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. 2023 Aug 29;43(4):409–418. doi: 10.1097/ICO.0000000000003363

Impact of Age on the Characteristics of Corneal Nerves and Corneal Epithelial Cells in Healthy Adults

Jia Ying Chin *, Chang Liu *,✉, Isabelle Xin Yu Lee *,✉, Molly Tzu Yu Lin *,✉, Ching-Yu Cheng †,‡,§, Jipson Hon Fai Wong *, Cong Ling Teo ‡, Jodhbir S Mehta *,†,, Yu-Chi Liu *,†,¶,║,✉
PMCID: PMC10906190  PMID: 37643477

Abstract

Purpose:

The aim of this study was to investigate age-related changes in corneal nerves and corneal epithelial cell parameters and to establish age-adjusted reference values.

Methods:

A total of 7025 corneal nerve images and 4215 corneal epithelial images obtained using in vivo confocal microscopy from 281 eyes of 143 healthy participants were included. Seven corneal nerve parameters and 3 corneal epithelial cell parameters were quantified using 2 automatic analytic software and analyzed across 6 age groups ranging from 21 to 80 years.

Results:

There was a declining trend in all 7 nerve parameters with advancing age. In particular, corneal nerve fiber length and corneal nerve fiber density demonstrated a significant decrease in subjects aged 65 years or older compared with subjects younger than 65 years (10.8 ± 2.6 mm/mm2 vs. 9.9 ± 2.0 mm/mm2, P = 0.011 in corneal nerve fiber length; 15.8 ± 5.2 fibers/mm2 vs. 14.4 ± 4.3 fibers/mm2, P = 0.046 in corneal nerve fiber density), whereas corneal nerve fractal dimension demonstrated a borderline significant decrease (P = 0.057). Similarly, there was a general declining trend in all epithelial cell parameters with advancing age. Corneal epithelial cell circularity was significantly lower in subjects aged 65 years and older as compared to subjects younger than 65 years (0.722 ± 0.021 μm2 vs. 0.714 ± 0.021 μm2; P = 0.011).

Conclusions:

Advancing age results in reduced corneal nerve metrics and alteration of corneal cell morphology. Aging effects should be considered when evaluating patients with corneal neuropathy.

Key Words: aging, corneal nerves, corneal epithelial cells, cornea, ocular surface

Corneal nerves play a pivotal role in the maintenance of ocular homeostasis and functional integrity of the corneal epithelium through the provision of sensory functions1 and neurotrophic support to ocular surface tissues.2 The corneal epithelium comprises basal cells, wing cells, and superficial cells held together by desmosomes forming tight junctions, which allows for the provision of a natural structural barrier against invasion of pathogens and loss of fluid.3 The preservation of the functional integrity of the corneal epithelium involves a dynamic process of epithelial self-renewal which is achieved by maintaining a pivotal balance between cell proliferation, differentiation, and death.4 Terminal branches of the subbasal nerve plexus richly innervate the corneal epithelium and regulate the dynamic process of epithelial self-renewal and wound healing by releasing epitheliotropic substances.5 Studies have shown that neuropeptides, such as substance P and calcitonin gene-related peptide, play a role in influencing epithelial cell migration, proliferation, differentiation, and adhesion in vivo. Neurotrophins such as nerve growth factor activate epithelial progenitor cells and modulate epithelial cell migration and proliferation.6 In addition, the corneal epithelium synthesizes corneal neuromediators, which in turn promote corneal nerve regeneration and maintain proper distribution of nerve fibers in addition to promoting corneal epithelial wound healing.7 Therefore, the maintenance of ocular homeostasis involves a notable intricate interplay between corneal nerves and the corneal epithelium, whereby both release neurotrophic factors that promote one another's regeneration and healing.

