# Injury-specific pathways shape corneal nerve regeneration dynamics

Léna Meneux, Sarah Pernot, Nadège Feret, Melissa Girard, Alicia Caballero
Megido, Marilou Decoudu, Naima Nhiri, Elea Miessen, Eric Jacquet, Vincent
Daien, et al.

► To cite this version:

Léna Meneux, Sarah Pernot, Nadège Feret, Melissa Girard, Alicia Caballero Megido, et al. Injury-specific pathways shape corneal nerve regeneration dynamics. Scientific Reports, 2025, 15 (1), pp.25755. 10.1038/s41598-025-10434-y. hal-05203818

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# Injury-specific pathways shape corneal nerve regeneration dynamics

Léna Meneux<fnref n="1" />,⁵, Sarah Pernot<fnref n="1" />,⁵, Nadège Feret<fnref n="1" />, Melissa Girard<fnref n="1" />, Alicia Caballero Megido<fnref n="1" />, Marilou Decoudu<fnref n="1" />, Naima Nhiri², Elea Miessen<fnref n="1" />, Eric Jacquet², Vincent Daien<fnref n="1" />,³,⁴ & Frederic Michon<fnref n="1" />,³✉

The cornea, a transparent tissue critical for clear vision, is uniquely characterized by a dense network of peripheral sensory nerves. This innervation not only enables sensation but also provides vital trophic support, ensuring corneal health and integrity. Damage or loss of these peripheral nerves leads to neurotrophic keratitis (NK), a debilitating condition that disrupts corneal healing and function, ultimately threatening vision. Remarkably similar to other peripheral neuropathies, NK arises from various injuries, diseases, or surgical interventions, yet its underlying regenerative mechanisms remain poorly understood, limiting effective treatment options. In this study, we investigated how corneal nerves regenerate following two different types of clinically relevant injuries—axotomy (mimicking nerve damage during corneal transplantation) and epithelial abrasion (resembling superficial corneal surgery). We found that the type of injury distinctly influences both the structural and functional recovery of corneal nerves. Axotomy resulted in delayed nerve regeneration, significant disruption of epithelial cell homeostasis, and increased risk of severe corneal damage. Conversely, epithelial abrasion induced rapid but structurally abnormal nerve recovery. Furthermore, we identified specific molecular pathways activated differently depending on injury type. Our findings highlight the complexity of peripheral nerve regeneration, underscoring the necessity for targeted therapeutic strategies tailored to individual injury scenarios. By illuminating the variability in nerve recovery, our study provides valuable insights applicable not only to NK but potentially to a broader spectrum of peripheral neuropathies, paving the way for novel therapeutic interventions.

**Keywords** Cornea, Innervation, Development, Regeneration

The unique structural organization of the cornea is critical for preserving both its optical transparency and mechanical strength. Accounting for approximately 75% of the ocular refractive power, the cornea also serves as a frontline defense against environmental insults<fnref n="1" />. Any disruption to its structural integrity can severely impair vision, potentially leading to blindness. Anatomically, the cornea is composed mainly of the stroma, which constitutes about 90% of its thickness and consists largely of extracellular matrix interspersed with keratocytes. This central layer is flanked by a stratified, self-renewing epithelium on the anterior surface and a monolayer of endothelial cells posteriorly². The maintenance of corneal transparency and function is critically dependent on homeostasis, orchestrated by a specialized microenvironment that minimizes damage and supports repair. Key components of this microenvironment include the tear film, the epithelial barrier, and an exceptionally dense network of corneal nerves that collectively deliver essential trophic support³⁻⁵.

Indeed, the cornea is among the most densely innervated tissues in the human body, predominantly by sensory C and Aδ fibers originating from the ophthalmic branch of the trigeminal ganglion⁶. During embryonic development in mice, these sensory axons emerge from the trigeminal ganglion, advance along the optic cup, and penetrate the corneal stroma. They branch extensively to form the subepithelial plexus, before turning perpendicularly through Bowman's membrane to establish the subbasal plexus—identified by its characteristic central vortex pattern—and ultimately terminate as free nerve endings at the epithelial surface⁷⁻⁹.

<footnote n="1">¹Institute for Neurosciences of Montpellier, INSERM, Univ Montpellier, Montpellier, France. ²Institut de Chimie des Substances Naturelles, CNRS UPR2301, Paris-Saclay University, Gif-sur-Yvette, France. ³Department of Ophthalmology, Gui de Chauliac Hospital, CHU Montpellier, Montpellier, France. ⁴Save Sight Institute, Sydney Medical School, The University of Sydney, Sydney, NSW, Australia. ⁵Léna Meneux and Sarah Pernot contributed equally to this work. ✉email: frederic.michon@inserm.fr</footnote>
In the mature cornea, nerve fibers exhibit functional specialization¹⁰: cold-sensitive fibers expressing TRPM8 respond to temperature changes, mechano-nociceptors activate through Piezo2 in response to mechanical stimuli, and polymodal nociceptors—by far the most prevalent—react to mechanical, thermal, and chemical stimuli. These fibers not only mediate protective reflexes such as blinking and tear production, but also release a suite of trophic factors that preserve corneal integrity¹¹⁻¹³. Disruption of this innervation network can lead to neurotrophic keratopathy (NK), a progressive degenerative condition marked by loss of sensation, impaired epithelial repair, and ultimately, vision-threatening corneal ulceration¹⁴,¹⁵.

