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

L&#233;na Meneux; Alicia Caballero; Hassan Boukhaddaoui; Frederic Michon

doi:10.3791/66182

JoVE Journal > Neuroscience

Neurociência

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

Published: December 08, 2023

doi:

10.3791/66182

Léna Meneux¹, Alicia Caballero¹, Hassan Boukhaddaoui¹, Frederic Michon^1,2

¹Institute for Neurosciences of Montpellier,University of Montpellier, INSERM, ²Department of Ophthalmology,Gui de Chauliac Hospital

Summary

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.

Abstract

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 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.

Introduction

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 Aδ 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'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' 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' 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' 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.

Protocol

All experiments were approved by the National Animal Experiment Board.

1. Preparations

Prepare an anesthetic solution of ketamine-xylazine for anesthesia. Inject ketamine at 80 mg/kg and xylazine at 10 mg/kg by diluting 200 µL of ketamine (100 mg/mL) and 125 µL of xylazine (20 mg/mL) in 2,175 mL of sterile 0.9% NaCl.
Prepare 0.02 mg/mL buprenorphine solution as an analgesic solution by adding 100 µL of 0.3 mg/mL buprenorphine to 1,400 mL of sterile 0.9% NaCl.
Prepare the fluorescent staining solution.
1. Use a fine scale to weigh 10 mg of fluorescein salt and dilute it in 10 mL of phosphate-buffered saline (PBS) to obtain a 0.1% fluorescein solution.
2. Protect the solution from the light and shake it for 5 min. Use a 10 mL syringe and a syringe filter of 0.2 µm to filter the solution.
  NOTE: The fluorescent solution has to be protected from the light and can be stored at +4 °C for 5 days.
Preparation of the mouse
1. Weight the mouse and induce anesthesia by performing an intraperitoneal injection of 10 µL/g of mouse weight (MW) of the anesthetic solution. When the mouse stops moving, place it on a heated plate (37 °C) and verify that it is completely anesthetized by pinching its toes.
2. Apply a drop of artificial tear on the eye undergoing the surgery and a drop of ocular gel on the contralateral eye.
3. Once the mouse is unresponsive to pinch, inject the analgesia (0.02 mg/mL buprenorphine) at 5 µL/g of MW to provide 0.1 mg/kg buprenorphine subcutaneously in the neck.

2. Abrasion of the cornea

Follow step 1.3 to prepare the fluorescent staining solution.
Follow step 1.4 to prepare the mouse.
Before doing the abrasion, dip the ocular burr in 70% EtOH and then in PBS to clean it.
Place the mouse on a heated plate (37 °C) on the side to easily access the eye undergoing the surgery.
1. Use a cotton swab to absorb the artificial tear and brush the eyelashes away without touching the eye.
2. Turn on the ocular burr, open the eyelid of the mouse with two fingers, and simultaneously block the vibrissae to avoid them getting stuck into the burr.
Localize the pupil or the center of the eye and apply the ocular burr on the surface of the eye, and do circular movements. By looking closely, the surface of the removed epithelium can be observed. Otherwise, do approximately 20 circular movements on the cornea.
Put the mouse back on its belly and check if the ocular gel is still present in the contralateral eye.
Apply a drop of fluorescent staining solution on the abraded eye and wait for 20 s. Absorb the drop with a tissue without touching the eye and rinse it with a drop of artificial tear. Absorb the drop of the artificial tear with a tissue and illuminate the eye with the blue cobalt lamp.
NOTE: The abrasion has to have a circular shape. It requires some training to obtain it.
Apply a drop of ocular gel on the abraded eye and let the mouse wake up on the heated pad. When the mouse is moving on its own, put it back in its cage.
Check the well-being of the animal during the following 2 days.
After the use of the ocular burr, use a soft tissue to remove epithelial cells stuck into the head of the burr and dip it in 70% EtOH and then PBS.
NOTE: Once the epithelial cells are removed with the soft tissue, make sure that no pieces of tissue are stuck in the head of the burr.