Over the years, in vivo corneal microscopy (IVCM) has served as a reliable tool for rapid, real-time assessment of corneal nerve morphology and distribution.8,9 The clinical relevance and development of quantitative tools in recent years for the evaluation of corneal nerve status further lends to its increasing popularity in research to explore the relationship between corneal nerve parameters and clinical manifestations in both healthy and diseased corneas.10–12 In addition to visualization of subbasal nerves, corneal epithelial morphometric analysis is becoming increasingly important with several studies demonstrating a clinical association between epithelial cell findings and diseases such as diabetes mellitus13,14 and meibomian gland dysfunction.15,16

Dysregulation or degeneration with age due to inflammatory processes associated with the exposure of the cornea to external insults over time disrupts homeostatic processes and protective mechanisms.17 Current studies have shown inconsistent results of the age effects on corneal nerves, with some demonstrating adverse effects of age on corneal nerve morphology such as decreasing subbasal nerve fiber density with age,18–20 while others demonstrate no statistically significant changes in nerve fiber density, branch density, and length with age.21–23 Regarding the corneal epithelium, Gambato et al and Niederer et al showed that there was no change in mean cell density of basal epithelial cells with age using IVCM evaluation.21,24 Another study showed that a decrease in the density of limbal basal epithelial cells was observed with age because of the deteriorating proliferative ability with age, leading to a decrease in corneal epithelial thickness, although the central corneal epithelium was less susceptible to these age-related changes.25 This is supported by the work published by Zheng and his colleagues, who demonstrated that peripheral basal epithelial cell density was significantly higher in the youngest age group compared with older age groups, whereas central basal epithelial cell density had no significant age-related changes.26

To fully appreciate and explore these relationships, it is essential to establish baseline standardized reference values adjusted for age for comparison. This will allow for the differentiation of age effects from pathological degenerative diseases in the cornea. The elimination of age effects will enable precise recognition of small changes in the subbasal nerve plexus and corneal epithelial cell parameters. This allows us to clearly monitor pathological changes, thus ensuring reliable early diagnosis and detection of disease progression. In this study, we aimed to investigate age-adjusted characteristics for corneal nerve and corneal epithelial parameters.

MATERIALS AND METHODS

Study Population

This is a cross-sectional, observational study including a total of 281 eyes of 143 healthy participants recruited from the Singapore Eye Research Institute. Patients who had the following conditions that may affect the analysis for corneal nerves and epithelial cells were excluded: previous corneal surgery or ocular surgery including cataract surgery, previous infectious keratitis, glaucoma on antiglaucoma medications, moderate or severe meibomian gland dysfunction, > 1-year use of contact lens, known corneal or ocular surface diseases, active ocular surface or intraocular inflammation, diabetes mellitus, and systemic autoimmune diseases. The study subjects were further divided into 6 age groups: 20 to 29 years, 30 to 39 years, 40 to 49 years, 50 to 59 years, 60 to 69 years, and 70 years or older. This study was approved by the Institutional Review Board of SingHealth, Singapore (reference number: 2020/2050), and conducted in accordance with the tenets of the Declaration of Helsinki.

In Vivo Confocal Microscopy Image Acquisition

All patients were examined using the Heidelberg HRT3 Rostock Cornea Module (Heidelberg Engineering GmbH, Heidelberg, Germany), a laser confocal scanning microscope, with the protocol as published.27 A drop of topical 0.5% proparacaine hydrochloride (Alcaine; Alcon, Country), an anesthetic, was instilled into each patient's cornea to be examined. Patients were instructed to fixate on the flashing light of the microscope with their contralateral eye to stabilize the scanning view. Subsequently, the objective tip, covered in gel, was advanced forward incrementally to establish contact with the cornea using lateral and axial controls. The central cornea was first scanned before the patient was instructed to change their gaze to scan the nasal, temporal, superior, and inferior parts of the cornea at an approximate distance of 3 mm away from the corneal apex. Both corneas of each patient were examined and 2-dimensional images depicting a 400 × 400 μm corneal area were acquired with a resolution of 384 × 384 pixels.