Clinically, NK advances through defined stages: from mild epithelial defects and tear film instability to persistent epithelial breakdown, stromal involvement, and potential perforation¹⁶. Etiologies include infectious diseases, systemic conditions such as diabetes, trauma, and ocular surgeries. Current treatment options range from pharmacological supports¹⁷⁻¹⁹ to surgical interventions like nerve grafts²⁰,²¹ or corneal transplantation²²⁻²⁴, but these often face limited efficacy and risk of complications²⁵,²⁶. Due to its well-defined pathology, accessible location, and relevance to broader nerve disorders, NK represents a powerful model for exploring peripheral neuropathy and developing novel regenerative therapies.

Despite its significance, our understanding of the molecular and cellular mechanisms governing corneal nerve regeneration remains limited—hindering therapeutic innovation. Addressing this gap, our study provides a comprehensive temporal characterization of corneal nerve development in mice during the slow corneal maturation²⁷, detailing the progression of axonal growth and fiber maturation. Moreover, we employ two distinct injury models—axotomy, simulating deep nerve disruption akin to transplantation, and epithelial abrasion, mimicking superficial clinical injuries such as those from refractive surgery.

Our data reveal that nerve regeneration is not a uniform process but is highly dependent on the nature of the injury. We demonstrate distinct regenerative trajectories, epithelial responses, and gene expression profiles between the two models. Notably, abrasion appeared to recapitulate aspects of developmental neurogenesis, while axotomy induced deeper tissue remodeling and inflammation. These findings offer novel insight into the complexity of corneal nerve repair and suggest that injury-specific regenerative pathways could be harnessed to design targeted therapies. Beyond NK, this research contributes broadly to our understanding of peripheral nerve regeneration, with implications for a range of neuropathic conditions.

# Results

## Corneal sensory innervation has a slow paced maturation

Given the cornea essential roles in maintaining transparency and mechanical integrity, elucidating the formation of its innervation during development is critical. While previous studies have primarily relied on different murine models, we first validated that embryonic corneal innervation morphogenesis in the Swiss model was consistent with these earlier findings⁷. This was achieved using light-sheet microscopy on cleared embryos combined with βIII-tubulin immunolabelling, a pan-neuronal marker (Figure S1).

The corneal innervation consists of diverse fiber types, each defined by specific functional roles, electrophysiological properties, and molecular markers¹⁰. To explore the developmental dynamics of these fibers, we performed volumetric analyses from birth (P0) to adulthood (12 weeks of age, 12 W), following established protocols²⁸. Our investigation focused on four key markers, representative of corneal innervation fiber identities¹⁰.

First, L1CAM—a multifunctional protein implicated in axon guidance—was used to identify a broad population of developing fibers. Second, we analyzed neurofilament 200 (NF200) + fibers, which correspond to large myelinated A-type fibers. Third, we examined substance P (SP) + fibers, indicative of C-type peptidergic nociceptive fibers. Finally, we included cold-sensing fibers marked by the expression of TRPM8, a member of the transient receptor potential cation channel subfamily M.

By quantifying the volume of each fiber subtype relative to the total βIII-tubulin + fiber volume—which represents close to the entirety of corneal innervation—we aimed to characterize the establishment and maturation of distinct fiber populations within the corneal epithelium.

To investigate the morphogenetic dynamics of corneal innervation, we performed βIII-tubulin immunolabelling, which broadly marks neuronal fibers (Fig. 1A). At birth (P0), βIII-tubulin staining revealed a sparse, uniformly distributed network of nerve fibers. No substantial changes were observed by eyelid opening (P14). However, between P14 and 4 weeks of age, we detected an approximately tenfold increase in innervation volume. From 4 to 12 weeks, a well-organized subbasal nerve plexus progressively emerged, forming the characteristic central vortex pattern that persists into adulthood.

To quantify these changes, we measured the volume of epithelial innervation within the central 50% of the total corneal area, as we described before²⁸. As anticipated, innervation volume was negligible before eyelid opening, followed by a sharp increase over the subsequent two weeks (Fig. 1B). This volume then plateaued and remained stable through to full corneal maturation at 12 weeks.

We then applied the same visualization strategy and epithelial volume quantification to fibers labeled with L1CAM, NF200, SP, and TRPM8. The epithelial volumes of these specific fiber subtypes were compared to βIII-tubulin + fibers to assess the evolving composition of corneal epithelial innervation during maturation.

From P0 to 12 weeks, L1CAM + fibers exhibited a distribution pattern largely overlapping with that of βIII-tubulin + fibers (Fig. 2A). However, volumetric analysis revealed distinct developmental kinetics: L1CAM + fiber volume peaked around eyelid opening (P14), coinciding with the onset of epithelial stratification, suggesting a possible role in innervation expansion during this critical phase (Fig. 2B). In contrast, NF200 + fibers—representing myelinated axons—progressively declined during development (Fig. 3A), consistent with established findings that mature corneas lack myelinated fibers⁸. Correspondingly, NF200 + epithelial volume steadily decreased over time (Fig. 3B). SP + fibers (Fig. 4A) were already abundant at birth (P0), and included fine fibers not detected by βIII-tubulin staining (Fig. 4B). Their epithelial volume decreased between P0 and 4 weeks
<figure><img src="image_3.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_4.png" /></figure>

**Fig. 1.** Visualization and volume analysis of the βIII-tubulin + innervation. (A) The visualization of βIII-tubulin + axons showed the progressive innervation pattern formation from P0 to 12 weeks (W) of age. The vortex-shaped structure formed by the epithelial fibers is visible at 4 W and 12 W. (B) The epithelial fiber volumes in the central area covering 50% of the whole corneal surface are then measured for each stage using Imaris software (n = 14 to 17). Volumes of βIII-tubulin + fibers are drastically increased from P14 to 4 weeks of age. Data are represented as mean ± SEM. Statistical analysis using Ordinary one-way statistic test and
***p***<0.0001. Scale bars = 300 µm.

but showed a marked increase in adulthood, from 5% at 4 W to 21% at 12 W (Fig. 4C), suggesting a delayed maturation or functional specialization of this nociceptive subpopulation. Finally, TRPM8+ fibers displayed a distinct organizational pattern, forming epithelial “bushes” that were notably devoid of L1CAM expression (Fig. 5A–B). Similar to SP+fibers, TRPM8+ fiber epithelial volume decreased during early development, followed by a significant increase later in corneal maturation, from 3 to 13% (Fig. 5C), indicating a unique temporal profile of sensory fiber integration.

