Neurofilaments in Tears as Early Biomarkers of Corneal Neurodegeneration in Diabetes Without Retinopathy

Abstract

Background:

Neurodegeneration in the cornea, particularly in diabetic patients, is an emerging area of research that highlights the potential of tear fluid as a source of biomarkers. Neurofilaments light chain (Nf-L), which are structural proteins found in neurons, have been identified as promising candidates for early biomarkers of neurodegeneration.

Objective:

This study investigates the correlation Nf-L levels in tears and corneal in vivo confocal microscopy (IVCM) parameters in patients with diabetes mellitus without retinopathy with the aim of associating Nf-L level with corneal neurodegeneration.

Design:

Case-control observational study.

Methods:

We enrolled 20 patients with diabetes mellitus type 2 (DM) and 20 healthy controls (HC): we studied the levels of Nf-L in serum and tears, and we looked for a correlation with the parameters detected on IVCM, including corneal nerve fiber length (CNFL), corneal nerve fiber density (CNFD) and corneal nerve branching density (CNBD).

Results:

Serum Nf-L levels showed no difference between DM (mean ± SD 14.00 ± 2.18 pg/mL) and HC (mean ± SD 15.18 ± 2.28 pg/mL). Instead we detected a significant increase in Nf-L levels in DM tears (right eye mean ± SD 321.23 ± 192.40 pg/mL; left eye mean ± SD 277.90 ± 254.23 pg/mL) compared to HC (right eye mean ± SD 25.80 ± 22.0 pg/mL; left eye mean ± SD 27.96 ± 20.21 pg/mL). Furthermore, a negative correlation was evident between Nf-L levels and IVCM parameters: CNFL/Nf-L r −.4234 P < .001; CNFD/Nf-L −.4959 P < .001; CNBD/Nf-L r −.5310 P < .0005.

Conclusion:

Our findings indicate that Nf-L levels are significantly elevated in the tears of DM patients and that high Nf-L levels correlate with lower corneal IVCM parameters, suggesting that tears Nf-L could be a reliable biomarker for early corneal neurodegeneration in diabetes without retinopathy.

Introduction

Diabetic retinopathy (DR) is a well-documented complication of diabetes mellitus (DM), characterized by damage to the retinal blood vessels, which can lead to vision impairment and blindness. ¹ However, the implications of diabetes extend beyond the retina, significantly affecting the corneal nerve structure and function. The cornea, being the most sensitive part of the eye, is innervated by a complex network of nerve fibers, primarily derived from the ophthalmic branch of the trigeminal nerve. ² In diabetic patients, alterations in corneal nerve morphology and function have been increasingly recognized as a critical aspect of diabetic neuropathy, which can precede the onset of diabetic retinopathy itself often leading to dry eye syndrome and impaired wound healing. ³ Studies have shown that diabetic patients, even those without retinopathy, exhibit significant corneal nerve damage, which can be assessed through techniques such as in vivo confocal microscopy (IVCM). ⁴

Research indicates that diabetic patients exhibit a marked reduction in corneal nerve fiber density (CNFD), corneal nerve fiber length (CNFL) and corneal nerve branching density (CNBD), which are critical indicators of corneal health. For instance, studies using IVCM have demonstrated significant losses in corneal nerve fibers in both type 1 and type 2 diabetes, independent of the presence of diabetic retinopathy.^5,6 This loss of corneal innervation correlates with the severity of diabetic neuropathy, suggesting that corneal nerve damage may serve as an early indicator of diabetic complications.^7,8 Furthermore, the reduction in corneal nerve fibers is associated with decreased corneal sensitivity, which can lead to impaired wound healing and increased risk of corneal ulcers. ⁹

The pathophysiological mechanisms underlying corneal nerve damage in diabetes are multifactorial. Chronic hyperglycemia is known to induce oxidative stress and inflammation, which contribute to nerve degeneration. Additionally, the role of neurotrophic factors, such as nerve growth factor (NGF) and pigment epithelium-derived factor (PEDF), has been highlighted in the context of diabetic corneal neuropathy. These factors are crucial for maintaining nerve health and promoting regeneration; their dysregulation in diabetes may exacerbate nerve damage. ¹⁰ Moreover, the presence of advanced glycation end-products (AGEs) in diabetic patients can further impair nerve function and integrity, leading to a vicious cycle of neuropathy and retinopathy. ⁹

Interestingly, the correlation between corneal nerve loss and diabetic retinopathy remains a subject of ongoing research. Some studies have suggested that while corneal nerve damage is prevalent in diabetic patients, it does not always correlate directly with the severity of diabetic retinopathy. ¹¹ For example, in a cohort of patients with type 1 diabetes, significant corneal nerve loss was observed even in the absence of retinopathy or microalbuminuria, indicating that corneal neuropathy can occur independently of retinal changes.^12,13 This finding underscores the importance of assessing corneal nerve health as part of a comprehensive evaluation of diabetic complications.

