OBJECTIVE: To evaluate and compare subbasal corneal nerve parameters of the inferior whorl in patients with dry eye disease (DED), neuropathic corneal pain (NCP), and controls using a novel deep-learning-based algorithm to analyze in-vivo confocal microscopy (IVCM) images.
METHODS: Subbasal nerve plexus (SNP) images of the inferior whorl of patients with DED (n = 49, 77 eyes), NCP (n = 14, 24 eyes), and controls (n = 41, 59 eyes) were taken with IVCM and further analyzed using an open-source artificial intelligence (AI)-based algorithm previously developed by our group. This algorithm automatically segments nerves, immune cells, and neuromas in the SNP. The following parameters were compared between groups: nerve area density, average nerve thickness, average nerve segment tortuosity, junction point density, neuroma density, and immune cell density.
RESULTS: 160 eyes of 104 patients (63 % females), aged 56.8 ± 15.4 years, were included. The mean nerve area density was significantly lower in the DED (P = 0.012) and NCP (P < 0.001) groups compared to the control group. The junction point density was lower in the NCP group compared with control (P = 0.001) and DED (P = 0.004) groups. The immune cell density was higher in the DED group compared with controls (P < 0.001).
CONCLUSIONS: Deep-learning-based analysis of IVCM images of the corneal SNP inferior whorl distinguished a decreased mean nerve area density in patients with DED and NCP compared with controls and an increased immune cell density in patients with oGVHD- and SS-associated DED. These findings suggest that the inferior whorl could be used as landmark to distinguish between patients with DED and NCP.

OBJECTIVE: The corneal cap thickness is a vital parameter designed in small incision lenticule extraction (SMILE). The purpose was to investigate the changes in corneal subbasal nerve plexus (SNP) and stromal cells with different cap thicknesses and evaluate the optimized design for the surgery.
METHODS: In this prospective, comparative, non-randomized study, a total of 108 eyes of 54 patients who underwent SMILE were allocated into three groups with different corneal cap thicknesses (110 μm, 120 μm or 130 μm group). The SNP and stromal cell morphological changes obtained from in vivo corneal confocal microscopy (IVCCM) along with their refractive outcomes were collected at 1 week, 1 month, 3 months and 6 months postoperatively. One-way analysis of variance (ANOVA) was used to compare the parameters among the three groups.
RESULTS: The SNPs in the three groups all decreased after surgery and revealed a gradual increasing trend during the 6-month follow-up. The values of the quantitative nerve metrics were significantly lower in the 110 μm group than in the 120 μm and 130 μm groups, especially at 1 week postoperatively. No difference was detected between the 120 μm and 130 μm groups at any time point. Both Langerhans cells and keratocytes were activated after surgery, and the activation was alleviated during the follow-up.
CONCLUSIONS: The SMILE surgeries with 110 μm, 120 μm or 130 μm cap thickness design achieved good efficacy, safety, accuracy and stability for moderate to high myopic correction while the thicker corneal cap was more beneficial for corneal nerve regeneration.

During life up to 70% of aniridia subjects develop aniridia-associated keratopathy (AAK). AAK is characterized by limbal stem cell insufficiency, impaired corneal epithelial cell differentiation and abnormal cell adhesion, which leads to centripetal spreading vascularization, conjunctivalization, and thickening of the cornea. Our aim was to examine the subbasal nerve plexus and central corneal stromal microstructure in subjects with congenital aniridia, using in vivo confocal laser scanning microscopy CLSM.
31 eyes of 18 patients (55.6% males, mean age: 25.22 ± 16.35 years) with congenital aniridia and 46 eyes of 29 healthy subjects (41.4% males, mean age 30 ± 14.82 years) were examined using the Rostock Cornea Module of Heidelberg Retina Tomograph-III. At the subbasal nerve plexus, corneal nerve fiber density (CNFD), corneal nerve fiber length (CNFL), corneal total branch density (CTBD), and corneal nerve fiber width (CNFW) were analyzed using ACCMetrics software. Keratocyte density in the anterior, middle and posterior stroma was assessed manually.
The CNFD (2.02 ± 4.08 vs 13.99 ± 6.34/mm2), CNFL (5.78 ± 2.68 vs 10.56 ± 2.82 mm/mm2) and CTBD (15.08 ± 15.62 vs 27.44 ± 15.05/mm2) were significantly lower in congenital aniridia subjects than in controls (p < 0.001 for all). CNFW was significantly higher in aniridia subjects than in controls (0.03 ± 0.004 vs 0.02 ± 0.003 mm/mm2) (p = 0.003). Keratocyte density was significantly lower in all stromal layers of aniridia subjects than in controls (p < 0.001 for all). Stromal alterations included confluent keratocytes, keratocytes with long extensions and hyperreflective dots between keratocytes in aniridia.
Decrease in CNFD, CNFL, and CTBD, as well as increase in CNFW well refer to the congenital aniridia-associated neuropathy. The decreased keratocyte density and the stromal alterations may be related to an increased cell death in congenital aniridia, nevertheless, stromal changes in different stages of AAK have to be further analyzed in detail.