Corneal Subbasal Nerve Image Analysis

The nerve images were analyzed as described previously.28 In brief, an experienced masked observer selected 5 best-focused and most representative images of the subbasal nerves from 5 areas: central, superior, inferior, nasal, and temporal quadrants at different depths from both eyes, with a total of 25 images for each eye. All images were evaluated using automatic image analysis software ACCMetrics (University of Manchester, Manchester, UK).29 Seven corneal nerve parameters were quantified: corneal nerve fiber length (CNFL; length of all nerve fibers in mm/mm2), corneal nerve fiber density (CNFD; number of main nerve fibers/mm2), corneal nerve branch density (CNBD; number of branch points on the main fibers/mm2), corneal nerve total branch density (CTBD; total number of branch points/mm2), corneal nerve fiber area (CNFA; total nerve fiber area mm2/mm2), corneal nerve fiber width (CNFW; average nerve fiber width mm/mm2), and corneal nerve fractal dimension (CNFrD; measure of corneal nerve complexity) (Figs. 1A, B). The mean values of these 25 representative images of each eye was obtained for each parameter.

FIGURE 1.

FIGURE 1.

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Illustrations of corneal nerve and epithelial analysis. A, Raw nerve image, (B) nerve image marked by ACCMetrics, (C) raw corneal epithelial cell image, and (D) epithelial cell image with cell annotation.

Corneal Epithelial Cell Image Analysis

An experienced masked observer selected 3 best images from the 4 quadrants and central corneas of each eye based on the following criteria: greater clarity of the epithelial border and larger area covered by epithelial cells within the confocal image. These 15 images were analyzed with an automated software AlConfocal Rapid Image Evaluation System (ARIES; ADCIS, France) to quantify epithelial cells before manual postprocessing was performed to deselect nonepithelial cells erroneously included and to outline epithelial cells missed previously. Epithelial cells were quantitatively evaluated with 3 parameters: cell density (cells/mm2), average size (μm2), and circularity (um) (Figs. 1C, D). Cell circularity is a measure of the how circular a cell's morphology is and is measured by the formula 4π(area/perimeter2). C = 1 indicates a perfect circle, and the value of C decreases with increasing deviation from a circle. The mean values of the 15 images of each eye was obtained for each parameter.

Statistical Analysis

Statistical analysis was performed using STATA 17 (STATACorp LP, College Station, TX), and all corneal nerve and epithelial data were presented as mean ± SD for each decade of age. The Kolmogorov–Smirnov test was used to analyze the normality of the data. Mixed linear model was used to determine the relationship between each nerve and epithelial parameter with age to account for the correlations between both eyes of each patient. The independent t test was used to compare the data between the younger than 65 years age group and aged 65 years and older age group. We considered P values ≤0.05 to be statistically significant.

RESULTS

Participants' Characteristics

The average age of the patients was 49.1 ± 17.2 years, and there was a total of 46.9% men and 53.1% women. Seventy-eight subjects were Chinese, 38 subjects were Indian, 7 subjects were Malay, and 20 subjects were other races. Patients were divided into 6 groups in incremental decades of age: 54 eyes of 20 to 29 years (27 patients; mean age 26.5 ± 1.3 years), 40 eyes of 30 to 39 years (20 patients; mean age 34.3 ± 2.6 years), 43 eyes of 40 to 49 years (22 patients; mean age 44.3 ± 3.3 years), 49 eyes of 50 to 59 years (25 patients; mean age 56.1 ± 3.0 years), 55 eyes of 60 to 69 years (29 patients; mean age 64.5 ± 3.6 years), and 40 eyes of aged 70 years and older (20 patients; mean age 74.8 ± 3.0 years).

