Morphological alterations of corneal basal nerves in adult patients with mild to moderate dry eye by in vivo confocal microscopy

doi:10.21203/rs.3.rs-2483847/v1

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Morphological alterations of corneal basal nerves in adult patients with mild to moderate dry eye by in vivo confocal microscopy

https://doi.org/10.21203/rs.3.rs-2483847/v1

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Background/Objectives: In previous studies, the morphological differences of corneal basal nerves in dry eye patients may be related to severity and aging.The aim of the study was to evaluate the morphological alterations of corneal basal nerves in adult patients with mild to moderate dry eye disease (DED) by in vivo confocal microscopy (IVCM).

Subjects/Methods:Prospective, cross-sectional study.Forty-two adult patients (19-44 years) with mild to moderate DED and 16 sex- and age-matched healthy controls were included in this study. All patients had a history of dry eye lasting less than 12 months.The Ocular Surface Disease Index (OSDI), tear film break-up time (TBUT), sodium fluorescein staining andSchirmer’s test, and IVCM were used to observe central corneal basal nerve parameters, including nerve density, main number, branch number, width, reflectivity, tortuosity and beading number.

Results: Compared with the 16 healthy subjects (16 eyes), the 42 patients (42 eyes) with DED showed higher nerve density (21.889±3.459 vs. 16.653±1.793, P<0.001) and branch numbers (9.694±2.817 vs. 4.403±1.539, P<0.001). The main nerve numbers (6.720±1.027 vs. 6.328±0.521, P=0.199) were not significantly increased. Moreover, greater nerve width, reflectivity, tortuosity and beading numbers were observed in the DED patients (P <0.001). In the DED group, nerve density was positively correlated with main numbers, branch numbers, width and reflectivity (r=0.513, P<0.000, r=0.790, P<0.000, r=0.420, P=0.006 and r=0.526, P<0.000, respectively) and negatively correlated with tortuosity (r=-0.473, P=0.02).

Conclusions: Adult patients with mild to moderate DED demonstrated higher nerve density, branch numbers, width, tortuosity, reflectivity and beading numbers than normal subjects. IVCM may be a sensitive indicator for assessing mild to moderate DED in the early stage.

Health sciences/Diseases/Eye diseases/Corneal diseases

Health sciences/Biomarkers/Predictive markers

Dry eye disease (DED) is a complex and multifactor chronic disease that affects the ocular surface. With the widespread use of mobile phones, computers and other devices, the incidence of DED has increased rapidly in young adults.¹ With further research on the pathogenesis of DED, the relationship between this disease and corneal nerve is receiving increased attention. Recent studies have shown that the painful symptoms of DED and continuous damage to the ocular surface are closely related to abnormal corneal neuromorphology. At present, in vivo confocal microscopy (IVCM) is the only device that can directly, continuously and dynamically observe corneal nerves in vivo.² In previous reports, the corneal nerves in patients with DED showed increased tortuosity, reflectivity, width and beading, although study results about the density of corneal nerves are not consistent. Most scholars believe that the density of corneal nerves decreases,^3–8 although some scholars have observed no change,^9,10 and a few scholars suggest that the density of corneal nerves increases.¹¹ These different results may be related to the type and severity of DED observed in different studies. Many previous studies have had varying proportions of patients with severe dry eye, which may have influenced the results.However, the morphology of corneal nerves in patients with mild to moderate DED is rarely reported. Moreover, other studies have found that the density of corneal nerves negatively correlate with age, and the nerves in adult patients with DED are less affected. Adults aged 19 through 44 years are the backbone of society. Dry eyes are common in this group of people due to excessive screen use, and it is important to investigate how corneal neuromorphology changes in individuals with DED, although the morphology of corneal nerves in this population of DED patients has not been studied. The purpose of this study was to observe the morphological characteristics of corneal basal nerves in adult patients with mild to moderate DED by IVCM.

2.1 Study design and patients

This prospective, cross-sectional study was conducted at the DED Clinic of the Eye Hospital of Shandong First Medical University. All the subjects recruited from April 2021 to May 2022 were informed of the study purpose and signed informed consent forms. This study was approved by the Ethics Committee of the Eye Hospital of Shandong First Medical University and was conducted according to the Declaration of Helsinki. The recruited subjects included adult patients with mild to moderate DED and healthy volunteers. Both groups were matched in sex and age.