Neurofilaments (NFs) are integral components of the neuronal cytoskeleton, primarily composed of 3 subunits: neurofilament light (Nf-L), medium (Nf-M), and heavy (Nf-H) chains. These proteins are crucial for maintaining neuronal structure, supporting axonal transport, and ensuring the stability of axons and dendrites. ¹⁴ Their role extends beyond structural support; Nf-L are also involved in the pathophysiology of various neurodegenerative diseases, including multiple sclerosis (MS), ¹⁵ amyotrophic lateral sclerosis (ALS), ¹⁶ and in general in neurodegenerative diseases as a marker of axonal damage. ¹⁷

Initially, Nf-L levels were measured in cerebrospinal fluid (CSF) using enzyme-linked immunosorbent assay (ELISA). However, with the development of ultrasensitive assays, it is now possible to quantify Nf-L in blood and other fluids, broadening its potential applications. ¹⁸

Recent studies have begun to explore the use of Nf-L as a biomarker of ocular involvement in primary neurological diseases. ¹⁹ For instance, elevated Nf-L levels have been detected in the tears of patients with Parkinson’s disease, where they correlate with alterations in retinal vascular structures. ²⁰

This finding suggests that Nf-L could serve as a useful biomarker for detecting early neurodegenerative changes in a range of conditions, including corneal pathologies.

Our study aims to evaluate Nf-L as a novel biomarker of corneal neurodegeneration, investigating, for the first time, the correlation between Nf-L levels in tears and IVCM parameters, in patients with type 2 diabetes without retinopathy. By focusing on these indicators of corneal health, the study seeks to improve our understanding of corneal neurodegeneration progression and open up new possibilities for early detection and intervention, ultimately enhancing patient outcomes.

Material and Methods

Study Design and Participants

The study included 40 participants, divided into 2 groups: 20 patients suffering from type 2 diabetes for less than 10 years without diabetic retinopathy and neuropathy (DM group) and 20 age- and sex-matched healthy controls (HC group). Due the limited number of the population and not being a trial study the power calculation it’s influent. Inclusion criteria for the DM group were based on clinical diagnosis. Exclusion criteria included the presence of retinopathy by optical coherence tomography angiography (OCT-A), diabetic neuropathy by Toronto Clinical Scoring System, ²¹ other ocular diseases, systemic neurodegenerative conditions, and recent ocular surgeries. All participants provided written informed consent, in line with local institutional review board requirements at the time of collection. The study was approved by the ethics committee of University of Messina (07/Q0512/26).

Demographic and Clinical Characteristics

The demographic characteristics of the study population are summarized in Table 1. No significant differences were found in age and gender distribution between the DM and HC groups.

Table 1.

Clinical Characteristics of Study Participants.

Characteristics of participants	HC	DM
Number	20	20
Age, years (mean ± SD)	52.6 ± 21.3	53.2 ± 12.3
Sex, male, n (%)	10 (50)	14 (70)
Diabetes duration (years)	N.A.	7.2 ± 2.3

Abbreviations: HC, healthy controls; DM, diabetes mellitus.

Data are expressed as means ± standard deviations.

In Vivo Confocal Microscopy

IVCM was performed using the slit scanning confocal microscope (Confoscan 4, Nidek Technologies^®, Vigonza, Italy) after topical instillation of unpreserved 0.4% oxybuprocaine (Novesina; Novartis Farma, Origgio, Italy). The examination was performed using the 40× contact objective, provided with Z-Ring probe to allow precise positioning of the lens over the central corneal area. An ophthalmic gel 0.2% carbomer (Viscotears; Novartis Farma, Italy) was used to improve the adhesion of the objective to the cornea. A masked assessor, without knowing which group they belonged to, evidenced the nerves that were afterward evaluated by the build-in software of the machine. Complete scanning of the cornea was carried out 3 times for each eye. ²² The intra and inter observer repeatability of this procedure show less than 1% of difference between observers and none intra observer.