The analysis of epithelial innervation composition from P0 to 4 weeks revealed a finely tuned maturation
process of corneal sensory fibers (Fig. 6). At birth (P0), the distribution of fiber types appeared relatively
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Visualization and volume analysis of the L1CAM+ innervation. (A) The visualization of L1CAM+ innervation showed the progressive innervation pattern formation from P0 to 12 weeks (W) of age. (B) The epithelial fiber volumes in the central area covering 50% of the whole corneal surface are then measured for each stage using Imaris software (n = 15 to 27). Volumes of L1CAM+ fibers in the epithelium are at their maximum at P14. Data are represented as mean ± SEM. Statistical analysis using one-way ANOVA.

**p**<0.01; ***p**<0.001; ****p**<0.0001. Scale bars = 300 µm.</figcaption></figure>

homogeneous. However, by eyelid opening (P14)—a stage coinciding with epithelial stratification— L1CAM+ fibers became predominant, suggesting a role in axonal guidance during the early phases of epithelial nerve integration. Between 4 and 12 weeks of age, the innervation pattern further refined, with a progressive increase in SP+ and TRPM8+ fibers, indicative of the functional maturation of nociceptive and thermosensitive pathways.

To correlate these morphological changes with functional outcomes, we assessed corneal sensory sensitivity throughout development (Fig. 7). Sensitivity increased steadily, reaching a plateau around 3 months of age, with only minor changes observed thereafter, up to 8 months.

Collectively, these findings demonstrate that corneal innervation undergoes a protracted developmental program, with critical remodeling and specialization occurring between 4 and 12 weeks of age. To further explore the relevance of this morphogenetic process, particularly its implications for physiological nerve regeneration, we next employed two clinically relevant models of corneal innervation loss: surgical axotomy and epithelial abrasion.

## Characterization of the murine axotomy model recapitulating penetrating keratoplasty

During corneal transplantation procedures, called penetrating keratoplasty, corneal innervation is inevitably severed around the transplanted region, necessitating subsequent nerve regeneration²⁹. To understand the effects of this innervation loss on corneal epithelial cells and to characterize the regenerative process, we employed our previously established axotomy model (Figs. 8 and 9).
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Visualization and volume analysis of the NF200 + innervation. (A) The visualization of NF200 + innervation showed the progressive innervation pattern formation from P0 to 12 weeks (W) of age. (B) The epithelial fiber volumes in the central area covering 50% of the whole corneal surface are then measured for each stage using Imaris software (n = 18 to 18). Volumes of NF200 + fibers in the epithelium are at their maximum at P0. Data are represented as mean ± SEM. Statistical analysis using one-way ANOVA.
\*p < 0.05; \*\*p < 0.001; \*\*\*p < 0.0001. Scale bars = 300 µm.</figcaption></figure>

Immediately following application of the biopsy punch to the cornea, we assessed epithelial integrity using fluorescein staining and βIII-tubulin immunolabelling (Fig. 8A). The weak fluorescein signal suggested that the blade had minimal impact on the epithelial layer, a finding further corroborated by high-resolution microscopy.

Peripheral transection of corneal stromal axons led to a gradual decline in innervation density; however, no region was found to be completely denervated (Fig. 8B). Between 1 and 2 weeks post-axotomy (WPAx), βIII-tubulin staining revealed a progressive decrease in innervation starting from the periphery and advancing toward the center, consistent with axonal dieback or retraction. By 6 WPAx, peripheral nerve fibers began to regenerate and progressively reinnervated the central cornea (Fig. 8B).

To characterize the dynamics of this regeneration, we compared epithelial innervation volumes before injury and at multiple stages post-axotomy. We observed a significant initial drop in epithelial innervation volume following axotomy, followed by a gradual recovery. By 10 WPAx, innervation volume had returned to pre-injury levels (Fig. 9A).

Interestingly, sensory function recovered earlier than anatomical reinnervation. Corneal sensitivity was restored by 6 WPAx—four weeks before structural innervation was fully reestablished (Fig. 9B). This observation underscores a striking dissociation between anatomical reinnervation and functional recovery, suggesting that sensory restoration may precede and potentially guide structural nerve regeneration.

To investigate the impact of nerve injury on corneal epithelial homeostasis, we assessed epithelial cell proliferation and apoptosis over a 4-week period following axotomy. Our results revealed a marked disruption in epithelial turnover: cell proliferation significantly increased throughout the first four weeks post-injury (Fig. 9C), while apoptosis was substantially suppressed during the same timeframe (Fig. 9D). This imbalance led to a disruption of normal epithelial homeostasis. In rare cases (7.5% of animals), this dysregulation culminated in severe epithelial damage, including corneal ulceration (Fig. 9E). Due to their infrequency and potential confounding effects, these extreme events were excluded from the quantitative analysis.