3. Axotomy of the corneal nerves

Follow step 1.4 to prepare the mouse.
Place the mouse under a binocular loupe on the side to access the eye undergoing the surgery. Put the bottom of a Petri dish under the head of the mouse for the eye to be horizontal.
NOTE: The following steps will be performed by looking through the binocular loupe.
Remove the artificial tear with a cotton swab and remove the eyelashes without touching the eye.
Place smooth curved pliers under the eye of the animal in order to pop it out from the orbit. Make sure to close the pliers enough for the eye not to be able to go back in the orbit once pressure is applied, but not excessively to prevent optic nerve damage.
Apply the biopsy punch of 2.5 mm vertically on the eye without pressure to ensure total contact of the punch with the surface of the cornea.
NOTE: Once the punch is applied against the cornea, the hand of the experimenter might interfere with the field of view.
Then, start applying pressure and twist the punch several times.
NOTE: Depending on the pressure, the number of twists may vary, but generally it is around 5. It requires training to feel the right pressure and movement. If too much pressure is applied, the eye ruptures. In this case, liquid can be observed exiting the lesion and the eye get soften. The animal has to be sacrificed.
Remove the biopsy punch. A circle must be visible on the cornea where the biopsy punch was applied.
NOTE: The next steps do not require the binocular loupe.
Check if the contralateral eye still has ocular gel and apply some on the axotomized eye.
Let the mouse wake up on a heated pad (37 °C) and put it back in its cage when it is moving on its own.
Check the well-being of the animal during the following 2 days.

4. Cornea whole-mount processing

Sample collection and fixation.
1. Weigh the mouse and induce anesthesia by performing an intraperitoneal injection of 10 µL/g of mouse weight (MW) of the anesthetic solution.
2. When the mouse stops moving and does not have reflexes by toe pinching, perform a cervical dislocation.
3. Pop the eye out of the orbit using two fingers and enucleate the eye by cutting the optic nerve using curved scissors.
4. Place the eye in a 2 mL plastic tube containing PBS.
5. Fix the eye by replacing the PBS with 4% paraformaldehyde and placing it on a shaker (30 rpm) at room temperature (RT) for 20 min.
6. Rinse 3 times for 10 min with PBS at RT on a shaker (30 rpm).
Dissection of the cornea
1. Place a drop of PBS on a piece of parafilm inside a Petri dish.
2. Place the eye in this drop of PBS.
3. Place the Petri dish under the binocular loupe.
4. Dissect the cornea using microdissection scissors and forceps. Cut above the ciliary body to keep the limbus.
5. Remove the lens, the ciliary body, and the iris with fine forceps.
6. Place the cornea back into a 2 mL plastic tube.
Immunofluorescence protocol
1. Incubate the cornea in 2 mL of 2.5% fish skin gelatine, 5% goat serum, and 0.5% detergent in PBS for 1 h at RT on a shaker (30 rpm).
2. Incubate the cornea in 150 µL of a solution of 2.5% fish skin gelatine, 5% goat serum, and 0.1% detergent in PBS containing the primary antibody diluted at 1/1000 (anti βIII tubulin antibody) overnight at 4 °C on a shaker (30 rpm).
3. Rinse 3 times for 1 h with 0.1% detergent in PBS at RT on a shaker (30 rpm).
4. Incubate the cornea in the same solution as for step 4.3.2, containing the secondary antibody diluted at 1/500 overnight at 4 °C on a shaker (30 rpm).
5. Rinse 3 times for 1 h with PBS at RT on a shaker (30 rpm).
6. Incubate the cornea for 10 min with 150 µL of DNA intercalator diluted at 1/5000 at RT on a shaker (30 rpm).
7. Rinse 2 times for 5 min with PBS.
Mounting of the cornea
1. Place the cornea on a slide, epithelium facing the slide, and use a scalpel to create four sections without reaching the center.
  NOTE: Do the sections opposite to each other to form a flower shape.
2. Place the cornea endothelium facing the slide and remove the PBS around the cornea using a tissue.
  NOTE: Do not remove the PBS under the cornea; otherwise, air bubbles may appear. If so, add PBS to the cornea and remove the air bubble. Then restart from step 4.3.2.
3. Add model paste on the four corners of a rectangular coverslip.
  NOTE: The model paste elevates the coverslip as the cornea is a thick tissue.
4. Drop 50 µL of a mounting medium on the cornea and lay carefully the coverslip above to avoid bubble formation.
5. Seal the coverslip with nail polish.
Imaging
1. Place the slide under an epifluorescent microscope.
2. Define the size of the mosaic and the depth of the image and start the acquisition.
3. Apply a deblurring and deconvolution program on the acquired image.
  NOTE: Step 4.6.3 is an option selected on the acquisition software (Large Volume Computational Clearing [LVCC])

