*2.2. Participants*

Patients over 18 years suffering from DED of any underlying etiology were eligible for inclusion. The patients had to be under stable, unchanged, dry eye therapy for at least two months (in case of concomitant cyclosporine therapy, three months) by the time of inclusion. Patients were excluded if they participated in any other clinical trial, suffered from eye diseases other than dry eyes, had ocular surgery less than three months prior to study inclusion, were using punctual plugs, or had masquerading conditions as identified by Karpecki [50]. Masquerading conditions are conjunctivochalasis, recurrent corneal erosions, epithelial basement membrane dystrophy, mucus fishing syndrome, floppy eyelid syndrome, giant papillary conjunctivitis, Salzmann's nodular degeneration, and ocular rosacea.

As inclusion criteria for severe dry eye, the primary criteria, according to Baudouin et al., were chosen [51]. The dry eye symptoms were assessed using the Ocular Surface Disease Index (OSDI) questionnaire, with an OSDI score of 33 or more being required for inclusion [52]. Corneal fluorescein staining (CFS) was selected as a dry eye sign [53]. For inclusion, patients had to have at least one eye

with CFS Oxford grade 3 or more, but no confluent CFS. The eyes with the higher staining score were defined as study eyes.

#### *2.3. Confocal Scanning Laser Microscopy*

The Heidelberg Retina Tomograph (HRT 3), in combination with the Rostock Cornea Module (Heidelberg Engineering GmbH, Heidelberg, Germany), was used for the in vivo assessment of the corneal subbasal nerve plexus (SNP), as described previously [54,55]. Both eyes were anesthetized with topical anesthetic and covered with artificial tears. To prevent eye movements, the patients were asked to fixate on a spotlight with the unexamined eye.

Five non-overlapping images were taken in the central region of the cornea, close to the apex and more than 0.5 mm apart from the inferior whorl (see Figure 1A for an example of an image and Figure 1B after image processing by the reading center).

**Figure 1.** Single image from the subbasal nerve plexus (SNP) in an individual (**A**) and automatically detected nerve fibers used for quantification (**B**).

Image processing and quantitative image analysis were performed by the reading center using Mathematica (Version 11.3, Wolfram Research Inc., Champaign, IL, USA), as previously described [56]. The following SNP parameters were calculated: corneal nerve fiber length (CNFL), defined as the total length of all nerve fibers per unit area (mm/mm2); corneal nerve fiber density (CNFD), defined as the number of nerve fibers per unit area (n/mm2); corneal nerve branch density (CNBD), defined as the number of branching points per unit area (n/mm2); average weighted corneal nerve fiber tortuosity (CNFTo), reflected variability of nerve fiber directions and defined as absolute nerve fiber curvature/nerve fiber length (μm<sup>−</sup>1); corneal nerve connection points (CNCP), defined as the number of nerve fibers crossing the area boundary (connections/mm2); average corneal nerve single-fiber length (CNSFL), defined as the average length of nerve fibers (μm); and average weighted corneal nerve fiber thickness (CNFTh), measured as mean thickness perpendicular to the nerve fiber course (μm).

#### *2.4. Statistical Analysis*

Statistical analysis was performed using IBM SPSS Statistics (Version 22, IBM Corp., Armonk, New York, NY, USA). Descriptive statistics were calculated, and box plots were generated. Data were examined for normal distribution using the Shapiro–Wilk test. Group comparisons were performed using the Wilcoxon Signed Rank Test and the Mann–Whitney U test, respectively. The significance level was determined to be *p* < 0.05.