Changes in Corneal Nerve Parameters with Age

A total of 7025 images were used for corneal subbasal nerve analysis. Representative images of the subbasal nerve for each age group are presented in Figure 2. With advancing age, the nerves noticeably decreased in density and length. There was also nerve loss at the inferior whorl with increasing age. Table 1 shows the results of the 7 nerve parameters for the 6 age groups. There is a general declining trend in all nerve parameters, including CNFL, CNFD, CNBD, CTBD, CNFA, CNFW, and CNFrD with advancing age. The corresponding regression plots demonstrating the relationship between age and each nerve parameter are depicted in Figure 3. The patients were further divided into 2 groups by age, with 210 eyes in the younger than 65 years age group and 71 eyes in the older or equal to 65 years age group. Subjects aged 65 years or older had significantly lower CNFL than subjects younger than 65 years (10.8 ± 2.6 mm/mm2 in the younger than 65 years group vs. 9.9 ± 2.0 mm/mm2 in the 65 years and older group; P = 0.011). In addition, subjects aged 65 years and older also had significantly lower CNFD than subjects younger than 65 years (15.8 ± 5.2 fibers/mm2 in the younger than 65 years group vs. 14.4 ± 4.3 fibers/mm2 in the 65 years and older group; P = 0.046). Similarly, those aged 65 years and older had borderline significantly lower CNFrD than those who are younger than 65 years (1.43 ± 0.04 in the younger than 65 years group vs. 1.42 ± 0.03 in the 65 years and older group; P = 0.057). There were no statistically significant differences between the 2 age groups for the other parameters: CNBD (16.2 ± 8.6 branch points/mm2 in the younger than 65 years group vs. 14.8 ± 7.2 branch points/mm2 in the 65 years and older group; P = 0.234), CTBD (29.4 ± 14.1 branch points/mm2 in the younger than 65 years group vs. 27.4 ± 10.7 in the 65 years and older group; P = 0.284), CNFA (0.005 ± 0.001 mm2/mm2 in the younger than 65 years group vs. 0.005 ± 0.001 mm2/mm2 in the 65 years and older group; P = 0.629), and CNFW (0.022 ± 0.001 mm/mm2 in the younger than 65 years group vs. 0.021 ± 0.001 mm/mm2 in the 65 years and older group; P = 0.110).

FIGURE 2.

FIGURE 2.

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Representative nerve images for each age group at peripheral cornea, central cornea, and corneal inferior whorl. (A1-3) 20 to 29 years, (B1-3) 30 to 39 years, (C1-3) 40 to 49 years, (D1-3) 50 to 59 years, (E1-3) 60 to 69 years, and (F1-3) 70 years and older. For each age group from left to right: peripheral cornea, central cornea, and corneal inferior whorl area.

TABLE 1.

Corneal Nerve Parameters in Normal Subjects for the 6 Age Groups

Age Group CNFL CNFD CNBD CTBD CNFA CNFW CNFrD
20–29 11.0 ± 2.5 16.2 ± 4.6 16.2 ± 8.3 29.2 ± 15.0 0.00508 ± 0.0015 0.0219 ± 0.001 1.44 ± 0.03
30–39 11.3 ± 2.3 16.3 ± 4.7 15.9 ± 8.7 29.3 ± 15.1 0.00530 ± 0.00158 0.0218 ± 0.0014 1.44 ± 0.03
40–49 10.4 ± 2.4 15.7 ± 5.5 15.5 ± 7.3 27.9 ± 9.7 0.00479 ± 0.00092 0.0213 ± 0.001 1.43 ± 0.04
50–59 10.4 ± 3.2 15.0 ± 6.2 16.3 ± 10.3 30.2 ± 16.0 0.00504 ± 0.00159 0.0216 ± 0.0011 1.42 ± 0.06
60–69 9.9 ± 2.0 14.3 ± 4.7 15.5 ± 7.8 27.8 ± 11.4 0.00512 ± 0.00131 0.0214 ± 0.0012 1.42 ± 0.03
70 and older 10.1 ± 2.2 14.9 ± 4.1 15.3 ± 7.4 28.7 ± 11.6 0.00503 ± 0.00139 0.0214 ± 0.0011 1.43 ± 0.03
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FIGURE 3.

FIGURE 3.

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Regression plots showing the relationship between age and each nerve parameter. A, Corneal nerve fiber length (CNFL), (B) corneal nerve fiber density (CNFD), (C) corneal nerve fractal dimension (CNFrD), (D) corneal nerve branch density (CNBD), (E) corneal nerve fiber width (CNFW), (F) corneal nerve fiber area (CNFA), and (G) corneal nerve total branch density (CTBD). The solid line in each plot represents the regression line, showing a declining trend with age.