5. Localized laser ablation of corneal nerves

Turn on the upright multiphoton microscope and the laser combined with an optical parametric oscillator. Activate the heated stage of the microscope (37 °C) and set the lasers at 850 nm and 1100 nm, corresponding to the required wavelengths for the mouse model.
Use a power meter to set the power of the lasers simultaneously at around 20 mW.
Follow step 1.4 to prepare the mouse and apply ocular gel on both eyes.
NOTE: The animal used for this protocol has to be from a transgenic mouse line (MAGIC-Marker transgenic mouse²¹) that expresses an endogenous fluorophore in the corneal nerve fibers.
Install the animal in a head holder. Turn the animal 90° for the eye of interest to face the ceiling.
Strap the vibrissae of the animal to clear the eye area. Strap the animal's body to lessen the head movement induced by breathing.
Position a circular coverslip on the eye above the ocular gel. The coverslip has to be horizontal. Do not hesitate to move the head of the animal for the coverslip to be positioned correctly.
Add a drop of PBS above the coverslip. Place the animal on the microscope stage carefully and set the objective turret at the right distance.
Use the aqueous 20x objective and epifluorescence to localize the eye and the innervation through the eyepiece.
Activate the lasers and define the depth that needs to be illuminated.
Acquire 1024 x 1024 (pixels) image at an acquisition speed of 70%-85% of the maximum scan speed.
Once the image is acquired, take the animal off the microscope and remove the coverslip and the straps. Remove the animal from the head holder, add more ocular gel if needed, and let it wake up in a cage on a heated plate.
NOTE: Destruction of the innervation can be observed 1 week after the imaging. This protocol can be repeated once a week for several weeks to increase the damage caused by the lasers.
When the animal is moving on its own, put the cage back in the stable and check its well-being for the following 2 days.

Representative Results

This study proposes several protocols for inflicting damage on corneal innervation in mice. While similar protocols have been used to investigate the physiopathology of the healing of the epithelium, we chose to adapt and develop new methods of investigating corneal innervation regeneration. To observe the innervation, we used two techniques. First, we employed an immunofluorescence technique to stain the nerve fibers using a pan-neuronal antibody (BIII tubulin) and the nuclei with an intercalator. Second, we took advantage of a transgenic mouse strain expressing fluorescent proteins in the nerve fibers. The corneas were imaged using an epifluorescent microscope.

The first method used an ophthalmic burr with a rust ring remover of 0.5 mm to remove the corneal epithelial layer (Figure 1A). This study focuses on the damage caused to the innervation located inside the epithelium as a means of understanding the cellular and molecular components involved in its regeneration. Before the surgery, the basal epithelium was intact. The nerve fibers in the subbasal plexus formed a typical pattern called the vortex (Figure 1B). The tip of the burr used in this technique removed only the first layer of the cornea but preserved both the stroma, where big bundles of fibers are located, and the endothelium. The part of the epithelium removed by the burr was easily visible, as there was a clear rupture of the basal epithelium and the subbasal plexus (Figure 1B). The nuclei of the keratocytes can be easily identified as they are sparsely arranged throughout the stroma. Regarding the innervation, the border was also clear, with a rupture of the subbasal plexus allowing the big bundles of fibers inside the stroma to be seen. While it takes around 3 days for the epithelium to heal, regeneration of the innervation takes more than 4 weeks after an abrasion. Figure 1 illustrates the regeneration of the epithelium 1 week after the surgery, in which the basal epithelium is smooth. However, the vortex is absent from the center of the cornea, given that the innervation has not yet returned to that point.

The second method consists of sectioning the nerves at the periphery of the cornea with a biopsy punch of 2.5 mm diameter rather than brushing them away with the burr (Figure 2A). This method does not disrupt the epithelium as much as the abrasion. After the surgery, a thin peripheral ring can be observed at the point where the biopsy punch was applied, damaging the basal epithelium (Figure 2B). However, the nerves at the periphery have been sectioned, triggering the Wallerian degeneration of the axons. This type of degeneration consists of the decay of nerves from the terminal ending of the neurons toward the cell body and, in this case, toward the section induced by the biopsy punch. Depending on the depth of the axotomy, damage can be severe. Here, we see that heavy degeneration has occurred, leading to a total loss of innervation in the center of the cornea in the epithelium (Figure 2B). As innervation is essential to the maintenance of epithelium homeostasis, a disintegration of the epithelial layer is initiated, creating an ulcer (Figure 2B). Ulcer formation only happened in heavily damaged corneas.