Neurofilament Analysis

Serum Blood samples were collected in Vacutainer EDTA tubesand centrifuged at 2000g for 10 minutes at room temperature and plasma samples were aliquoted and stored at −80°C. Tears Nf-L analysis in tears was performed following the method described by Lin et al. ²⁰ Tear fluid samples were collected from both eyes using Schirmer test strips (Ophtechnics unlimited, Haryana, India). Strips were placed at the inferior eye lid margin. Participants’ eyes were closed for 5 minutes. After 5 minutes, the participants were asked to open their eyes and look upward, and the strips were removed. The results of the Schirmer test were determined by the length of the moistened area of the strips. In all subjects examined, the Schirmer strip was wet between 5 and 10 mm, so the volume of tears is more or less the same. The strips were immediately put into polypropylene tubes and stored at − 80°C until further analysis. Tear fluid proteins were eluted by adding RIPA buffer (Thermo Fisher Scientific, Waltham, MA) with protease and phosphatase inhibitors (cOmplete and PhosStOP; Roche, Basel, Switzerland) on ice overnight, with subsequent centrifugation at 36 000g for 1 hour.

Nf-L was measured in serum and tears of all participants using a single-molecule array (SIMOA) detection system (SR-X, Quanterix, Billerica, MA).

Statistical Analysis

Numerical variables were expressed as the mean ± standard deviation of the mean. The groups were compared with the nonparametric Mann–Whitney U test. Pearson correlation coefficient test was applied to determine the correlation between tear N-fL and IVCM parameters. A P-value <.05 was considered to indicate significance. The statistical analyses were performed in in Prism 10 (GraphPad Software, La Jolla, CA, USA)

Results

Nf-L Analysis

Serum Nfl levels showed no significant differences between the 2 groups, confirming the absence of systemic neuroaxonal damage signs in patients in the DM group (Table 2; Figure 1).

Table 2.

Serum Nf-L Levels.

Serum Nf-L Levels	HC	DM	P value
Nf-L pg/mL	15.18 ± 2.28	14.00 ± 2.18	NS

Abbreviations: HC, healthy controls; DM, diabetes mellitus; Nf-L, neurofilament light chain.

Mann-Whitney U test. Data are expressed as means ± standard deviations.

No significant differences in serum Nf-L levels were present between the 2 groups.

NS = non statistical significance.

Figure 1.

Expression levels of Nf-L in serum in DM patients and healthy controls. No significant differences in serum Nf-L levels were present between the 2 groups. Mann-Whitney U test.

Abbreviations: HC, healthy controls; DM, diabetes mellitus; Nf-L, neurofilament light chain.

Tears Nf-L levels were significantly higher in the DM group compared to the HC group (P < .0001) in both eyes (Figure 2). These signs of neuroaxonal damage confirm the evidence of recent studies on the proteomics of tears in corneal alterations and accredit Nf-L as a reliable marker of corneal nerve pathologies. ²³

Figure 2.

Expression levels of Nf-L in tears in DM patients. The scatterplots display data density and means with standard deviations of tears levels of Nf-L in DM patients and healthy controls. Tears Nf-L levels were significantly higher in the DM group compared to the HC group (P < .0001) in both eyes. Mann-Whitney U test.

Abbreviations: HC, healthy controls; DM, diabetes mellitus; Nf-L, neurofilament light chain.

*P < .05; **P < .005; ***P < .0005; ****P < .0001; NS = non statistical significance.

There were no significant correlations between disease duration or glycated hemoglobin levels and NfL values (Table 3).

Table 3.

Disease Duration/Nf-L Tears Correlation and HbA1c/ Nf-L Tears Correlation.

Correlation	R	P value
Disease duration/Nf-L	.1360	NS
HbA1c/Nf-L	.1820	NS

Abbreviations: HbA1c, glycated hemoglobin; Nf-L, neurofilament light chain.

Spearman’s rank correlation coefficient.

No correlation was evident between tears Nf-L levels and disease duration or HbA1c levels.

NS = non statistical significance.

IVCM

Representative images from both groups are reported in Figure 3.

Figure 3.

In Vivo Confocal Microscopy image of Corneal Sub-Basal Nerve Plexus. HC: The source image of the SBNP fibers (A) and the software elaboration (B). DM: The source image of the SBNP fibers (C) and the software elaboration (D). DM sample shows evident reduction of CNFD and CNFL and CNBD.