Together, these findings underscore a clear dissociation between structural nerve regeneration and functional recovery, and demonstrate that nerve injury induces profound and sustained alterations in epithelial proliferation and apoptosis dynamics.
<figure><img src="image_7.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_9.png" /></figure>

**Fig. 4.** Visualization of the SP + innervation. (A) The visualization of SP + innervation showed the progressive innervation pattern formation from P0 to 12 weeks (W) of age. (B) The close-up on some small terminal fibers at P0 showed the absence of βIII-tubulin in SP + domains (yellow cercles) and the absence of SP + in βIII-tubulin domains (blue cercles). (C) The epithelial fiber volumes in the central area covering 50% of the whole corneal surface are then measured for each stage using Imaris software (n = 5 to 9). Volumes of SP + fibers in the epithelium are at their maximum at P0, and increase from 4 W to 12 W. Data are represented as mean ± SEM. Statistical analysis using one-way Kruskal-Wallis test (black stars) and t-test (red hashes). \* and \#p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001. Scale bars = 300 µm in (A) and 600 µm in (B).

**Characterization of the murine abrasion model recapitulating photorefractive keratitis model**

To further investigate corneal nerve regeneration, we employed a clinically relevant mouse model of photorefractive keratitis, induced via mechanical epithelial abrasion, as we previously described³,²⁸,³⁰. To visualize the extensive damage caused by this injury, we performed fluorescein staining and βIII-tubulin immunolabelling (Fig. 10A). The large fluorescein-positive area was completely devoid of epithelial innervation.
<figure><img src="image_10.png" /><figcaption>**Fig. 5.** Visualization of the TRPM8 + innervation. (A) The visualization of TRPM8 + innervation showed the progressive innervation pattern formation from P0 to 12 weeks (W) of age. (B) The close-up on some small terminal fibers at P0 in the epithelium as well as in the stroma showed the absence of L1CAM in TRPM8 + fibres of the epithelium and the absence of TRPM8 in L1CAM + positive fibres in the stroma. (C) The epithelial fiber volumes in the central area covering 50% of the whole corneal surface are then measured for each stage using Imaris software (n = 5 to 10). Volumes of TRPM8 + fibers in the epithelium are at their maximum at P0, and increase from 4 W to 12 W. Data are represented as mean ± SEM. Statistical analysis using one-way Kruskal-Wallis test (black stars) and t-test (red hashes). \*p < 0.05; \*\* and \*\*\*p < 0.01; \*\*\*\*p < 0.001. Scale bars = 300 µm in (A) and 600 µm in (B).</figcaption></figure>
A

P0

<figure><img src="image_11.png" /></figure>

B

P14

<figure><img src="image_12.png" /></figure>

C

4 weeks

<figure><img src="image_13.png" /></figure>

D

12 weeks

<figure><img src="image_14.png" /></figure>

**Fig. 6.** Dynamics of epithelial innervation composition from P0 to 12 weeks of age. The epithelial fiber volumes in the central area covering 50% of the whole corneal surface are used to interrogate the proportion of each fiber type at any given timepoint, when compared to the average βIII-tubulin + volumes of the same area. (A) At P0 each marker represented the same proportion of the innervation. (B) At P14, L1CAM + volume was close to βIII-tubulin, showing a strong expression of this marker. (C) At 4 weeks, L1CAM was still the most expressed marker, but NF200, SP and TRPM8 were very lowly expressed. (D) At 12 weeks, while NF200 was close to not being detected, L1CAM, SP and TRPM8 represented a similar volume (n = 5 to 27). Data are represented as mean ± SEM. Statistical analysis using Kruskal-Wallis test. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.0001.

Our analysis commenced one week post-abrasion—once epithelial wound closure was confirmed—and extended over several weeks. Unlike axotomy (Fig. 8), central epithelial abrasion caused immediate innervation loss, which remained evident at 1 week post-abrasion (1WPAb) (Fig. 10B). By 2 weeks post-abrasion, regenerated nerve fibers appeared morphologically abnormal, displaying dense, highly branched, and disorganized endings resembling “nerve bushes.” These aberrant structures persisted and were still visible six weeks after injury.

Epithelial innervation volume dropped sharply following abrasion, reaching a nadir at 2WPAb (Fig. 11A). Interestingly, corneal sensitivity was most reduced at 1WPAb—preceding the lowest structural innervation
<figure><img src="image_15.png" /><figcaption>**Fig. 7.** Corneal sensitivity evolution from 6 to 32 weeks of age. Central corneal sensitivity is monitored up with Von Frey filaments. The sensitivity increases gradually from 6 to 18 weeks of age. Then, the sensitivity reaches a plateau by 26 weeks of age (n = 13 to 15). Data are represented as mean ± SEM. Statistical analysis using one-way ANOVA test \*p < 0.05 ; \*\*p < 0.01.</figcaption></figure>

volume—suggesting a temporal mismatch between functional and anatomical recovery (Fig. 11B). Sensitivity fully recovered within three weeks, while structural regeneration lagged behind.

We also evaluated epithelial homeostasis during the four-week regenerative window. Basal epithelial cell proliferation, assessed by phospho-histone H3 + cell counts, remained stable except at 3WPAb, where a significant increase was observed (Fig. 11C). Although apoptosis showed a downward trend, ANOVA analysis indicated no statistically significant variation over time (Fig. 11D).

Together, these findings highlight a clear divergence between the timelines of structural and functional nerve regeneration. Despite the gradual regrowth of nerve fibers over five weeks post-abrasion, histological analysis at 4WPAb revealed incomplete central reinnervation, with persistent denervated zones interspersed with dense nerve clusters (Fig. 11E).

