This study was aimed at observing the morphological changes of the cornea with ocular in vivo confocal microscopy (IVCM) in patients with Terrien’s marginal degeneration (TMD). Ten patients (20 eyes) with TMD treated in the Department of Ophthalmology, Xiangya Hospital, and 10 healthy controls (20 eyes) were included in the current study. A detailed slit lamp microscopy, anterior segment photography, and corneal IVCM examination were performed for each eye. The density of central and marginal corneal epithelial cells, stromal cells, and subepithelial nerve fibers was compared between the two groups using the Wilcoxon rank sum test. Compared with the control group, the corneal epithelial and endothelial cells in the TMD group showed granular highly reflective substances and thinner subepithelial nerve fibers. The uneven dot-like highly reflective substances without cell structures appeared in the stromal layer of the cornea. The density of central and marginal corneal epithelial cells, stromal cells, and subepithelial nerve fibers was lower in the TMD group (
Terrien’s marginal degeneration (TMD) is a chronic and bilateral marginal keratopathy [
At present, the objective examination methods of TMD in the clinic include ultrasound biomicroscopy of the anterior segment, optical coherence tomography (OCT) of the anterior segment, corneal topography, and IVCM examination. Among all the above-mentioned methods, IVCM is the only one that can observe the normal and pathological corneal characteristics at the living corneal cell level. At present, there are only a few reports that IVCM is used to observe TMD. Some studies have found that TMD had hyperreflective substances deposition in the corneal epithelium, abnormal aberration of nerve fibers, activation of stromal cells, no cellular structural substances, and deposition of lipid layer particles by IVCM [
The clinical diagnosis for some early and atypical cases is difficult, which results in poor therapeutic effect in severe cases. In the present study, we investigated the corneal characteristic pathological changes of TMD on the in vivo level of cells by IVCM, which provides a clinical basis for clarifying the pathogenesis of TMD and meanwhile provides a sensitive and specific detection method for the clinical diagnosis and follow-up of TMD patients.
Patients diagnosed with TMD for the first time from July 2015 to June 2017 in the Department of Ophthalmology, Xiangya Hospital, Central South University, were included in the study. No gender or age restrictions were applied. Patients with history of ocular surgery, other corneal lesions by any causes, systemic diseases, immune system diseases, and psychological or mental illness were excluded from the study. Meanwhile, ten healthy volunteers matched by age and gender with no ocular or systemic diseases were recruited as controls.
Patients with TMD were classified into Stage I, Stage II, Stage III, and Stage IV by the Francois staging method [
Anterior segment photography of TMD at various stages. (a) Normal eyes; (b) TMD at Stage I: the gray-white turbidity at the corneal margin parallel to the corneal limbus (black arrow); (c) TMD at Stage II: the sulcus shape of the cornea was thinned, and there was lipid deposition in the sulcus (white arrow); (d) TMD at Stage III: the cornea in the lesion zone became thinner to form one or more bulging zones with a width of 1.5–3 mm or above (red arrow); (e) TMD at Stage IV: the marginal cornea was further thinned (blue arrow), with protruded central cornea.
The anterior segment was checked using a slit lamp.
The corneal photos were taken before and after corneal fluorescein staining.
An in vivo laser confocal microscope (Heidelberg Engineering, Laser class1) was used to perform IVCM. For each eye, different layers of corneal cells in both the central and peripheral cornea as well as the corneal subepithelial sensory plexus were scanned. All examinations were finished by one skilled physician with the same machine. The most affected portion of the cornea in all patients was examined.
