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, &#34;Investments for the future&#34;). This research was supported by the ATIP-Avenir program, Inserm, R&#233;gion Occitanie, the University of Montpellier, the French National Research Agency (ANR-21-CE17-0061), the Fondation pour la Recherche M&#233;dicale (FRM Regenerative Medicine, REP202110014140), and the Groupama Foundation.

All experiments were approved by the National Animal Experiment Board.
1. Preparations
<ol>
	<li>Prepare an anesthetic solution of ketamine-xylazine for anesthesia. Inject ketamine at 80 mg/kg and xylazine at 10 mg/kg by diluting 200 &#181;L of ketamine (100 mg/mL) and 125 &#181;L of xylazine (20 mg/mL) in 2,175 mL of sterile 0.9% NaCl.</li>
	<li>Prepare 0.02 mg/mL buprenorphine solution as an analgesic solution by adding 100 &#181;L of 0.3 mg/mL buprenorphine to 1,400 mL of sterile 0.9% NaCl.</li>
	<li>Prepare the fluorescent staining solution.
	<ol>
		<li>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.</li>
		<li>Protect the solution from the light and shake it for 5 min. Use a 10 mL syringe and a syringe filter of 0.2 &#181;m to filter the solution. 
		NOTE: The fluorescent solution has to be protected from the light and can be stored at +4 &#176;C for 5 days.</li>
	</ol>
	</li>
	<li>Preparation of the mouse
	<ol>
		<li>Weight the mouse and induce anesthesia by performing an intraperitoneal injection of 10 &#181;L/g of mouse weight (MW) of the anesthetic solution. When the mouse stops moving, place it on a heated plate (37 &#176;C) and verify that it is completely anesthetized by pinching its toes.</li>
		<li>Apply a drop of artificial tear on the eye undergoing the surgery and a drop of ocular gel on the contralateral eye.</li>
		<li>Once the mouse is unresponsive to pinch, inject the analgesia (0.02 mg/mL buprenorphine) at 5 &#181;L/g of MW to provide 0.1 mg/kg buprenorphine subcutaneously in the neck.</li>
	</ol>
	</li>
</ol>
2. Abrasion of the cornea
<ol>
	<li>Follow step 1.3 to prepare the fluorescent staining solution.</li>
	<li>Follow step 1.4 to prepare the mouse.</li>
	<li>Before doing the abrasion, dip the ocular burr in 70% EtOH and then in PBS to clean it.</li>
	<li>Place the mouse on a heated plate (37 &#176;C) on the side to easily access the eye undergoing the surgery.
	<ol>
		<li>Use a cotton swab to absorb the artificial tear and brush the eyelashes away without touching the eye.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Put the mouse back on its belly and check if the ocular gel is still present in the contralateral eye.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Check the well-being of the animal during the following 2 days.</li>
	<li>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. 
	&#8203;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.</li>
</ol>
3. Axotomy of the corneal nerves
<ol>
	<li>Follow step 1.4 to prepare the mouse.</li>
	<li>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.</li>
	<li>Remove the artificial tear with a cotton swab and remove the eyelashes without touching the eye.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Check if the contralateral eye still has ocular gel and apply some on the axotomized eye.</li>
	<li>Let the mouse wake up on a heated pad (37 &#176;C) and put it back in its cage when it is moving on its own.</li>
	<li>Check the well-being of the animal during the following 2 days.</li>
</ol>
4. Cornea whole-mount processing
<ol>
	<li>Sample collection and fixation.
	<ol>
		<li>Weigh&#160;the mouse and induce anesthesia by performing an intraperitoneal injection of 10 &#181;L/g of mouse weight (MW) of the anesthetic solution.</li>
		<li>When the mouse stops moving and does not have reflexes by toe pinching, perform a cervical dislocation.</li>
		<li>Pop the eye out of the orbit using two fingers and enucleate the eye by cutting the optic nerve using curved scissors.</li>
		<li>Place the eye in a 2 mL plastic tube containing PBS.</li>
		<li>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.</li>
		<li>Rinse 3 times for 10 min with PBS at RT on a shaker (30 rpm).</li>
	</ol>
	</li>
	<li>Dissection of the cornea
	<ol>
		<li>Place a drop of PBS on a piece of parafilm inside a Petri dish.</li>
		<li>Place the eye in this&#160;drop of PBS.</li>
		<li>Place the Petri dish under the binocular loupe.</li>
		<li>Dissect the cornea using microdissection scissors and forceps. Cut above the ciliary body to keep the limbus.</li>
		<li>Remove the lens, the ciliary body, and the iris with fine forceps.</li>
		<li>Place the cornea back into a 2 mL plastic tube.</li>
	</ol>
	</li>
	<li>Immunofluorescence protocol
	<ol>
		<li>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).</li>
		<li>Incubate the cornea in 150 &#181;L 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 &#946;III tubulin antibody) overnight at 4 &#176;C on a shaker (30 rpm).</li>
		<li>Rinse 3 times for 1 h with 0.1% detergent in PBS at RT on a shaker (30 rpm).</li>
		<li>Incubate the cornea in the same solution as for step 4.3.2, containing the secondary antibody diluted at 1/500 overnight at 4 &#176;C on a shaker (30 rpm).</li>
		<li>Rinse 3 times for 1 h with PBS at RT on a shaker (30 rpm).</li>
		<li>Incubate the cornea for 10 min with 150 &#181;L of DNA intercalator diluted at 1/5000 at RT on a shaker (30 rpm).</li>
		<li>Rinse 2 times for 5 min with PBS.</li>
	</ol>
	</li>
	<li>Mounting of the cornea
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Drop 50 &#181;L of a mounting medium on the cornea and lay carefully the coverslip above to avoid bubble formation.</li>
		<li>Seal the coverslip with nail polish.</li>
	</ol>
	</li>
	<li>Imaging
	<ol>
		<li>Place the slide under an epifluorescent microscope.</li>
		<li>Define the size of the mosaic and the depth of the image and start the acquisition.</li>
		<li>Apply a deblurring and deconvolution program on the acquired image. 
		&#8203;NOTE: Step 4.6.3 is an option selected on the acquisition software (Large Volume Computational Clearing [LVCC])</li>
	</ol>
	</li>
</ol>
5. Localized laser ablation of corneal nerves
<ol>
	<li>Turn on the upright multiphoton microscope and the laser combined with an optical parametric oscillator. Activate the heated stage of the microscope (37 &#176;C) and set the lasers at 850 nm and 1100 nm, corresponding to the required wavelengths for the mouse model.</li>
	<li>Use a power meter to set the power of the lasers simultaneously at around 20 mW.</li>
	<li>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.</li>
	<li>Install the animal in a head holder. Turn the animal 90&#176; for the eye of interest to face the ceiling.</li>
	<li>Strap the vibrissae of the animal to clear the eye area. Strap the animal&#39;s body to lessen the head movement induced by breathing.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Use the aqueous 20x objective and epifluorescence to localize the eye and the innervation through the eyepiece.</li>
	<li>Activate the lasers and define the depth that needs to be illuminated.</li>
	<li>Acquire 1024 x 1024 (pixels) image at an acquisition speed of 70%-85% of the maximum scan speed.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