The last technique consists of locally burning in vivo the corneal nerves of anesthetized transgenic mice with a multiphoton microscope (Figure 3A). The first image is acquired at the location where the burn will be made. This study used a transgenic mouse whose innervation expresses fluorescent proteins. The image was acquired in the center of the cornea, where the vortex is located (Figure 3B), from the top of the epithelium to the mid-stroma. The vortex is no longer visible 1 week after the first image acquisition. Instead, a group of immune cells has migrated to the illumination site, where we can identify the bundle of fibers in the stroma, which have thinned following the first acquisition (Figure 3B).

Figure 1: Ocular burr and example of corneal innervation following abrasion. (A) An ophthalmologic burr with a rust ring remover of 0.5 mm was used to make the abrasion. (B) Examples of dissected and immunostained corneas. Pre-abrasion images illustrate the smooth basal epithelium and the vortex (white arrowhead) in the center. Post-abrasion images show a clear delimitation of the subbasal plexus, which was removed (red arrowheads). Images acquired at 1 week post-abrasion (1WPAbr) show where the fibers are regenerating (orange arrowhead). The innervation is stained with anti-BIII tubulin antibody (green). The nuclei are stained with a DNA intercalator (purple). The images were acquired with an epifluorescent microscope. Scale bars = 500 µm. Please click here to view a larger version of this figure.

Figure 2: Biopsy punch and example of corneal innervation following axotomy. (A) A sterile biopsy punch of 2.5 mm diameter is applied to the eye to induce an axotomy. (B) Examples of dissected and immunostained corneas. Pre-axotomy images illustrate the presence of the vortex in the center of the cornea (white arrowhead). Post-axotomy images show the ring (red arrowheads) created by the punch in the epithelium. Images acquired at 2 weeks post-axotomy (2WPAx) illustrate the formation of the ulcer (orange arrowhead). The innervation is stained with BIII tubulin (green). The nuclei are stained with an intercalator (purple). The images were acquired with an epifluorescent microscope. Scale bars = 500 µm. Please click here to view a larger version of this figure.

Figure 3: Set up for corneal nerve cauterization by lasers and an example (A) The anesthetized MAGIC-Marker transgenic mouse²¹ is maintained by the head-holder, and its eye is facing the 20x objective of the biphoton. The body of the mouse is strapped to avoid breathing movement during acquisition. (B) Three-dimensional (3D) view of the first acquisition of the vortex using the wavelengths 850 nm and 1100 nm simultaneously before the photodamage and 1 week post photodamage (1WPPhDa) at the same location of the vortex. The lasers induced migration of immune cells (arrowhead) where the vortex use to be located. Scale bars = 80 µm. Please click here to view a larger version of this figure.

Technique	Impacted structure	Degree of impact (+/-)	Type of study	Healing period
Abrasion	Epithelium	++	Epithelium and innervation regeneration	Epithelium <1 week Innervation >2 months
	Innervation	++
Axotomy	Epithelium	+/-	Innervation regeneration Neurotrophic keratitis	Innervation >2 months NK >2 weeks
	Stroma	+/-
	Innervation	++
Nerve cauterization	Epithelium	+ (local)	Local innervation regeneration Neurotrophic keratitis	Depending on the frequence of cauterization
	Stroma
	Innervation

Table 1: Comparison of the three proposed strategies.

Discussion

Neurotrophic keratitis is considered a rare disease, affecting 5 in 10,000 individuals. However, people suffering from NK due to a physical injury such as chemical burns, or syndromes such as diabetes or multiple sclerosis are not included in those statistics³. Furthermore, this condition remains significantly underdiagnosed²², and the prevalence of the disease is underestimated. There is a strong need for new treatments and therapy that would promote axon regeneration as a means of preserving patients' sight. Currently, the only available treatment offered to patients suffering from the early stages of NK is the application of pharmacological eyedrops²³^,²⁴^,²⁵ that support corneal nerve regeneration. These, however, are effective only to a certain point. In cases of more severe alteration of the cornea, such as ulcers, amniotic membrane transplant or keratoplasty³^,⁴ offer a temporary solution to close the wound and avoid infection. However, those grafts must be replaced after a limited time, with the risk of rejection increasing at each additional procedure.

This report presents several models recapitulating different forms of NK commonly encountered in the clinic (Table 1). These can be used to deepen our knowledge of corneal innervation and to investigate alternatives to current treatments. The abrasion model can be associated with PRK surgery, given that it recapitulates the slow regrowth of the innervation, which occurs after the healing of the abraded epithelium. To its advantage, this technique can be taught and reproduced relatively easily when the correct pressure with the ophthalmic burr is applied, and a regular abraded area is created. Furthermore, this technique can be adapted to each patient's specific needs or to suit the user's interest by choosing the size of the abraded area.

