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 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.
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.
All experiments were approved by the National Animal Experiment Board.
1. Preparations
2. Abrasion of the cornea
3. Axotomy of the corneal nerves
4. Cornea whole-mount processing
5. Localized laser ablation of corneal nerves
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.
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.
The authors have nothing to disclose.
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.
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 |