Corneal Sensory Fiber Damage Protocols for Axon Regeneration Studies

<ol>
	<li>Marfurt, C. F., Cox, J., Deek, S., Dvorscak, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+human+corneal+innervation.">Anatomy of the human corneal innervation.</a> Exp Eye Res. 90 (4), 478-492 (2010).</li><li>Al-Aqaba, M. A., Dhillon, V. K., Mohammed, I., Said, D. G., Dua, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+nerves+in+health+and+disease.">Corneal nerves in health and disease.</a> Prog Retin Eye Res. 73, 100762 (2019).</li><li>Dua, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophic+keratopathy.">Neurotrophic keratopathy.</a> Prog Retin Eye Res. 66, 107-131 (2018).</li><li>Bonini, S., Rama, P., Olzi, D., Lambiase, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophic+keratitis.">Neurotrophic keratitis.</a> Eye. 17 (8), 989-995 (2003).</li><li>Barrientez, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+Injury:+Clinical+and+molecular+aspects.">Corneal Injury: Clinical and molecular aspects.</a> Exp Eye Res. 186, 107709 (2019).</li><li>Willmann, D., Fu, L., Melanson, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+Injury.">Corneal Injury.</a> StatPearls. , (2023).</li><li>Kalha, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bmi1++progenitor+cell+dynamics+in+murine+cornea+during+homeostasis+and+wound+healing.">Bmi1+ progenitor cell dynamics in murine cornea during homeostasis and wound healing.</a> Stem Cells. 36 (4), 562-573 (2018).</li><li>Park, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+roles+of+nitrate+and+nitrite+in+nitric+oxide+metabolism+in+the+eye.">Potential roles of nitrate and nitrite in nitric oxide metabolism in the eye.</a> Sci Rep. 10 (1), 13166 (2020).</li><li>Ikkala, K., Stratoulias, V., Michon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unilateral+corneal+insult+in+Zebrafish+results+in+a+bilateral+cell+shape+and+identity+modification,+supporting+wound+closure.">Unilateral corneal insult in Zebrafish results in a bilateral cell shape and identity modification, supporting wound closure.</a> bioRxiv. , (2021).</li><li>Yang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substance+P+promotes+diabetic+corneal+epithelial+wound+healing+through+molecular+mechanisms+mediated+via+the+Neurokinin-1+receptor.">Substance P promotes diabetic corneal epithelial wound healing through molecular mechanisms mediated via the Neurokinin-1 receptor.</a> Diabetes. 63 (12), 4262-4274 (2014).</li><li>Zhao, W., He, X., Liu, R., Ruan, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerating+corneal+wound+healing+using+exosome-mediated+targeting+of+NF-&#954;B+c-Rel.">Accelerating corneal wound healing using exosome-mediated targeting of NF-&#954;B c-Rel.</a> Inflamm Regen. 43 (1), 6 (2023).</li><li>Downie, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+the+sub-basal+nerve+plexus+and+superficial+nerve+terminals+after+corneal+epithelial+injury+in+mice.">Recovery of the sub-basal nerve plexus and superficial nerve terminals after corneal epithelial injury in mice.</a> Exp Eye Res. 171, 92-100 (2018).</li><li>He, J., Pham, T. L., Kakazu, A. H., Bazan, H. E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+of+substance+P+sensory+nerves+and+transient+receptor+potential+melastatin+8+(TRPM8)+cold+receptors+after+corneal+experimental+surgery.">Remodeling of substance P sensory nerves and transient receptor potential melastatin 8 (TRPM8) cold receptors after corneal experimental surgery.</a> Invest Ophthalmol Vis Sci. 60 (7), 2449-2460 (2019).</li><li>Bandeira, F., Yusoff, N. Z., Yam, G. H. -. F., Mehta, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+reinnervation+following+refractive+surgery+treatments.">Corneal reinnervation following refractive surgery treatments.