Abbreviations: HC, healthy controls; DM, diabetes mellitus; CNFL, corneal nerve fiber length; CNFD, corneal nerve fiber density; CNBD, corneal nerve branching density.

The IVCM parameters and Nf-L tears concentration for both groups are presented in Table 4 and Figure 4. All IVCM values were significantly reduced in the DM group compared to controls in both eyes: these data confirm that early corneal nerve damage could be present in diabetic patients even before retinopathy occurs. ¹²

Table 4.

IVCM Parameters and Nf-L Tears Level of Study Participants.

IVCM and Nf-L	HC	DM	P value	HC	DM	P value
	Right eye (n = 20)	Right eye (n = 20)		Left eye (n = 20)	Left eye (n = 20)
CNFL mm/mm2	24.76 ± 6.8	13.16 ± 7.93	****	25.24 ± 5.17	14.53 ± 5.73	****
CNFD fibers/mm2	33.42 ± 10.58	21.67 ± 11.38	***	31.70 ± 6.90	20.98 ± 4.66	****
CNBD n/mm2	53.85 ± 14.66	21.75 ± 13.61	****	45.80 ± 18.28	19.95 ± 9.78	****
Nf-L pg/mL	25.80 ± 22.0	321.23 ± 192.40	****	27.96 ± 20.21	277.90 ± 254.23	****

Abbreviations: HC, healthy controls; DM, diabetes mellitus; CNFL, corneal nerve fiber length; CNFD, corneal nerve fiber density; CNBD, corneal nerve branching density; IVCM, in vivo confocal microscopy; Nf-L, neurofilament light chain.

Mann-Whitney U test. Data are expressed as means ± standard deviations.

Tears Nf-L levels in both eyes were significantly higher in the DM group compared to the HC group. IVCM parameters in both eyes were significantly worse in the DM group compared to the HC group.

^***P < .0005. ****P < .0001.

Figure 4.

IVCM parameters. Data are expressed as means ± standard deviations. Mann-Whitney U test. IVCM parameters in both eyes are all significant worse in DM respect HC.

Abbreviations: HC, healthy controls; DM, diabetes mellitus; CNFL, corneal nerve fiber length; CNFD, corneal nerve fiber density; CNBD, corneal nerve branching density; IVCM, in vivo confocal microscopy; Nf-L, neurofilament light chain.

*P < .05. **P < .005. ***P < .0005. ****P < .0001. NS = non statistical significance.

Correlation Between NfL Levels and IVCM Parameters

Significant negative correlations were observed between tears Nf-L levels and IVCM values in the DM group (Table 5; Figure 5). In small fiber neuropathies, the CNFL, CNFD, and CNBD observed with IVCM are decreased, ²⁴ : our findings strictly correlate these corneal alterations with Nf-L increased level in tears.

Table 5.

IVCM/Nf-L Tears Correlations.

IVCM/Nf-L Tears	r	P value
CNFL/Nf-L	−.4234	**
CNFD/Nf-L	−.4959	**
CNBD/Nf-L	−.5310	***

Abbreviations: CNFL, corneal nerve fiber length; CNFD, corneal nerve fiber density; CNBD, corneal nerve branching density; IVCM, in vivo confocal microscopy; Nf-L, neurofilament light chain.

Spearman’s rank correlation coefficient.

A significant negative correlation was evident between tears Nf-L levels and IVCM parameters.

**P < .005. ***P < .0005.

Figure 5.

Pearson correlation coefficient test between Nf-L tear level and IVCM parameters. Significant inverse correlation between Nf-L tear level and IVCM parameters in DM group.

Abbreviations: CNFL, corneal nerve fiber length; CNFD, corneal nerve fiber density; CNBD, corneal nerve branching density; IVCM, in vivo confocal microscopy; Nf-L, neurofilament light chain.

*P < .05. **P < .005. ***P < .0005. ****P < .0001. NS = non statistical significance.

Discussion

The assessment of corneal nerve morphology using IVCM has emerged as a valuable tool in clinical practice. This non-invasive imaging technique allows for detailed visualization of corneal nerve fibers, enabling early detection of neuropathy in diabetic patients. ²⁵ Studies have shown that corneal nerve fiber density can serve as a predictive marker for the development of diabetic neuropathy, potentially allowing for earlier intervention and management strategies. ²⁶

Recently, corneal nerve morphology assessed by IVCM has been proposed as an early marker for small nerve fiber damage in DM because corneal nerve fiber length correlates with clinical and electrophysiological measures of diabetic peripheral neuropathy.^27,28 The exact temporal relationship between neuropathy and retinopathy however remains unclear. The ability to identify corneal nerve damage before the onset of overt neuropathy or retinopathy could significantly impact patient outcomes by facilitating timely therapeutic measures.