Distinct regenerative process after abrasion or axotomy

Given that axonal regeneration appeared to differ depending on the type of corneal nerve injury, we analyzed the expression of 12 neural genes commonly associated with neurogenesis: *Atf3*, *Calca*, *L1cam*, *Nf200*, *Ngfr*, *Nrp1*, *Ntrk1*, *Piezo2*, *Robo1*, *SP*, *Trpm8*, and *Tubb3*. Despite the relatively small subset of trigeminal ganglion neurons innervating the cornea, we chose this tissue to evaluate transcriptional changes by qRT-PCR (Fig. 12). We first quantified *βIII-tubulin* expression at time points corresponding to reduced corneal sensitivity—two weeks post-axotomy (Figs. 9B, 12A) and one week post-abrasion (Figs. 11B, 12B). Both injury models were associated with elevated *βIII-tubulin* expression.

Given that the 6-week time point appeared critical for active nerve regeneration, we next assessed expression of the 12 selected genes at this stage. Of these, seven showed a significant change compared to uninjured controls. Four genes were differentially expressed following axotomy, namely *L1cam*, *Nrp1*, *Sp* and *βIII-tubulin* (Fig. 12C), whereas only two were modulated after abrasion, i.e. *L1cam* and *Nf200* (Fig. 12D). *L1cam* was the only gene upregulated in both injury models, underscoring its potential role as a common molecular mediator in corneal nerve regeneration across distinct injury contexts.

Discussion

The health and function of the cornea rely heavily on its dense peripheral sensory innervation. These nerves are not only critical for sensing environmental stimuli but also provide essential trophic support that preserves corneal transparency, structural integrity, and wound healing capacity. Injury to this delicate network can lead to neurotrophic keratitis (NK)—a severe, vision-threatening condition marked by impaired epithelial repair, progressive corneal degeneration, and ulceration. Despite its clinical importance, our limited understanding of corneal nerve regeneration mechanisms has hindered the development of effective therapies.
<figure><img src="image_16.png" /><figcaption>**Fig. 8.** Visualization of the impact of axotomy on corneal innervation. (A) Visualization of the minimal injury caused by the biopsy punch injury on the cornea via fluorescein staining, and immunostaining, immediately after axotomy. (B) Visualization of bIII tubulin staining before and during 6 weeks post-axotomy (WPAx), and close-up of the corneal innervation fibers, oriented towards the central cornea at 6WPAx. (n = 12). Scale bars: 300 µm.</figcaption></figure>

In this study, we demonstrate for the first time that corneal nerve regeneration is highly context-dependent and varies markedly with the type of injury. Through a comparative analysis of axotomy and epithelial abrasion models, we reveal injury-specific differences in regenerative dynamics, epithelial responses, and underlying molecular programs, thereby providing novel mechanistic insights into peripheral nerve repair.

We show that corneal nerve development in mice begins at embryonic day 12 (E12) and continues until 12 weeks postnatally. While overall central corneal innervation density increases steadily until eyelid opening, fiber-type specification and functional maturation primarily occur afterward. Between 2 and 12 weeks, shifts in fiber composition align with increases in sensory sensitivity, indicating progressive functional specialization of the corneal sensory network.

Our marker-specific analysis uncovered a sequential remodeling of the central corneal innervation landscape. L1CAM, a key axonal guidance molecule, was broadly expressed early but declined significantly by 4 and 12 weeks as fibers thinned. NF200, indicative of myelinated fibers, was rapidly downregulated postnatally and nearly absent in adults. SP, a nociceptive neuropeptide, decreased after eyelid opening but rebounded modestly in adulthood. TRPM8+cold-sensing fibers followed a similar trend—initially reduced, then localized predominantly to epithelial nerve endings later in development.

These changes were reflected in fiber-type proportions: at birth, distributions were uniform, but by P14, NF200+fibers declined sharply. Between 4 and 12 weeks, L1CAM+fibers also diminished, while SP+and TRPM8+fibers increased, reaching equal representation with L1CAM+fibers by adulthood. This shift highlights a dynamic specialization of the corneal innervation network. Notably, our findings challenge previous reports suggesting age-related loss of corneal sensitivity³¹, showing instead that fiber identity—not density—may be the key to functional preservation. In line with prior work, we underscore the critical role of SP+fibers during early maturation, consistent with their established importance in corneal wound healing³².
<figure><img src="image_17.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_21.png" /></figure>

**Fig. 9.** Characterization of the corneal parameters after axotomy. (A) Fiber volume, (B) corneal sensitivity, and (C) the number of proliferating and (D) apoptotic cells in the basal epithelium were measured at various time points after axotomy. (E) Example of a cornea exhibiting an ulceration in the epithelium 2 weeks after surgery. (n = 5 to 20). WPAx: weeks post-axotomy. Data are represented as mean ± SEM. Statistical analysis using Kruskal-Wallis test. ns = non significant, \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.0001. Scale bars: 400 µm.
<figure><img src="image_22.png" /><figcaption>**Fig. 10.** Visualization of the impact of abrasion on corneal innervation. (A) Visualization of the large injury caused by the corneal epithelial abrasion via fluorescein staining, and immunostaining, immediately after axotomy. (B) Visualization of βIII tubulin staining before and during 6 weeks post-abrasion (WPAb). (C) Close-up of the regenerated corneal innervation fibers, lacking any orientations at 6WPAb. (n = 14) Scale bar: 300 µm.</figcaption></figure>

We also reveal that the regenerative response and epithelial homeostasis differ significantly based on injury type. Axotomy, which induces axonal dieback³³, resulted in prolonged suppression of apoptosis, disrupted epithelial turnover, and occasional ulceration. In contrast, epithelial abrasion, which affects only terminal endings, triggered milder epithelial changes and no ulceration. These observations suggest distinct regenerative environments shaped by injury depth and severity. We propose that abrasion promotes epithelial plasticity, creating a permissive niche that supports both tissue repair and nerve regeneration, as we observed before³⁴.