</a> Neural Regen Res. 14 (4), 557-565 (2019).</li><li>Erie, J. C., McLaren, J. W., Hodge, D. O., Bourne, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+corneal+subbasal+nerve+density+after+PRK+and+LASIK.">Recovery of corneal subbasal nerve density after PRK and LASIK.</a> Am J Ophthalmol. 140 (6), 1059-1064.e1 (2005).</li><li>Coleman, M. P., Freeman, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wallerian+degeneration,+WldS,+and+Nmnat.">Wallerian degeneration, WldS, and Nmnat.</a> Annu Rev Neurosci. 33, 245-267 (2010).</li><li>Arenas, E., Esquenazi, S., Anwar, M., Terry, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lamellar+corneal+transplantation.">Lamellar corneal transplantation.</a> Surv Ophthalmol. 57 (6), 510-529 (2012).</li><li>Niederer, R. L., Perumal, D., Sherwin, T., McGhee, C. N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+innervation+and+cellular+changes+after+corneal+transplantation:+An+in+vivo+confocal+microscopy+study.">Corneal innervation and cellular changes after corneal transplantation: An in vivo confocal microscopy study.</a> Invest Ophthalmol Vis Sci. 48 (2), 621-626 (2007).</li><li>Icha, J., Weber, M., Waters, J. C., Norden, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototoxicity+in+live+fluorescence+microscopy,+and+how+to+avoid+it.">Phototoxicity in live fluorescence microscopy, and how to avoid it.</a> BioEssays. 39 (8), 1700003 (2017).</li><li>Volatier, T., Schumacher, B., Cursiefen, C., Notara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UV+protection+in+the+cornea:+Failure+and+rescue.">UV protection in the cornea: Failure and rescue.</a> Biology. 11 (2), 278 (2022).</li><li>Loulier, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+cell+and+lineage+tracking+with+combinatorial+labels.">Multiplex cell and lineage tracking with combinatorial labels.</a> Neuron. 81 (3), 505-520 (2014).</li><li>Dana, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expert+consensus+on+the+identification,+diagnosis,+and+treatment+of+neurotrophic+keratopathy.">Expert consensus on the identification, diagnosis, and treatment of neurotrophic keratopathy.</a> BMC Ophthalmol. 21 (1), 327 (2021).</li><li>Matsumoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+serum+application+in+the+treatment+of+neurotrophic+keratopathy.">Autologous serum application in the treatment of neurotrophic keratopathy.</a> Ophthalmology. 111 (6), 1115-1120 (2004).</li><li>Bonini, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+II+randomized,+double-masked,+vehicle-controlled+trial+of+recombinant+human+nerve+growth+factor+for+neurotrophic+keratitis.">Phase II randomized, double-masked, vehicle-controlled trial of recombinant human nerve growth factor for neurotrophic keratitis.</a> Ophthalmology. 125 (9), 1332-1343 (2018).</li><li>Aggarwal, S., Colon, C., Kheirkhah, A., Hamrah, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+autologous+serum+tears+for+treatment+of+neuropathic+corneal+pain.">Efficacy of autologous serum tears for treatment of neuropathic corneal pain.</a> Ocul Surf. 17 (3), 532-539 (2019).</li><li>Singh, N. P., Said, D. G., Dua, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lamellar+keratoplasty+techniques.">Lamellar keratoplasty techniques.</a> Indian J Ophthalmol. 66 (9), 1239-1250 (2018).</li><li>Gautier, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV2/9-mediated+gene+transfer+into+murine+lacrimal+gland+leads+to+a+long-term+targeted+tear+film+modification.">AAV2/9-mediated gene transfer into murine lacrimal gland leads to a long-term targeted tear film modification.</a> Mol Ther Methods Clin Dev. 27, 1-16 (2022).</li></ol>