The random number method was used to select 3 frames of pictures randomly at each level, each frame of picture was divided into 36 equal parts on average, and the random number method was used to select 10 equal parts randomly. The average corneal cell density was calculated using the built-in cell counting software [
At least 3 images of subepithelial nerve fibers were randomly selected for analysis in each case. The density of nerve fibers (the total length of nerve fibers in a picture) was measured by semiautomatic neural analysis and the tracking system (Neuron J) [
The experiment results underwent a descriptive statistical analysis based on the software SPSS 22.0 (IBM, Armonk, NY). After the normality test, the data of two groups showed a skewed distribution. Therefore, the median (median,
There were six TMD female patients and four TMD male patients, with a mean age of 51.9 ± 20.3 years. Among them, eight patients had binocular disease, accounting for 80% of the group. There were three patients (5 eyes) with TMD in the Stage-I period, three patients (5 eyes) with TMD in the Stage-II period, two patients (4 eyes) with TMD in the Stage-III period, and two patients (4 eyes) with TMD in the Stage-IV period (Figure
The corneal epithelial layer in the marginal and central cornea of both eyes in 10 TMD patients showed a decrease in cell density (
Corneal structures under IVCM. a1–a6 are the central corneal structures of TMD patients, b1–b6 are the central corneal structures of the controls, c1–c6 are the marginal corneal structures of TMD patients, and d1–d6 are the marginal corneal structures of the controls. The density of epithelial cells in the central and marginal cornea of TMD patients was decreased, the cell boundaries were blurred, and the dot-like highly reflective substances appeared; the density of the subepithelial nerve fiber plexus was significantly reduced and even disappeared (blue arrow), while the density of inactivated Langerhans cells (green arrow) and activated Langerhans cells (black arrow) was increased. The number of anterior, medium, and deep stromal cells of the cornea was decreased, the dot-like (yellow arrow), needle-like (white arrow), and flaky highly reflective substances without cell structures (red arrow) were presented, and the boundaries of endothelial cells were blurred.
The affected eyes of 10 TMD patients showed a significant decrease in the density of subepithelial nerve fibers (
The corneal stroma of TMD patients showed a variety of morphological changes by IVCM, including stromal neovascularization, bright cell nuclei, enhanced cytoplasmic reflex, activated stromal cells, a decrease or absence of stromal cells, dot-like, needle-like, highly reflexive, and uneven fiber tissue without cell structures. Besides, the morphological change was more significant in the marginal corneal stromal zone than that in the central corneal zone (
The corneal endothelial cells of 11 eyes in TMD patients showed dot-like reflections and blurred boundaries of endothelial cells, accounting for 55% of all cases (Figure
A large number of dendritic activated Langerhans cells were found in the Bowman membrane and subepithelial nerve plexus of two TMD patients (4 eyes), accounting for 20% of all cases. A large number of inactivated Langerhans cells without dendritic were found in the Bowman membrane and subepithelial nerve plexus of one TMD patient (1 eye), accounting for 5% of all cases (Figure
The density of central and marginal corneal epithelial cells; superficial, medium, and deep stromal cells; and subepithelial nerve fibers in patients of TMD group was significantly lower than that of the control group, and the difference was statistically significant (
Comparison of the density of central corneal cells between Terrien’s marginal degeneration patients and the control group (
Group | Case | Density of central corneal cells | ||||
---|---|---|---|---|---|---|
Epithelium | Anterior stroma | Medium stroma | Deep stroma | Endothelium | ||
TMD group | 10 | 5068.0 ± 1185.0 | 477.5 ± 744.0 | 250.0 ± 510.0 | 250.0 ± 529.0 | 2686.5 ± 314.0 |
Control group | 10 | 5930.0 ± 924.0 | 688.0 ± 519.0 | 521.5 ± 476.0 | 484.0 ± 465.0 | 2864.0 ± 422.0 |
|