Our study also helps reinterpret prior work. For example, Bech et al.³⁵ focused specifically on TRPM8+fibers using electrophysiological recordings, while we used βIII-tubulin to visualize total innervation and assessed functional recovery behaviorally. Since TRPM8+fibers represent only a subset of total corneal innervation³¹, methodological differences likely explain the contrasting outcomes. Moreover, even when nerves appear structurally restored, sensory integration at the central level may lag, explaining delayed functional recovery measured by Von Frey testing.

We also compared our findings to the live imaging study by Namavari et al.³⁶, which used a Thy-YFP mouse line to assess partial axotomy. Despite differences in models and methodologies, our data align with their overall regeneration timeframe, reinforcing the robustness of our conclusions.

Gene expression analyses further clarified these patterns. Nrp1, a regulator of corneal nerve development³⁷, was downregulated specifically after axotomy—possibly accounting for the increased ulceration seen in this model. In contrast, abrasion upregulated L1cam and Nf200, suggesting a reactivation of developmental programs. This may reflect a shift toward a plastic, embryonic-like state of innervation, which then progresses toward full maturation and functionality.

In summary, our study provides a comprehensive, side-by-side characterization of corneal nerve regeneration in distinct injury contexts. We show that structural regeneration and functional recovery are not tightly
<figure><img src="image_23.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_25.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_26.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_27.png" /></figure>

**Fig. 11.** Characterization of the cornea and its innervation after abrasion. (A) Fiber volume, (B) corneal sensitivity, and (C) the number of proliferating and (D) apoptotic cells in the basal epithelium were measured at various time points after abrasion. (E) Example of a cornea exhibiting an ulceration in the epithelium 4 weeks after surgery. (n = 5 to 20). WPAb: weeks post-abrasion. Data are represented as mean ± SEM. Statistical analysis using Kruskal-Wallis test. ns = non significative, \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.0001. Scale bars: 400 µm.

correlated, and highlight the importance of nerve fiber identity and plasticity in successful repair. By identifying specific cellular and molecular responses tailored to different injury types, we propose novel therapeutic targets for NK and related peripheral neuropathies. Our findings advance the current understanding of corneal nerve biology and lay a foundation for precision-guided nerve regeneration strategies.

## Materials and methods

### Animals included in this study

All the experiments conducted on mice were approved by the local ethical committee and the Ministère de la Recherche et de l'enseignement Supérieur (authorization 2016080510211993 version2). All of the procedures were carried out in accordance with the French regulation for the animal procedure (French decree 2013-118)
<figure><img src="image_28.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_29.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_30.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_31.png" /><figcaption>**Fig. 12.** Neural gene expression is modulated by innervation injury. (A) *bIII tubulin* expression is over expressed 2 weeks post axotomy (2WPAx), similarly to 1 week post abrasion (1WPAb) (B). (C) *L1cam*, *Nrp1*, *Sp* and *bIII tubulin* expression are modulated 6WPAx, but not *Nf200*. (D) *L1cam* and *Nf200* expression are modulated 6WPAb, but not *Nrp1*, *Sp*, or *bIII tubulin*. (n=5). Data are represented as mean ± SEM. Statistical analysis using t-test.ns, non significant, \*p < 0.05.</figcaption></figure>

and with specific European Union guidelines for the protection of animal welfare (Directive 2010/63/EU). The study is reported in accordance with ARRIVE guidelines. Swiss/CD1 female mice, RjOrl: SWISS, from Janvier Labs, France, were housed in plastic boxes, on a standard light cycle (12 h light, 12 h dark), with food and water ad libitum, in a 40-60% relative humidity environment and a 21-22 °C ambient temperature. Embryos were collected on ice between E11 and E14, newborn animals were ethanazied by beheading, and adult animals were euthanazied by cervical dislocation.
## Sensitivity

Sensitivity of the central cornea was assessed in vivo using Von Frey filaments (Bioseb, France, #vio-VF-M) as previously described³⁸. Succinctly, the nylon filaments of increasing strength were applied until the mouse blinked.

## Tissue collection and processing

For each sample, two trigeminal ganglions from the wounded side of two mice were collected and pooled in lysis solution, snap frozen then stored at -80 °C. Eyes were collected using curved scissors to cut the optic nerve. They were then fixed in 4% PFA (Antigenfix) for 20 min, then washed in PBS. The eyes were then dehydrated for 2 H in 50% ethanol/PBS and stored in 70% ethanol/PBS at 4 °C.

## Corneal abrasion

Corneal abrasions were performed as previously described³⁰,³⁹. Mice were anesthetized by intraperitoneal injection with a mix of ketamine (80 mg/kg, Imalgene® 1000, Centravet) and medetomidine (1 mg/kg, Domitor®, Centravet). The center of the corneal epithelium of the righteye was circulary removed using an ocular burr (Algerbrush II, reference BR2-5 0.5 mm, The Alger Company, USA). Immediatly after, the abrasion was checked using a fluorescein solution (1% in PBS, Sigma-Aldrich) under cobalt blue light. After abrasion, a drop of Ocrygel (TVM) was applied on both eyes, mice were treated with an analgesic solution of buprenorphine (0.1 mg/kg, Burprecare®, Centravet). The wellbeing of the animals was monitored in the following days.