The inclusion criteria were as follows: (1) age from 19 to 44 years; (2) complaints of symptoms less than 12 months; (3) Ocular Surface Disease Index (OSDI) ≥ 13; (4) tear film break-up time (TBUT) < 10 seconds or > five spots of corneal fluorescein staining; and (5) no other systemic diseases, including rheumatic immune diseases such as Sjögren's syndrome.^12,13 In the inclusion criteria, to exclude the influence of old age on nerve density, adult dry eye patients aged 19–44 years, determined from the international definition of adult, were selected.

In addition, the symptoms and signs of patients with DED are often inconsistent in clinical practice. Therefore, we used the severity classification criteria in the Chinese Expert Consensus on Dry Eye: Definition and Classification (2020)¹⁴; that is, the classification of DED severity was mainly based on the signs of dry eye.

1. Mild: No obvious signs of ocular surface injury were observed under a slit lamp microscope (fluorescein staining spots on the cornea < 5), and the breakup time (BUT) was ≥ 2 s.

2. Moderate: The range of corneal injury observed under a slit lamp microscope was not more than 2 quadrants and/or corneal fluorescein staining spots ≥ 5 and < 30, and the BUT was ≥ 2 s.

For the control subjects, there were no complaints of ocular surface irritation and no anterior segment abnormalities observed by biomicroscopic examination or ocular surface tests.

The control group consisted of sex- and age-matched healthy volunteers with TBUT > 5 seconds, Schirmer’s test values > 10 mm and no symptoms of dry eye or other ocular conditions.

The exclusion criteria for both groups were as follows: (1) inability to complete the DED questionnaire independently; (2) history of ocular and systemic diseases, including diagnosis of rheumatoid immune-related diseases, such as Sjögren's syndrome and rheumatoid arthritis; and (3) previous eye surgery or contact lens use.

2.2 Clinical evaluation

All the subjects completed the OSDI questionnaire under the guidance of the same physician.¹⁵ Then, all the subjects underwent examination in the following order: TBUT, corneal fluorescein staining, and Schirmer’s test with anesthesia. The TBUT refers to the time from eye opening to the appearance of the first black spot on the cornea stained with fluorescein sodium.¹² Corneal staining was evaluated using the National Eye Institute (NEI) scale after application of fluorescein.¹⁶ Schirmer’s test was performed under topical anesthesia for 5 minutes with the patient’s eyes closed.¹²

2.3 IVCM

Confocal microscopy was performed in the center of the cornea. All the subjects were examined by the same skilled physician. The central corneal tissue was observed using the Rostock Corneal Module (RCM) by IVCM (HRT-3, Heidelberg Engineering, Germany). The instrument parameters were as follows: laser wavelength, 670 nm; field of view, 400 µm×400 µm; image resolution, 384 pixels×384 pixels; magnification, 800 times; and transverse and longitudinal resolution, 1 µm.¹⁷ Before examination, the eyes of the subject were treated with topical anesthesia (0.4% bupivacaine eye drops) once. The heads of the subjects were fixed while sitting, and an adjusting handle was used to move the objective lens forward and align the corneal central area. The images of the corneal subbasal nerve layer were selected by a masked observer. Approximately 20 images were collected from the central cornea of each eye, and the 5 images with the most representative subbasal nerve fibers were selected for quantitative analysis. Images of corneal subbasal nerves were retrospectively analyzed using Neuron J (Biomedical Imaging Group) by two researchers who were blinded to patient identity and the ocular surface investigation results. The parameters used to evaluate the subbasal corneal nerves were the following: (1) density of corneal basal nerves (total length of the nerves visible within a frame); (2) main number of corneal basal nerves (sum of the long nerve fiber bundles observed within a frame); (3) branch number of corneal basal nerves (number of fine fibers connecting to long nerve fibers within a frame); (4) width of corneal basal nerves (mean width of all long nerve fibers within a frame); (5) tortuosity (graded by simultaneously considering the frequency and amplitude of the changes in the nerve fiber direction^4,18); (6) reflectivity (graded with respect to the background^4,18); and (7) number of beadings (number of beadings measured on all long nerves within a frame). The parameters for each eye are reported as the average values of the five images selected at the beginning.

2.4 Statistical analysis

Statistical analysis was performed using SPSS 25.0 (Chicago, IL). For each patient, one eye was chosen randomly for statistical analysis. Quantitative variables are presented as the mean (M) and SD. The Kruskal–Wallis H test was applied to assess differences between groups and among the three groups. Variables were compared using the nonparametric Mann–Whitney test. Spearman correlation coefficients were utilized to assess correlations among age, clinical indicators and IVCM parameters in the DED group. P values were considered statistically significant at less than 0.05.