— | −11.436 | −4.962 | −6.889 | −4.072 | −1.876 |
|
— | <0.001 |
<0.001 |
<0.001 |
<0.001 |
0.061 |
Comparison of the density of marginal corneal cells between Terrien’s marginal degeneration patients and the control group (
Group | Case | Density of marginal corneal cells | ||||
---|---|---|---|---|---|---|
Epithelium | Anterior stroma | Medium stroma | Deep stroma | Endothelium | ||
TMD group | 10 | 4679.0 ± 2662.0 | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.0 ± 0.0 | 2765.0 ± 435.0 |
Control group | 10 | 6139.0 ± 1082.0 | 412.0 ± 693.0 | 229.0 ± 606.0 | 216.0 ± 509.0 | 2814.0 ± 366.0 |
|
— | −13.740 | −9.948 | −6.632 | −7.569 | −1.568 |
|
— | <0.001 |
<0.001 |
<0.001 |
<0.001 |
0.117 |
Comparison of the density of corneal cells between the central corneal zone and marginal corneal zone in Terrien’s marginal degeneration patients (
Group | Case | Density of corneal cells | ||||
---|---|---|---|---|---|---|
Epithelium | Anterior stroma | Medium stroma | Deep stroma | Endothelium | ||
Central cornea | 10 | 5068.0 ± 1185.3 | 477.5 ± 744.0 | 250.0 ± 510.0 | 250.0 ± 529.0 | 2708.0 ± 319.0 |
Marginal cornea | 10 | 4679.0 ± 2001.5 | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.0 ± 0.0 | 2655.5 ± 362.3 |
|
— | −3.672 | −10.991 | −7.407 | −9.681 | −0.289 |
|
— | <0.001 |
<0.001 |
<0.001 |
<0.001 |
0.773 |
Density of subepithelial nerve fibers (
Group | Case | Marginal cornea | Group | Case | Central cornea | Group | Case | Subepithelial nerve fibers |
---|---|---|---|---|---|---|---|---|
TMD group | 10 | 0.0 ± 0.0 | TMD group | 10 | 2082.7 ± 10174.0 | TMD marginal cornea | 10 | 0.0 ± 0.0 |
Control group | 10 | 11555.0 ± 7800.6 | Control group | 10 | 12657.6 ± 6657.4 | TMD central cornea | 10 | 2082.7 ± 10174.0 |
|
— | −6.203 | — | — | −4.301 | — | — | −3.765 |
|
— | <0.001 |
— | — | <0.001 |
— | — | <0.001 |
Correlation analysis of the density of corneal cells and epithelial nerve fibers in patients at stages I, II, III, and IV (
Staging | Case | Density of corneal cells and epithelial nerve fibers | |||||
---|---|---|---|---|---|---|---|
Epithelium | Anterior stroma | Medium stroma | Deep stroma | Endothelium | Subepithelial nerve fibers | ||
Stage I | 3 | 5329.0 ± 1025.0 | 258.5 ± 756.0 | 257.5 ± 529.0 | 250.0 ± 529.0 | 2819.0 ± 349.0 | 8045.9 ± 14435.1 |
Stage II | 3 | 4847.5 ± 1275.0 | 0.0 ± 274.0 | 0.0 ± 253.0 | 0.0 ± 0.0 | 2654.0 ± 544.0 | 0.0 ± 1427.7 |
Stage III | 2 | 4611.5 ± 1341.0 | 0.0 ± 231.0 | 0.0 ± 250.0 | 0.0 ± 0.0 | 2462.0 ± 643.0 | 0.0 ± 4707.5 |
Stage IV | 2 | 0.0 ± 4020.0 | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.0 ± 0.0 | 2645.0 ± 323.0 | 0.0 ± 0.0 |
|
— | −0.499 | −0.353 | −0.395 | −0.190 | −0.418 | −0.397 |
|
— | <0.001 |
<0.001 |
<0.001 |
<0.001 |
>0.99 | 0.020 |
Marginal corneal degeneration, also known as Terrien’s corneal degeneration (TMD), was first reported by Trumpy in 1881 as “corneal hyaline degeneration” and described by Terrien in 1990. Suveges and associates demonstrated TMD as a disease of asymmetric progression in both eyes, while Austin and Brown classified TMD into quiescent period and advanced period through the histological presence of inflammation in TMD patients. Moreover, many researchers believed that inflammation played a key role in the progression of TMD and the inflammatory cells could promote the progression of TMD. TMD has been recognized to a certain extent, but it is still restricted to understand the tissue and cytological features of TMD. TMD presents no specific changes in pathological morphology, and it is mainly characterized by sulcus thinning of noninflammatory corneal stroma in the lesion zone, accompanied by superficial neovascularization and lipid deposition. With integral corneal epithelium, the epithelial cells can proliferate to more than 10 layers, accompanied by abnormal proliferation of basal membrane tissues. Historically, the cytologic examination of TMD could only rely on the pathologic and other invasive examination methods. The invention and application of IVCM made it possible to perform the noninvasive biopsy of corneal conjunctival lesions at the cellular level. However, there are still few studies on the characteristic pathological changes of TMD using IVCM.