The authors have no conflicts of interest to disclose.

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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<tr>
			<td>0.2 &#181;m seringe filter</td>
			<td>CLEARLINE</td>
			<td>51733</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mm rust ring remover</td>
			<td>Alger Equipment Company</td>
			<td>BU-5S</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL plastic tubes</td>
			<td>Eppendrof&#160;</td>
			<td>30120094</td>
			<td></td>
		</tr>
		<tr>
			<td>Algerbrush burr, Complete instrument</td>
			<td>Alger Equipment Company</td>
			<td>BR2-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-beta III Tubulin antibody</td>
			<td>Abcam</td>
			<td>ab18207</td>
			<td></td>
		</tr>
		<tr>
			<td>Antigenfix</td>
			<td>Diapath</td>
			<td>P0016</td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial tear</td>
			<td>Larmes artificielles Martinet</td>
			<td>N/A</td>
			<td></td>
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		<tr>
			<td>Buprecare</td>
			<td>Animalcare</td>
			<td>N/A</td>
			<td></td>
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		<tr>
			<td>Cotton swab</td>
			<td>Any provider</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting tools</td>
			<td>Fine Science Tools</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein</td>
			<td>Merck</td>
			<td>103887</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin from cold water fish skin</td>
			<td>Sigma</td>
			<td>G7765</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Merck</td>
			<td>S26</td>
			<td></td>
		</tr>
		<tr>
			<td>Head Holder</td>
			<td>Narishige</td>
			<td>SGM 4</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated plate</td>
			<td>BIOSEB LAB instruments</td>
			<td>BIO-HE002</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Thermo Fisher Scientific</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Imalgene 1000</td>
			<td>BOEHRINGER INGELHEIM ANIMAL HEALTH France</td>
			<td>N/A</td>
			<td>French marketing authorization numbre: FR/V/0167433 4/1992</td>
		</tr>
		<tr>
			<td>LAS X software</td>
			<td>Leica</td>
			<td>N/A</td>
			<td>Large volume computational clearing (LVCC) process</td>
		</tr>
		<tr>
			<td>Laser Chameleon Ultra II</td>
			<td>Coherent</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser power meter</td>
			<td>Coherent</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Thunder Imager Tissue microscope</td>
			<td>Leica</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-photon Zeiss LSM 7MP upright microscope</td>
			<td>Zeiss</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ocry-gel</td>
			<td>TVM lab</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Parametric oscillator</td>
			<td>Coherent</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Penlights with blue cobalt filtercap</td>
			<td>Bernell</td>
			<td>ALPEN</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Thermo Scientific</td>
			<td>150318</td>
			<td>Axotomy protocol</td>
		</tr>
		<tr>
			<td>Petridish</td>
			<td>Thermo Scientific</td>
			<td>150288</td>
			<td>Cornea whole-mount processing</td>
		</tr>
		<tr>
			<td>Rompun 2%</td>
			<td>Elanco</td>
			<td>N/A</td>
			<td>French marketing authorization numbre: FR/V/8146715 2/1980</td>
		</tr>
		<tr>
			<td>Sterile biopsy punch 2.5 mm</td>
			<td>LCH medical</td>
			<td>LCH-PUK-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>VWR</td>
			<td>0694</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>EuroBioSciences</td>
			<td>H-1000</td>
			<td>Mounting medium</td>
		</tr>
	</tbody>
</table>