This study included 42 eyes of 42 patients with mild to moderate DED (20 males and 22 females; age, 30.452 ± 6.371 years; range, 21–44 years) and 16 eyes of 16 normal control subjects (9 males and 7 females; mean age, 29.250 ± 8.095 years; range, 21–44 years). The demographic data are presented in Table 1. There was no significant difference in terms of age (P = 0.413) or sex (χ2 = 0.557) between the DED group and the control group.

Table 1

Demographic Data and Clinical Test Results
Parameters	DED group	Control Group	P Value
Patients, n	42	16
Sex
Male, n (%)	20(47.6)	9(56.2)	0.557
Female, n (%)	22(52.4)	7(43.8)
Age, y	30.452 ± 6.371	29.250 ± 8.095	0.413
OSDI	38.694 ± 17.219	7.805 ± 3.063	< 0.001
TBUT,s	4.599 ± 2.437	12.246 ± 2.671	< 0.001
NEI cornea score	2.333 ± 1.455	0.156 ± 0.239	< 0.001
Schirmer’s test score	6.404 ± 4.221	17.750 ± 2.620	< 0.001
DED = dry eye disease; OSDI = Ocular Surface Disease Index; TBUT = tear film break-up time; NEI = National Eye Institute

Compared with the control group, clinical evaluation in the DED group revealed significant changes: higher OSDI (P < 0.001) and NEI corneal scores (P < 0.001) and lower TBUT (P < 0.001) and Schirmer’s test score (P < 0.001). The clinical data are presented in Table 1.

The density and branch number of corneal basal nerves were significantly increased in the DED group compared to those in the control group (P < 0.001). Moreover, the main number of corneal basal nerves was higher in the DED group, but the difference was not statistically significant (P = 0.199) (Figs. 1 and 2). The width, tortuosity, reflectivity and number of beadings were also increased in the DED group (P < 0.001) (Fig. 3). Among them, tortuosity and reflectivity show different degrees (Fig. 4). The IVCM analysis of corneal basal nerves is shown in Table 2.

Table 2

IVCM Analysis of Corneal Basal Nerves Parameters
Parameters	DED group	Control group	P Value
Density of corneal basal nerves, mm/mm²	21.889 ± 3.459	16.653 ± 1.793	< 0.001
Main number of corneal basal nerves, number/mm²	6.720 ± 1.027	6.328 ± 0.521	0.199
Branch number of corneal basal nerves, number/mm²	9.694 ± 2.817	4.403 ± 1.539	< 0.001
Width, µm	3.132 ± 0.590	2.011 ± 0.314	< 0.001
Reflectivity, arbitrary units	3.003 ± 0.351	2.301 ± 0.324	< 0.001
Tortuosity, arbitrary units	2.702 ± 0.287	2.096 ± 0.272	< 0.001
Number of beadings, number/100 µm of nerve length	12.246 ± 1.070	9.154 ± 1.909	< 0.001
IVCM = in vivo confocal microscopy; DED = dry eye disease

In addition, the density of corneal basal nerves was positively correlated with the main numbers (r = 0.513 P < 0.000), reflectivity (r = 0.526 P < 0.000), width (r = 0.420 P = 0.006), and branch numbers (r = 0.790 P < 0.000) and negatively correlated with tortuosity (r=-0.473 P = 0.02). The statistical results of the correlations of the IVCM analysis results are presented in Table 3. Furthermore, the beading number was positively associated with the NEI score (r = 0.558, P = 0.000) and negatively associated with TBUT (r=-0.496, P = 0.001).

Table 3

Statistical Results of Correlations of the IVCM Analysis Results
IVCM Parameter	Density of corneal basal nerves	Main number of corneal basal nerves	Width, µm	Number of beadings	Branch number of corneal basal nerves	Tortuosity	Reflectivity
Density of corneal basal nerves
r	--	.513**	.420**	.189	.790**	− .473**	.526**
P	--	.001	.006	.230	.000	.002	.000
Main number of corneal basal nerves
r	--	--	.164	-0.015	.354*	− .598**	.149
P	--	--	.299	.927	.021	.000	.346
Width
r	--	--	--	− .041	.439**	− .054	.382*
P	--	--	--	.798	.004	.733	.013
Number of beadings
r	--	--	--	--	.117	− .089	.338*
P	--	--	--	--	.459	.573	.029
Branch number of corneal basal nerves
r	--	--	--	--	--	− .147	.410**
P	--	--	--	--	--	.353	.007
Tortuosity
r	--	--	--	--	--	--	− .090
P	--	--	--	--	--	--	.571
At the 0.05 level (two-tailed), the correlation was significant; *At the 0.01 level (two-tailed), the correlation was significant; IVCM = in vivo confocal microscopy