OBJECTIVE: To investigate corneal neuropathy and corneal nerve alterations in type 2 diabetes mellitus (DM) patients with different diabetic retinopathy (DR) status.
METHODS: A total of 87 eyes of 87 patients with DM and 28 eyes of 28 healthy control subjects were included in the study. DM patients were further classified into 3 groups: patients without DR (NDR), patients with non-proliferative DR (NPDR), and patients with proliferative DR (PDR). PDR patients were classified into 2 groups regarding having undergone retinal argon laser photocoagulation treatment (ALP). Ocular surface disease index score (OSDI), average tear break-up time (A-BUT), corneal sensitivity and cornea nerve fiber length (CNFL), cornea nerve fiber density (CNFD), and cornea nerve branch density (CNBD) of the cornea subbasal nerve plexus (SBNP) were measured using in vivo confocal microscopy (IVCM).
RESULTS: OSDI scores increased and A-BUT decreased in DM patients compared to the control group, but no significant difference was found between DM patient groups. Corneal sensitivity decreased in DM patients who developed DR, compared to both the controls and the NDR group. CNFD and CNFL decreased in NPDR and PDR patients compared to controls. CNFD and CNBD decreased in patients who had developed PDR, compared to all three groups. All IVCM parameters decreased with DR progression.
CONCLUSIONS: IVCM can detect early structural corneal nerve changes in diabetic patients. The presence of DM affects ocular surface parameters, especially in long-term DM patients. Corneal sensitivity loss is increased with the presence of DR. All IVCM parameters decrease with DR development and its progression.

OBJECTIVE: We sought to investigate alterations in the corneal subbasal nerve plexus and endothelium in patients with Behçet\'s disease (BD).
METHODS: This cross-sectional study included 64 patients with BD and 30 age- and gender-matched healthy control subjects. Those with BD were classified as having ocular or non-ocular disease. All subjects underwent a corneal endothelial and subbasal nerve density evaluation using in vivo confocal microscopy (IVCM). The differences among groups were analyzed using the Kruskal-Wallis test followed by Dunn\'s multiple comparison procedure.
RESULTS: The mean age of study participants was 35.7 ± 10.2 years (16-58) in the ocular BD group, 39.6 ± 14.9 years (11-66) in the non-ocular BD group, and 34.1 ± 11.2 years (21-55) in the control group. No statistical significance was found in terms of age (p = 0.259) or sex (p = 0.560) between groups. The mean endothelial cell density determined with IVCM was 2124.9 [Formula: see text] 417.4 cells/mm2 (1811-3275) in the ocular group and 2546 [Formula: see text] 335 cells/mm2 (1798-3280) in the control group (p = 0.000). In the ocular group, the mean density of the subbasal nerve plexus was significantly lower (p = 0.004), and nerve tortuosity was significantly higher (p = 0.002).
CONCLUSIONS: Ocular BD could be responsible for changes in the corneal layers, especially endothelial and corneal nerve structures. Nerve density and tortuosity differences could be inflammatory indicators for BD.

OBJECTIVE: To describe the pattern of the nerves in the inferocentral whorl region of the human corneal subbasal nerve plexus (SBNP) in health and diseases known to affect the subbasal nerves.
METHODS: Laser-scanning in vivo confocal microscopy (IVCM) was used to image the SBNP bilaterally in 91 healthy subjects, 39 subjects with type 2 diabetes mellitus (T2DM), and 43 subjects with Parkinson\'s disease (PD). Whorl regions were classified according to nerve orientation relative to age and health/disease status.
RESULTS: Of 346 examined eyes, 300 (86.7%) had an identifiable whorl pattern. In healthy subjects, a clockwise nerve orientation of the whorl was most common (67.9%), followed by non-rotatory or \'seam\' morphology (21.4%), and counterclockwise (10.7%). The clockwise orientation was more prevalent in healthy subjects than in T2DM or PD (P < 0.001). Healthy individuals below 50 years of age had a predominantly clockwise orientation (93.8%) which was reduced to 51.9% in those over 50 years (P < 0.001). Age but not disease status explained whorl orientation in T2DM and PD groups. Moreover, whorl orientation is bilaterally clockwise in the young, but adopts other orientations and becomes asymmetric across eyes with age. Finally, we report reflective \'dot-like\' features confined to the whorl region of the subbasal plexus, sometimes appearing in close association with subbasal nerves and present in 84-93% of examined eyes regardless of disease status, eye or sex.
CONCLUSIONS: Subbasal nerves in the inferocentral whorl region are predominantly clockwise in young, healthy corneas. With aging and conditions of T2DM and PD, counterclockwise and non-rotatory configurations increase in prevalence, and bilateral symmetry is lost. Mechanisms regulating these changes warrant further investigation.