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.

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.

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 advant...

Watch this Scientific Journal Video about Advancing Corneal Innervation Research Through Innovative Models at JoVE.com

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 ...

three-strategies-to-induce-neurotrophic-keratitis-nerve-regeneration

Research

JoVE Journal

Neuroscience

Murine Corneal Nerve Abrasion for the Induction of Neurotrophic Keratitis

To begin corneal abrasion, place an anesthetized mouse on a table. Apply a drop of artificial tear on the eye undergoing the surgery. Apply a drop of ocular gel on the contralateral eye.Next, position the mouse on its side on a heated plate. Gently use a cotton swab to absorb the artificial tear, and brush aside the mouse's eyelashes without making contact with the eye. Turn on the ocular burr, and carefully open the mouse's eyelid with two fingers.Simultaneously shield the vibrancy to prevent them from entangling in the burr. Locate the pupil or the eye center and gently apply the ocular burr to the eye's surface in circular motions until the epithelium is removed. Alternatively, perform about 20 circular movements on the cornea.Now apply a drop of fluorescent staining solution on the abraided eye. After 20 seconds, dab the eye with a tissue to absorb the solution. Then rinse it with a drop of artificial tear.Finally, absorb the artificial tear with a tissue, and illuminate the eye using a blue cobalt lamp. Prior to the surgery, the basal epithelium was intact and the nerve fibers in the sub basal plexus formed a vortex. The burr tip removed the outer corneal layer, leaving the stroma and endothelium intact with a visible gap at the basal epithelium and sub basal plexus.Regeneration of the epithelium occurred over a week post-surgery. However, the vortex remained absent from the corneal center.

Murine Corneal Nerve Axotomy for the Induction of Neurotrophic Keratitis

To begin corneal nerve axotomy, place an anesthetized mouse on a table. Apply a drop of artificial tear on the eye undergoing the surgery. Apply a drop of ocular gel on the contralateral eye.Position the mouse under a binocular loupe to assess the eye that is being operated on. Place the bottom of a Petri dish under the mouse's head to keep the eye horizontal. With a cotton swab, carefully dad the artificial tear from the eye and remove the eyelashes without touching the eye.Place a pair of smooth curved pillars under the animal's eye and gently pop it out of the orbit. Gently place a biopsy punch vertically against the cornea surface, apply pressure, and twist the punch several times. Then, remove the biopsy punch and a circular mark should be visible on the cornea.A thin peripheral ring was seen at the point where the biopsy punch was applied, which damaged the basal epithelium. The peripheral nerves appeared to be sectioned, triggering the Wallerian axon degeneration. Heavy degeneration was observed, leading to total loss of innervation in the cornea center.This resulted in the disintegration of the epithelial layer, creating an ulcer.

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.

987 Views.  University of  Montpellier, INSERM.  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.

Corneal Sensory Fiber Damage Protocols for Axon Regeneration Studies

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