The axotomy is similar to the lamellar keratoplasty undergone by patients suffering from late-stage NK²⁶. It enables investigation of the degeneration of the fibers (Wallerian degeneration) and the recolonization of the axons in the central cornea. While the material required to perform the surgery is relatively simple, the practitioner must be trained in the proper technique, notably the correct application of pressure and the correct number of turns with the biopsy punch. In the context of a keratoplasty, the depth of the incision is precisely determined through the use of a trephine device and intraoperative optical coherence tomography, which techniques are not adaptable to murine models¹⁷. Another dissimilarity to keratoplasty is the absence of donor transplant in the technique described here, as we only performed the incision of the cornea. This model is of interest because of the presence of a healthy epithelium in which the innervation must grow back. This differentiates it from the abrasion model, where the epithelium is removed with the innervation.

The nerve cauterization technique, using a multiphoton microscope, mimics the damage caused by sunburn but does so on a localized area of the cornea, thus triggering DNA damage and ROS formation¹⁹^,²⁰. With this method, it is possible to study the regeneration of the fibers from different locations on the cornea and even to repeat cauterization in the same area several times, enabling the impact on axon growth and immune cell migration to be evaluated.

These three protocols offer the possibility of studying the fundamental aspects of axonal regrowth by investigating the regenerative mechanisms, as well as the interactions between the different cell types present in the cornea and the innervation. These methods also offer a means of promoting axon regeneration by applying new pharmacological molecules²⁴ or gene therapy²⁷.

Declarações

The authors have nothing to disclose.

Acknowledgements

The authors thank Dr. Karine Loulier for the access to the transgenic mouse line MAGIC-Markers. The authors also thank the RAM-Neuro animal core facility and the imaging facility MRI, a member of the France-BioImaging national infrastructure supported by the French National Research Agency (ANR-10-INBS-04, "Investments for the future"). This research was supported by the ATIP-Avenir program, Inserm, Région Occitanie, the University of Montpellier, the French National Research Agency (ANR-21-CE17-0061), the Fondation pour la Recherche Médicale (FRM Regenerative Medicine, REP202110014140), and the Groupama Foundation.

Materials

0.2 µm seringe filter	CLEARLINE	51733
0.5 mm rust ring remover	Alger Equipment Company	BU-5S
2 mL plastic tubes	Eppendrof	30120094
Algerbrush burr, Complete instrument	Alger Equipment Company	BR2-5
Anti-beta III Tubulin antibody	Abcam	ab18207
Antigenfix	Diapath	P0016
Artificial tear	Larmes artificielles Martinet	N/A
Buprecare	Animalcare	N/A
Cotton swab	Any provider	N/A
Dissecting tools	Fine Science Tools	N/A
Fluorescein	Merck	103887
Gelatin from cold water fish skin	Sigma	G7765
Goat serum	Merck	S26
Head Holder	Narishige	SGM 4
Heated plate	BIOSEB LAB instruments	BIO-HE002
Hoechst 33342	Thermo Fisher Scientific	H3570
Imalgene 1000	BOEHRINGER INGELHEIM ANIMAL HEALTH France	N/A	French marketing authorization numbre: FR/V/0167433 4/1992
LAS X software	Leica	N/A	Large volume computational clearing (LVCC) process
Laser Chameleon Ultra II	Coherent	N/A
Laser power meter	Coherent	N/A
Leica Thunder Imager Tissue microscope	Leica	N/A
Multi-photon Zeiss LSM 7MP upright microscope	Zeiss	N/A
Ocry-gel	TVM lab	N/A
Parametric oscillator	Coherent	N/A
Penlights with blue cobalt filtercap	Bernell	ALPEN
Petri dish	Thermo Scientific	150318	Axotomy protocol
Petridish	Thermo Scientific	150288	Cornea whole-mount processing
Rompun 2%	Elanco	N/A	French marketing authorization numbre: FR/V/8146715 2/1980
Sterile biopsy punch 2.5 mm	LCH medical	LCH-PUK-25
Triton X-100	VWR	0694
Vectashield	EuroBioSciences	H-1000	Mounting medium

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Meneux, L., Caballero, A., Boukhaddaoui, H., Michon, F. Three Strategies to Induce Neurotrophic Keratitis and Nerve Regeneration in Murine Cornea. J. Vis. Exp. (202), e66182, doi:10.3791/66182 (2023).