The present study included adult DED patients between 19 and 44 years of age as a case group and healthy volunteers of the same age group as controls. In previous studies, negative correlations between the density of corneal basal nerves and age in both DED subjects and healthy people were reported.^3,19 In addition, to determine whether mice show changes in cornea nerves with aging, M.A. Stepp²⁰compared cornea nerve axon density of 6 to 8 week old mice to that of 20 to 24 months, and the results showed that the corneal nerve axon density of older mice was significantly lower than that of younger mice.Nevertheless, there have been no reports of age classifications or observations of corneal neuromorphology in adult DED patients. Therefore, to exclude the influence of aging on the corneal nerve, adult patients with DED were selected as research subjects for this study. Moreover, as adult patients with DED tend to have a short disease course, we were able to observe morphological changes in corneal nerves in the early stage of DED.

Furthermore, we included DED patients with mild to moderate DED. Previous studies have found that the density of corneal basal nerves in DED patients with Sjögren's syndrome are lower than those in DED patients without Sjögren's syndrome,²¹ and such cases tend to be more serious, indicating that the severity of the disease has an impact on corneal neuromorphology.⁴ Indeed, we selected mild to moderate DED patients without Sjögren's syndrome to reduce influencing factors.

As expected, the corneal nerves of adult patients with mild to moderate DED displayed obvious morphological changes. Compared with healthy subjects, the density of corneal basal nerves was significantly increased, which was not consistent with most previous reports. Most assert that the density of corneal basal nerves decrease,^3–8 some believe that the density does not change,^9,10 and a few purport that the density increases.¹¹ Some researchers suggest that the decreased corneal basal nerve density in patients with DED is a sign of nerve injury^22–25 Labbe et al.⁴ also reported that the density of corneal basal nerves in DED correlates negatively with the severity. Nonetheless, regeneration of the corneal nerve is a key point that cannot be ignored. The pathological process of DED is likely a coexisting process of corneal nerve injury and regeneration,^4,11 with inflammation being the driving force.^26–28

In addition, two interesting results were obtained in this study: although the main number of corneal basal nerves in the DED group was higher than that in the control group, the difference was not statistically significant. Regardless, the number of nerve branches in the DED group was significantly higher than that in the control group. As the density of subbasal nerves refers to the length of all visible nerves in the image,⁴ namely, the main nerve and branch of corneal basal nerves, they will directly affect nerve density measurement results. It is suggested that the increased density of the corneal basal nerve in the DED group may be caused by the increase in nerve branches, with possible regeneration of corneal nerves during the pathological changes of DED.

Dry eye is a chronic inflammatory ocular surface disease, and a decrease in corneal nerve density might be considered a marker of nerve injury.^22–25 Previous studies have found that inflammation correlates negatively with nerve density.^16,29−31 For instance, higher levels of inflammation and lower nerve density have been observed in patients with severe dry eye, such as Sjögren's syndrome.^3,4,30,32 In addition, using IVCM, Cox et al.³³ observed higher inflammation levels and lower nerve density in aqueous-deficient dry eye than in evaporative dry eye. Some researchers have proposed that cytokines produced by immune cells are recognized by receptors on nerves, which may result in nerve degradation.³⁴ In the process of chronic inflammatory injury in DED, overexpression of inflammatory factors not only causes damage to the corneal epithelium and nerves but also activates corneal stromal cells, causing an increase in nerve growth factor (NGF) levels, and overexpressed NGF further promotes nerve regeneration.^6,9 Villani et al.⁷ reported an increase in the number of hyperreflective corneal stromal cells in dry eye with Sjögren's syndrome based on IVCM. Studies have also found increased levels of interleukin-1 (IL-1) and tumor necrosis factor (TNF) as well as NGF in the tears of dry eye patients.^6,35 In the process of injury and regeneration, different densities and numbers of corneal basal nerves in different processes are likely.