Changes in Corneal Epithelial Parameters with Age

A total of 4215 of IVCM micrographs were used for corneal epithelial cell analysis. The epithelial cells form a homogeneous, regular, and compact arrangement with bright cell boundaries and small cell bodies, with no dysmorphia (Fig. 4). Table 2 summarizes the epithelial cell parameters for the 6 age groups. There was a general declining trend in epithelial cell parameters, including the circularity, cell density, and cell size with advancing age. The corresponding regression plots showing the relationship between the epithelial cell parameters and age are depicted in Figure 5. Similarly, the patients were further divided into 2 groups by the age of 65. Subjects aged 65 years and older had significantly lower epithelial cell circularity than subjects older than 65 years (0.722 ± 0.021 μm2 in the younger than 65 years group vs. 0.714 ± 0.021 μm2 in the 65 years and older group; P = 0.011). There were no significant statistical differences between the 2 age groups for the cell density (7781 ± 101 cells/mm2 in the younger than 65 years group vs. 7747 ± 48 cells/mm2 in the 65 years and older group; P = 0.789) and the average size (131 ± 10 μm in the younger than 65 years group vs. 130 ± 8 μm in the 65 years and older group; P = 0.489).

FIGURE 4.

FIGURE 4.

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Representative epithelial images for each age group. A, 20 to 29 years, (B) 30 to 39 years, (C) 40 to 49 years, (D) 50 to 59 years, (E) 60 to 69 years, and (F) 70 years and older. The cells are in a regular compact arrangement with clear cell borders in these normal subjects.

TABLE 2.

Corneal Epithelial Cell Parameters for the 6 Age Groups

Age Group Circularity Cell Density (Cells/mm3) Average Size (μm)
20–29 0.727 ± 0.019 7828 ± 1591 132.4 ± 10.6
30–39 0.728 ± 0.022 7635 ± 651 131.1 ± 15.3
40–49 0.718 ± 0.021 7620 ± 504 132.8 ± 7.4
50–59 0.716 ± 0.023 7763 ± 504 128.3 ± 8.3
60–69 0.716 ± 0.020 7799 ± 421 129 ± 7.4
70 and older 0.714 ± 0.020 7719 ± 479 130.3 ± 8.2
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FIGURE 5.

FIGURE 5.

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Regression plots showing the relationship between age and epithelial cell parameters: (A) circularity, (B) cell density, and (C) cell size. The solid line in each plot represents the regression line.

DISCUSSION

In this study, we demonstrated that aging significantly and negatively affected corneal innervation, evidenced by the decrease in CNFL and CNFD, as well as corneal epithelial cell morphology. With rapidly aging populations looming ahead for countries around the world, the need to explore age-associated degenerative processes in the cornea to develop a better understanding and allow for novel research outcomes is more essential than ever. This will also allow for the differentiation of aging effects from other degenerative or pathological corneal neuropathies.

In ophthalmology, IVCM has become a reliable tool to guide and assist clinical diagnosis, management, and research because of its noninvasive nature, reproducibility, repeatability, sensitivity, specificity, and ability to directly visualize the subbasal nerve plexus. Considerable research in recent years has been targeted on exploring the relationship between the subbasal nerve plexus and ocular surface diseases such as neurotrophic keratopathy, dry eyes,30 and systemic diseases such as diabetic neuropathy,31 in particular their early diagnosis, prognosis, and treatment outcomes. Yet, the process of aging complicates this as it is believed to be associated with structural and functional changes in the cornea, which must be accounted for. We thus endeavored to investigate the impact of age on corneal nerves and corneal epithelial cells to establish a reliable database of baseline age-adjusted reference values. Conventionally, medical research defines a person of chronological age including and older than 65 as elderly.32 We have adopted this definition in our present study to allow for a standardized comparison of the characteristics of corneal nerves and epithelial cells between the nonelderly population and the elderly population.