## Corneal nerves axotomy

Axotomy of the corneal nerves were performed as previously described. Mice were anesthetized by intraperitoneal injection with a mix of ketamine (80 mg/kg, Imalgene® 1000, Centravet) and medetomidine (1 mg/kg, Domitor®, Centravet). Under a binocular loupe, lift the eye out of the orbit using smooth, curved forceps. Next, a 2.5 mm biopsy punch is applied vertically to the cornea with gentle pressure and twisted several times. If the eye ruptures, the animal must be euthanized. Once the punch is removed, a visible circle should be seen on the cornea. Ocular gel is applied to both eyes while the mouse recovers on a heated pad at 37 °C. The animal is monitored for the next two days.

## RT-qPCR on trigeminal ganglion

Samples composed of two pooled trigeminal ganglions were used for the RNA extraction. Tissue grinding was performed with 0.65 ml RA1 reagent (Macherey Nagel) using CKMix lysing kit and Precellys homogenizer (Bertin Technologies) followed by RNA purification and DNase treatment with the Nucleospin RNA Kit (Macherey Nagel). RNA samples were quantified using the NanodropOne spectrophotometer (ThermoFisher Scientific) and the integrity of the RNAs was verified using the Agilent 2100 bioanalyzer with the RNA 6000 Nano kit (Agilent Technologies), the RIN values obtained were between 7.5 and 8.5. 300 ng of total RNA was reverse transcribed in a 20 µL final reaction volume using the High-Capacity Reverse Transcription kit (Applied Biosystems) using random primers and the presence of RNase inhibitor following the manufacturer's instructions. The quantitative PCR experiments were performed using a TaqMan detection protocol on the QuantStudio 12 K Flex Real-Time PCR System (Life Technologies). The selected TaqMan assays (ThermoFisher Scientific) for each target and reference gene are listed in Table 1. For qPCR reactions, 3ng of cDNA was mixed with TaqMan FastAdvance Master Mix and the corresponding TaqMan assay in a final volume of 10 µL. Samples were then loaded onto 384-well microplates, and PCR was carried out for 40 cycles (50 °C for 2 min; (95 °C for 1 s, 60 °C for 20 s) ×40). To verify the absence of genomic DNA contamination, negative controls lacking reverse transcriptase were included. Data normalization was performed using three reference genes (Hprt1,

<table><caption>**Table 1.** List of genes and their identification used for the qPCR analysis.</caption><thead><tr><td>Gene</td><td>ID</td></tr></thead><tbody><tr><td>Atf3</td><td>Mm00476033_m1</td></tr><tr><td>Calca</td><td>Mm00801463_g1</td></tr><tr><td>Hprt</td><td>Mm00446968_m1</td></tr><tr><td>L1cam</td><td>Mm00493049_m1</td></tr><tr><td>Nf200</td><td>Mm01191455_m1</td></tr><tr><td>Ngfr</td><td>Mm00446296_m1</td></tr><tr><td>Nrp1</td><td>Mm00435372_m1</td></tr><tr><td>Ntrk1</td><td>Mm01219406_m1</td></tr><tr><td>Piezo2</td><td>Mm01265861_m1</td></tr><tr><td>Ppia</td><td>Mm02342430_g1</td></tr><tr><td>Robo1</td><td>Mm00803879_m1</td></tr><tr><td>SP</td><td>Mm01166996_m1</td></tr><tr><td>Trpm8</td><td>Mm01299593_m1</td></tr><tr><td>Tubb3</td><td>Mm00727586_s1</td></tr><tr><td>Ubc</td><td>Mm01198158_m1</td></tr></tbody></table>
<table><caption>**Table 2.** List of primary and secondary antibodies used for the immunostaining.</caption><thead><tr><td>Antibody name</td><td>Host</td><td>Supplier</td><td>Reference</td><td>Dilution</td></tr></thead><tbody><tr><td>Anti-beta III Tubulin</td><td>Rabbit</td><td>Abcam</td><td>ab18207</td><td>1/1000</td></tr><tr><td>Anti-NFH (Neurofilament Heavy Chain) Antibody</td><td>Chicken</td><td>Aves labs</td><td>AB_2313552</td><td>1/500</td></tr><tr><td>Calcitonin Gene-Related Peptide 1 (alpha-CGRP)</td><td>Rabbit</td><td>BMA Biomedicals</td><td>T-4032</td><td>1/500</td></tr><tr><td>Anticorps Substance P (NC1/34HL)</td><td>Rat</td><td>Santa Cruz</td><td>sc-21715</td><td>1/500</td></tr><tr><td>Anti-TRPM8 (extracellular) Antibody</td><td>Rabbit</td><td>Alomone labs</td><td>ACC-049</td><td>1/500</td></tr><tr><td>Anti-Neural Cell Adhesion Molecule L1 Antibody, clone 324</td><td>Rat</td><td>Merck</td><td>MAB5272</td><td>1/500</td></tr><tr><td>Anti-Histone H3 (phospho S28) antibody [HTA28]</td><td>Rat</td><td>Abcam</td><td>ab10543</td><td>1/500</td></tr><tr><td>Goat Anti-Rat IgG H&amp;L (Alexa Fluor® 647) preadsorbed</td><td>Goat</td><td>Abcam</td><td>ab150167</td><td>1/500</td></tr><tr><td>Anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td><td>Goat</td><td>ThermoFisher</td><td>A-11008</td><td>1/500</td></tr><tr><td>Anti-Chicken IgY H&amp;L (Alexa Fluor® 568) preadsorbed</td><td>Goat</td><td>Abcam</td><td>ab175711</td><td>1/500</td></tr></tbody></table>

*Ppia*, and *Ubc*), with the two most stable genes (*Hprt/Ppia*) identified using GenEx software (MultiD). Each measurement was conducted in duplicate, and Ct values were used for analysis. The relative gene expression ratio was calculated using the $\Delta\Delta$Ct method.