The main parameters considered for morphological and densitometric analysis of corneal nerves are CNFL related to a total length of nerve fibers within image and expressed in mm/mm², CNFD (fibers/mm²) responding to the total number of fibers per frame divided by the area of the frame in square millimeters, CNBD (n/mm²) reporting the total number of branches divided by the area of frame in mm². ²⁹

In addition to its diagnostic utility, understanding the relationship between corneal nerve health and diabetic retinopathy may inform therapeutic strategies. For instance, interventions aimed at improving glycemic control have been associated with enhancements in corneal nerve morphology and function. ³⁰ Moreover, emerging treatments targeting neuroprotection and nerve regeneration hold promise for mitigating corneal nerve damage in diabetic patients. ¹⁰

Tear fluid analysis has gained attention as a promising approach for identifying biomarkers associated with diabetes and its complications. Elevated levels of inflammatory cytokines and neurotrophic factors have been reported in the tears of diabetic patients, indicating ongoing neurodegenerative processes.^31,32 Specifically, neurotrophins such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) have been implicated in the pathophysiology of diabetic neuropathy. ³³ The analysis of neurofilament levels in tears could complement these findings, providing a more comprehensive understanding of the neurodegenerative changes occurring in the cornea.

In healthy individuals, small quantities of Nf-L, light chain of primary structural proteins of the neuronal cytoskeleton are released from axons into the blood and cerebrospinal fluid (CSF), with this release increasing with age. However, in response to traumatic brain injury, stroke, and various neuroinflammatory and neurodegenerative conditions, much larger amounts of Nf-L are released.^34,35

Initially, Nf-L levels were measured using enzyme-linked immunosorbent assay (ELISA), a method that, while effective, lacked the sensitivity needed for detecting extremely low concentrations in complex biological matrices. However, advances in assay technology, such as the development of chemiluminescent immunoassays (CLIA), electrochemiluminescent assays (ECL), and single molecule array (Simoa^®), have significantly improved the ability to measure Nf-L levels in various fluids, including blood.^{36 -38} These ultrasensitive assays have even made it possible to measure Nf-L in tear fluid, a novel and non-invasive medium for monitoring corneal neurodegeneration.

Our aim was then to look for an association between Nf-L levels in tears and signs of corneal nerve damage, such as reduction in IVCM parameters and we were successful in finding this association for the first time. The significant inverse correlation between CNFL, CNFD, and CNBD and Nf-L levels in tears can not only confirm early corneal neurodegeneration in DM but also represent an innovative possibility for monitoring the DM stage. We did not find significant differences in serum Nf-L levels between HC and DM, and this on the one hand reassures us about the absence of subclinical systemic neurodegenerative diseases in both groups, on the other hand that the high levels of Nf-L in tears represent a sign of local neuronal damage. The limitations of our study are mainly represented by the limited number of participants and the known variability of the clinical manifestation of diabetes, although our population was carefully selected.

The use of tear fluid as a medium for Nf-L analysis presents several advantages. Tear fluid is easily accessible, and its collection is non-invasive, making it a practical option for repeated measurements over time. This could allow for the continuous monitoring of neurodegeneration in DM, providing valuable insights into disease progression and the effectiveness of therapeutic interventions. As research progresses, the incorporation of tear-based Nf-L measurements into clinical practice could revolutionize the early diagnosis and management of diabetic corneal neuropathy, ultimately improving patient outcomes and preserving vision. These strategies may not only improve corneal health but also potentially influence the progression of diabetic retinopathy.

Conclusions

The intricate relationship between corneal nerve integrity and diabetic retinopathy highlights the need for a holistic approach to managing diabetes-related complications. The assessment of corneal nerve fibers through IVCM provides critical insights into the early manifestations of diabetic neuropathy, which can precede and potentially predict the onset of diabetic retinopathy. As research continues to elucidate the mechanisms underlying corneal nerve damage in diabetes, there is hope for the development of targeted therapies that can preserve corneal health and improve overall patient outcomes.