UNASSIGNED: Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory autoimmune disorder that damages optic nerves, brainstem, and spinal cord. In vivo corneal confocal microscopy (IVCM) is a noninvasive technique that provides corneal images with dendritic cells (DCs) and corneal subbasal nerve plexus (SBP), which arises from the trigeminal nerve.
UNASSIGNED: We investigated corneal SBP changes in NMOSD and proposed IVCM as a potential new disease severity biomarker for NMOSD.
UNASSIGNED: Seventeen age-sex matched NMOSD patients and 19 healthy participants underwent complete neurologic and ophthalmologic examinations. The duration of disease, first symptom, presence of optic neuritis attack, antibody status, Expanded Disability Status Scale(EDSS) score and disease severity score(DSS) were recorded. Retinal nerve fibre layer (RNFL) thickness was measured with optical coherence tomography, and corneal SBP images were taken with IVCM.
UNASSIGNED: NMOSD patients had significantly reduced corneal nerve fibre lenght-density and corneal nerve branch lenght-density compared with controls, while DC density was increased. NMOSD patients also showed significantly reduced RNFL thickness compared with controls. EDSS,DSS levels were inversely correlated with IVCM parameters.
UNASSIGNED: We observed significant corneal nerve fibre loss in NMOSD patients in relation to disease severity. IVCM can be a candidate noninvasive imaging method for axonal damage assessment in NMOSD that warrants further investigation.

In vivo large-area confocal laser scanning microscopy (CLSM) of the human eye using EyeGuidance technology allows a large-scale morphometric assessment of the corneal subbasal nerve plexus (SNP). Here, the SNP of a patient suffering from diabetes and associated late complications was analyzed. The SNP contained multiple clusters of large hyperintense, stellate-shaped, cellular-like structures. Comparable structures were not observed in control corneas from healthy volunteers. Two hypotheses regarding the origin of these atypical structures are proposed. First, these structures might be keratocyte-derived myofibroblasts that entered the epithelium from the underlying stroma through breaks in Bowman\'s layer. Second, these structures could be proliferating Schwann cells that entered the epithelium in association with subbasal nerves. The nature and pathophysiological significance of these atypical cellular structures, and whether they are a direct consequence of the patient\'s diabetic neuropathy/or a non-specific secondary effect of associated inflammatory processes, are unknown.

OBJECTIVE: To compare the reliability of the whorl-like region with that of the central cornea for accurate assessment of corneal subbasal nerve plexus (SNP) by analyzing the parameter variability of these two anatomical regions in repeated measurements.
METHODS: Participants were scanned in the central cornea and whorl-like region with in vivo confocal microscopy on three occasions by two examiners within a time span of one week. Coefficients of repeatability (CoR), intra-class correlation coefficient (ICC), and Bland-Altman scatter plots with 95% limits of agreement (LOA) in the central cornea and whorl-like region were calculated, respectively, based on the nerve fiber length, then the inter-observer and intra-observer agreement were compared between these two anatomical regions.
RESULTS: The inter-observer ICC was 0.945, the inter-observer CoR was 0.052, the intra-observer ICC was 0.936, and the inter-observer CoR was 0.046, with narrow 95% LOA within 1 standard deviation in the whorl-like region, whereas the inter-observer ICC was 0.600, the inter-observer CoR was 0.207, the intra-observer ICC was 0.206, and the intra-observer CoR was 0.253, with 95% LOA nearly threefold wider than the standard deviation in the central cornea.
CONCLUSIONS: Nerve parameter in the whorl-like region showed higher inter-observer and intra-observer agreement than that of the central cornea. The whorl-like region is a more reliable site for accurate assessment of SNP.

To evaluate corneal subbasal nerve plexus by in vivo confocal microscopy (IVCM) following punctal occlusion in patients with moderate to severe dry eye disease (DED).
Patients with grade 3 or 4 severity of DED based on Delphi Panel dry eye severity grading scheme were enrolled in the study. Permanent inferior punctal occlusion was performed. A comprehensive ophthalmic evaluation, including Ocular Surface Disease Index (OSDI) questionnaire, tear break-up time (TBUT), corneal fluorescein staining, conjunctival Rose bengal staining, Schirmer\'s test, and corneal sensation by Cochet-Bonnet esthesiometry, were performed at baseline, and 1 and 3 months after punctal occlusion. Furthermore, density and number of corneal subbasal nerves were evaluated by IVCM.
Forty-one eyes of 23 patients with a mean age of 46.3 ± 9.0 years were enrolled. Corneal fluorescein staining, Rose bengal staining, and TBUT significantly improved at 3 months following punctal occlusion (p < .015). Corneal esthesiometry significantly increased at both postoperative visits (p < .03), and OSDI scores improved only at 3-month follow-up (p < .005). Nerve density and total number significantly increased 3 months after punctal occlusion (p < .045). Baseline nerve density had significant correlations with TBUT, fluorescein staining, Rose bengal staining (p < .012), but not with esthesiometry, Schirmer scores, or OSDI scores (p > .329).
Corneal subbasal nerve density and total number increased following punctal occlusion in patients with moderate to severe DED. These findings were associated with improvements in corneal sensation, and signs and symptoms of DED. This emphasizes the effect of punctal occlusion in regeneration of corneal subbasal nerve plexus.