IVCM is an instrument equipped with the laser scanning device based on the ordinary microscope imaging, and it uses the laser to stimulate the fluorescence probe to perform the in vivo noninvasive examination and obtain the fluorescence images of cells or cell structures within tissues through image processing of the computer. IVCM has been widely applied in many fields, such as cell biology, microbiology, and neurobiology, and its application field has gradually expanded from the basic disciplines to the clinical applications. IVCM is also getting popularity in ophthalmology, and the IVCM can be used in almost all lesions involving the ocular surface to observe the microstructure change of the ocular surface resulted from the lesions. The concept of confocal microscopy was first patented by Marvin Minsky in 1957 to study the brain neural cells [
Ferrari’s IVCM examination of a patient with early TMD showed the highly reflective substances in the lesion of the patient, the activated anterior stroma for honeycomb changes, the decreased density of subepithelial nerve plexus, and the abnormality of the epithelial basement membrane and Bowman membrane. Meanwhile, Ferrari et al. also found the abnormality of the subepithelial nerve plexus and the activated anterior stromal cells and dendritic cells in the contralateral eye of the patient [
In this study, we also found a decreased density of marginal and central corneal epithelial nerve fibers in both eyes of all TMD patents, the broken subepithelial nerve fibers, the absence of subepithelial nerve fibers for severe patients, the bent nerve trend, the thinned nerve fibers, activated dendritic cells in nerve fiber layers of some patients, the increased density, and dot-like, massive, and cord-like highly reflective substances. Moreover, the density reduction of subepithelial nerve fiber layer was positively corrected with the severity of TMD. And the corneal degeneration of the marginal zone was more severe than that of the central zone in all patients. The decrease in the density of the subepithelial nerve fiber plexus is found in many diseases, and our study has further demonstrated that TMD can also be involved in the corneal subepithelial nerve fibers.
TMD is a corneal stromal atrophy disease. Our study further confirmed the gradual decrease of corneal stromal cells of TMD patients and the loss of normal stromal structure with the development of the disease. We also found that the anterior stromal cells in TMD patients were not necessarily in an activated state, while they were associated with the severity and location of the disease. The central and corneal anterior stromal cells in TMD patients at Stages I and II could be activated, and the activated anterior stromal cells were mainly located in the central cornea. However, the central and marginal anterior stromal cells in TMD patients at Stages III and IV mainly showed a blurred or fuzzy structure of stromal cells, and even the dot-like, needle-like, and uneven reflections without cell structures were presented. We speculated that the anterior stromal cells in the early TMD period showed an activated state, while, with the development of the disease, the density of stromal cells gradually decreased and disappeared eventually, showing the dot-like, needle-like, and flaky uneven reflections without cell structures. Meanwhile, the similar changes were found in the medium and deep stromal cells.
Our studies showed the dot-like reflections in the corneal endothelium layer, blurred boundary of corneal endothelial cells, and uneven highly reflective deposition in 11 eyes among all 20 eyes. We infer that the corneal degeneration of the TMD patients can also be involved in the corneal endothelium. In previous IVCM studies, the abnormality of the corneal endothelium was not found in TMD patients. This is maybe because there is no comparative analysis of corneal endothelial cells in TMD patients with different pathological changes due to a small number of study cases.
Previous studies showed that TMD was an immune-related disease, and the activated anterior stromal cells and dendritic cells were found in the IVCM examination of TMD patients [
We hypothesized that TMD was a degenerative disease involving all the layers of cornea and the pathogenesis may be associated with the immunity, but there may be other pathogenesis mechanisms which need to be further studied. The degeneration was the first to be involved in the corneal stroma; TMD was a commonly degenerative disease for the marginal and peripheral cornea, and the degeneration of the marginal cornea was earlier and more severe than that of the central cornea. Apart from the corneal endothelial cells in TMD patients, the decrease in the density of corneal stromal cells and the density of subepithelial nerve fibers was negatively corrected with the severity of the disease. As a noninvasive examination tool to observe characteristics of corneal degeneration, IVCM will be of great significance for the diagnosis and follow-up of the TMD patients.
The data are available from Ting Chen to researchers who meet the criteria for access to the confidential data.
The authors declare that they have no conflicts of interest.
Ting Chen and Min Liao curated the data; Hua Wang were involved in formal analysis; Qiangxiang Li and Hua Wang were responsible for funding acquisition; Xiangbo Tang and Hua Wang were responsible for methodology; Qiangxiang Li was involved in collection of resources; Ting Chen and Xiangbo Tang analyzed the data using software; and Ting Chen wrote the original draft.
This work was supported financially by the grants from the National Natural Science Foundation of China (81170823), Science and Technology Program Project of Hunan Provincial Science & Technology Department (nos. 2015JC3011 and 2015JC3118), Science and Technology Plan Project of Hunan Provincial Administration of Traditional Chinese Medicine (201855), Science and Technology Plan Project of Changsha Municipal Science and Technology Bureau (kq1701054), Scientific research project of Hunan Province Education Department (16 C0965), and Project of Hunan Traditional Chinese Medicine Administration (201856).