Therefore, we propose the following hypothesis for the mechanism of increased density and branch number of corneal basal nerves in adult patients with mild to moderate DED. Because inflammation has not caused significant nerve damage in adult patients with DED, overexpression of NGF promotes the regeneration of compensatory hyperplasia of the corneal basal nerve branches such that the regeneration rate of corneal nerves is greater than the damage rate, resulting in an increase in the density of corneal basal nerves. The increase in bead number, width, tortuosity and reflectivity also indicates metabolic activity in the nerve. The above theory suggests that timely intervention in the stage of corneal neurocompensatory hyperplasia may avoid corneal nerve injury. In a randomized clinical trial, Kheirkhah et al.³⁶ showed the effect of the corneal nerve on DED: after a month of therapy, only patients with near-normal corneal nerve density experienced improvements in both symptoms and signs, whereas patients with low corneal nerve density did not show changes in signs or symptoms. This study provides theoretical support for the need for early treatment of mild to moderate DED.

In addition, we found that the density of corneal basal nerves correlated negatively with tortuosity. In fact, tortuosity may be intuitive evidence of corneal nerve injury and regeneration, and morphological changes become more obvious with the prolongation and aggravation of disease.^30,37 Studies have found that the tortuosity of the corneal basal nerve in the dry eyes of patients with Sjögren's syndrome is significantly higher than that in the dry eyes of patients without Sjögren's syndrome.³⁸ Therefore, we propose the following hypothesis: in the early or mild to moderate stage of DED, the frequency of nerve injury is low, the cornea undergoes compensatory hyperplastic change, and the tortuosity of the cornea changes little; with progression of the disease, nerve damage gradually increases, the frequency of damage increases correspondingly, and obvious tortuosity morphological changes gradually appear. Hence, tortuosity may be used as an indicator of the course and severity of DED, and the corresponding corneal basal nerve density and quantity can be used as an indicator of early DED.

To evaluate the impact of the course of DED on corneal nerves, our patients were also grouped according to the time of chief complaint in this study, although we found no significant differences in corneal nerve parameters among the three groups. Thus, the time of chief complaint cannot objectively reflect the actual course of DED.

There are some limitations to the present methodology that must be mentioned. First, we excluded patients with immune-related DED, such as Sjögren's syndrome, and subjectively considered adult DED patients to have milder disease without grouping them according to disease severity. Second, although we discuss the influence of the course of DED on corneal neuromorphology, we used no statistical data on the specific course of DED. Third, staging based on the time of chief complaint in this study is subjective and cannot objectively distinguish the course of DED in patients.

In summary, adult patients with mild to moderate DED showed significant changes in corneal basal nerve morphology. The density of corneal basal nerves increased significantly, which may be related to compensatory hyperplasia of the number of nerve branches in the early stage of DED. The increased nerve density and branch numbers observed in IVCM may be sensitive indicators for assessing DED in the early stage.

Acknowledgments

The authors would like to thank Ms. Tong Liu for her editorial assistance.

Data availability statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflict of interest

No conflicting relationship exists for any author.

Funding

This study was supported by the National Natural Science Foundation of China(82271059), Natural Science Foundation of Shandong Province (ZR2020QH146), Taishan Scholars Program for Young Scholars(20161059) and the Medical and Health Science and Technology Development Project of Shandong Province (202007021324). The funding organization had no role in the design or conduct of this research.

Author Contribution Statement

Li G.W.was responsible for setting of overarching research goals and aims, designing of methodology, applying statistical methods, conducting a research and investigation process, writing original draft.Dong M.C.was responsible for conducting a research and investigation process, managing activities to annotate, designing computer programs. Zhong X.W.was responsible for provisioning of study materials and instrumentation, making pictures.Wang S.T. was responsible for provisioning of study materials and instrumentation, making pictures.Shi W.Y. was responsible for setting of overarching research goals and aims, writing - review & editing.Li S.X. was responsible for setting of overarching research goals and aims, verificationing of the overall replication and reproducibility of experiments, oversighting responsibility for the research activity planning and execution, managing responsibility for the research activity planning and execution, acquiting of the financial support for the project leading to this publication.

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Morphological alterations of corneal basal nerves in adult patients with mild to moderate dry eye by in vivo confocal microscopy

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1. Introduction

2. Methods

2.1 Study design and patients

2.2 Clinical evaluation

2.3 IVCM

2.4 Statistical analysis

3. Results

4. Discussion

5. Conclusion

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