All corneal nerve parameters showed a decreasing trend with age. In particular, elderly participants had significantly lower CNFD and CNFL compared with nonelderly participants. Aging exerts detrimental effects on corneal nerves; it is associated with dysregulated inflammatory processes and changes in metabolites that alter corneal homeostasis,17 impair nerve protection and regeneration,33 and reduce the regenerative capacity of nerves, eventually leading to a decrease in corneal nerve fiber parameters.33 Substance P, one of the most abundant neuromediators in the cornea involved in the maintenance of normal corneal nerve morphology, is expressed in high levels by corneal nerves. It promotes corneal nerve regeneration,34 and its expression decreases with advancing age, which parallels the decrease in corneal nerve density.35 In combination with insulin growth factor-1, substance P harnesses the synergistic ability to improve epithelial wound healing in age-denervated corneas.35,36 Although not yet studied in the cornea, research has shown that insulin growth factor-1 decreases with age in the cerebrovascular system.37 Similarly, although there are no existing studies of the effects of aging on nerve growth factor (NGF) production in corneal nerves, studies have shown that NGF expression is significantly higher postoperatively in younger age groups in peripheral nerves, suggesting that aging might be associated with reduced NGF production.38 In addition, deficits in retrograde transport of NGF precursor and expression of NGF receptor TrkA, as well as the alterations in NGF signaling, may further impair the effect of NGF with age.39 NGF production normally occurs at a low basal level but is upregulated in inflammatory states induced in response to nerve damage and is crucial in the maintenance and survival of corneal nerves. It stimulates keratocyte migration, promoting neurite growth through elongation, branching, and sprouting, facilitating corneal nerve regeneration in response to nerve damage.40,41 Thus, reduced expression of NGF with increased age may lead to a reduction in corneal nerve parameters, including corneal nerve length, density, and branch complexity. Taken together, aging impairs the ocular surface homeostasis and causes corneal nerve loss in 2 ways: first, due to the accumulation of toxic molecules with age, generating a neurotoxic milieu causing neuronal death, and second, due to the loss of regenerative capacity in corneal nerves with age, associated with lower substance P35 and NGF levels. CNFD is the most commonly studied nerve parameter,18–20,24 and we found there was a statistically significant decrease in CNFD between subjects aged65 years and older compared with subjects younger than65 years. On the contrary, Chirapapaisan et al22 reported no statistical difference in CNFD between 4 age classes ranging from 20 to 60 years. However, this difference can be easily accounted for by the exclusion of subjects older than 60 years in the study by Chirapapisan et al,22 which is in stark contrast to our study, in which we aimed to compare the elderly age group older and including 65 years and the nonelderly age group. Furthermore, in their study, nerve images were only taken from the central cornea and extrapolated to the whole cornea, whereas our study used images from the central cornea and 4 quadrants of the cornea, therefore enhancing the accuracy of our nerve analysis. CNFL was the next most commonly studied parameter, with 2 studies demonstrating negative correlations between CNFL and advancing age, which is in line with our results.20,23 CNFrD is a rather new automated nerve metric, which measures the topological complexity of main nerve fibers and branches.42 It is expressed as a ratio of the logarithm of the total number of boxes that contain nerve fibers to the logarithm of the number of boxes of the image width. The more complex a nerve structure, the higher the value of CNFrD. It has been evaluated to be of comparable diagnostic utility to CNFD and CNFL in diagnosing patients with and without diabetic neuropathy,43 and has been used in previous research to study alterations in the corneal nerve plexus in various types of peripheral neuropathy, providing additional value in differentiating distinct patterns associated with different corneal nerve pathologies.44 In our study, CNFrD was borderline significantly reduced in the elderly population, suggesting that aging not only results in nerve loss (CNFD and CNFL) but also negatively affects corneal nerve morphology. There were no significant differences in CNBD and CTBD with age, possibly due to the degeneration of nerve branches with age,20 rendering nerve branching points too faint to be accurately annotated by the automatic analytic software.