## Immunofluorescence labeling on whole cornea

Previously collected eyes were rehydrated in 50% ethanol for two hours and rinsed in PBS. Corneas were dissected just above the ciliary body, along the limbal region using microdissection scissors. They were then immersed in blocking and permeabilization solution in FSG 5% (Sigma-Aldrich, reference G7765) and goat serum (GS) 5% (Thermo Fisher Scientific, reference 16,210,064) in 0,5% PBS/Triton X-100 at room temperature. Then the corneas were incubated overnight at 4 °C with primary antibodies and then secondary antibodies (Table 2). After washing, nuclei were stained with Hoechst for 10 min. The corneas were incised at four cardinal points using a 15 C carbon steel surgical blade (reference 0221, Swann-Morton) and then flat-mounted in Vectashield medium (Vector Laboratories, H-1000), with the epithelium positioned against the coverslip. Apoptotic cells were labeled using the DeadEnd™ Fluorometric TUNEL System (Promega, reference G3250) on whole corneas. Briefly, freshly fixed corneas were incubated with proteinase K for 10 min at room temperature. They were then incubated in the rTdT reaction mix, consisting of 10% Fluorescein dUTP Nucleotide mix and 2% rTdT enzyme in Equilibration Buffer, for 60 min at 37 °C with gentle shaking (70 rpm). To stop the reaction, samples were immersed in 2X SCC Buffer for 3 min at room temperature. If the TUNEL assay is combined with another marker, immunofluorescence is subsequently performed as usual.

## Images acquisition and processing

The acquisition of the images was performed as previously described²⁸. A Leica Thunder Imager Tissue microscope was used to acquire the whole-cornea images, using the navigator module with the large volume computational clearing (LVCC) process. The LAS X software (version 3.7.4) was used to obtain the images using a 20X/0.55 objective. Then, to process the images Imaris Bitplane software (version 10.1) was used. All of the images from a single panel were acquired and processed with the same parameters.

## Innervation volume measurement and cell counting

A centered circle covering 50% of the total corneal area was drawn using the Fiji measurement plug-in (FIJI (RRID: SCR_002285)). The corneal diameter was measured twice for each sample, and the mean value was used to determine the circle's radius. A 50% circular crop of each cornea was then generated using the Crop plug-in, followed by the *Clear Outside* plug-in. The cropped images were converted to .ims format using the Imaris converter. Epithelial innervation density was quantified using the surface tool in Imaris. Once the surface was defined, volume data (expressed in µm³) were extracted for statistical analysis. Apoptotic or proliferating cells were manually counted on Fiji using the *Cell Counter* plug-in.

## Statistical analysis

Data analysis was performed using GraphPad Prism software (version 10.2.2). Results are presented as mean ± SEM. Significant p-values were represented as *p*<0.05, **p*<0.01, ***p*<0.001, ****p*<0.0001. Statistical analyses comparing mean values were performed using one of the following tests: the Friedman test with a Dunn's multiple comparison test, the Kruskal-Wallis test with a Dunn's multiple comparison test, or an Ordinary one-way ANOVA followed by Dunnett's multiple comparison test. Simple comparisons were performed using an unpaired t-test. Data were cleaned by removing the highest and lowest values.

## Data availability

All data generated or analyzed during this study are included in this published article. The raw datasets supporting the conclusions of this work are available from the corresponding author upon request.

Received: 27 March 2025; Accepted: 3 July 2025

Published online: 16 July 2025
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## Acknowledgements

This research was supported by ATIP-Avenir program, Inserm, the Région Occitanie, ANR (ANR-21-CE17-0061, TeFiCoPa; ANR-23-CE14-0036-02, INNERCOR), FRM (REP202110014140), Support for research: I-SITE 2024 - program of excellence of the University of Montpellier, CBS2 Doctoral School, the Fondation Groupama. The RT-qPCR service was provided by the Dr Eric Jacquet at the QPCR plateform from Réseau des plateformes de Génomique Paris-Saclay at the Institut de Chimie des Substances Naturelles at Gif-sur-Yvette. We thank the MRI-DBS imaging facility, member of the France-BioImaging national infrastructure supported by the French National Research Agency (ANR-10-INBS-04, «Investments for the future»). We thank the personnel of the INM
animal core facility, member of the Animal Facility Network in Montpellier (RAM).

**Author contributions**

Conceptualization: L.M., S.P., F.M.; Methodology: L.M., S.P., N.F., M.G., A.C.M., M.D., N.N., E.M., E.J., V.D., F.M.; Validation: L.M., S.P., F.M.; Formal analysis: L.M., S.P., F.M.; Data analysis: L.M., S.P., E.J., F.M.; Writing: L.M., S.P., E.J., V.D., F.M.; Supervision: F.M.; Project administration: F.M.; Funding acquisition: F.M.

**Declarations**

**Competing interests**

The authors declare no competing interests.

**Additional information**

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1038/s41598-025-10434-y.

**Correspondence** and requests for materials should be addressed to F.M.

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