around the eye. Secure the animal's body to minimize head movements caused by breathing.Place a circular cover slip horizontally over the eye on top of the ocular gel. Adjust the animal's head as necessary to correctly position the cover slip. Add a drop of PBS on top of the cover slip, then carefully place the animal onto a microscope stage.Adjust the objective turret of the microscope to the appropriate distance for observation. Activate the lasers to induce corneal nerve ablation. Once nerve ablation is complete, remove the animal from the head holder and place it in a cage on a heated plate.The vortex was visibly absent one week after laser ablation. A group of immune cells were observed to have migrated to the illumination site. The stromal fiber bundles appeared to have thinned out.

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Research

JoVE Journal

Neuroscience

98 Views.  University of  Montpellier, INSERM. To begin, place an anesthetized mouse on a table. Apply a drop of ocular gel on both eyes. Place the animal in a head holder to secure its position.Rotate the animal by 90 degrees so that the eye being operated on faces the ceiling. Secure the animal's vibrancy to clear the area around the eye. Secure the animal's body to minimize head movements caused by breathing.Place a circular cover slip horizontally over the eye on top of the ocular gel. Adjust the animal's head as necessa...