We also found that the circularity of corneal epithelial cells decreased with age, and it was significantly lower in aged65 years and older group compared with the younger than 65 years group. In the epithelial regenerative and proliferative cycle, the newly regenerated basal epithelial cells are of an ovoid shape. As these basal cells mature, they change their shape to a more cuboidal and more columnar shape.45 NGF plays a crucial role in the epithelial healing process, accelerating epithelial resurfacing by promoting development, proliferation, differentiation, and migration of corneal cells.6 Reduced NGF production with age is associated with reduced regeneration capacity in older participants, hence lower percentages of ovoid-shaped epithelial cells are present, accounting for lower cell circularity. There were no obvious trends in corneal epithelial cell size and density with increasing age. This is consistent with the results of several studies in which the authors demonstrated no association between basal epithelial cell density and age.21,24 However, younger age was reported to have smaller epithelial cell sizes in addition to increased peripheral cell densities in 1 study.26 This difference may be attributed to different image acquisition methods; in the abovementioned study, the authors only scanned the inferior peripheral cornea for convenience and comfort, whereas in our study, we analyzed 4 peripheral quadrants and central corneas for more comprehensive analysis. In our study, the density of corneal basal epithelial cells ranged from 7620 to 7828 cells/mm2, which is in agreement with previous literature reporting the density at the range of 3600 to 8996 cells/mm2.46

Corneal nerve endings transduce stimuli into nerve signals that are directly responsible for physiological processes fundamental to ocular surface homeostasis. These include the activation of reflex blinking, tear reflex, neuropeptide secretion, and migratory and mitogenic effects of corneal epithelial cells.17 Aging leads to the disruption of tear mechanisms including lower tear volume, tear flow, meniscus height of tears, thickness of lipid layer, and tear composition.47 Accordingly, studies have observed a decrease in tear film break-up time and Schirmer test values, and an increasing prevalence of dry eye disease in the aging population.47 Understanding the aging process in corneal nerves and cells would help us delineate the age-related alterations in ocular surface diseases.

Although our study presented new insight into the association of advancing age with deteriorating corneal nerve and epithelial cell parameters, we did not examine the underlying molecular mechanisms responsible for the phenomenon, as well as the clinical manifestations associated, as the aim of the present study was to investigate whether a trend with age exists. Future studies will be conducted to determine the underlying physiological processes at play to further our understanding of the age-related degenerative processes in the cornea. In addition, although the influence of race on age-adjusted reference values is beyond the scope of this study, significant differences may arise in corneal nerve metrics and epithelial morphology between different races in view of different genetic predispositions and exposures to varying environments. Studies have shown variations among races in corneal characteristics such as thinner corneas in African Americans compared with Whites48 and higher corneal endothelial cell densities in Japanese compared with Americans.49 In addition, reference values of corneal nerve metrics and epithelial morphology seem to vary across previous individual studies with populations of different racial compositions.19,22,50 These suggest that age-adjusted reference values might be influenced by race. It would be useful for future research to establish age-adjusted reference values among different races.

In conclusion, we have demonstrated that advancing age is associated with reduced corneal nerve plexus and alterations of epithelial cell characteristics. At a fundamental level, it is essential to differentiate pathological changes from physiological degenerative processes during aging. Our study of age-related changes in the characteristics of corneal subbasal nerve and epithelial cells allows for the differentiation of the effects of aging ensuring more precise, reliable age-adjusted outcomes in future research. These aging effects should be considered when evaluating patients with corneal neuropathy. Defining the relationship between corneal nerve and epithelial cell characteristics with age will also help to establish age-stratified thresholds for screening and diagnostic protocols.

Footnotes

Supported by Clinician Scientist Award Grant from the Singapore National Medical Research Council (MOH-CSAINV21jun-0001).

The authors have no funding or conflicts of interest to disclose.

This study was approved by the Institutional Review Board of SingHealth, Singapore (reference number: 2020/2050), and conducted in accordance with principles expressed in the Declaration of Helsinki. Informed consent was obtained from all participants.

Contributor Information

Jia Ying Chin, Email: jiaying.chin@mohh.com.sg.

Chang Liu, Email: chang1226@qq.com.

Isabelle Xin Yu Lee, Email: isabelleleexy.96@gmail.com.

Molly Tzu Yu Lin, Email: lgmolly24@gmail.com.

Ching-Yu Cheng, Email: chingyu.cheng@duke-nus.edu.sg.

Jipson Hon Fai Wong, Email: jipson.wong.h.f@seri.com.sg.

Cong Ling Teo, Email: teo.cong.ling@singhealth.com.sg.

Jodhbir S. Mehta, Email: jodmehta@gmail.com.

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