Characterization of dry eye disease in a mouse model by optical coherence tomography and fluorescein staining

: A custom-built ultrahigh-resolution optical coherence tomography (UHR-OCT) system and ﬂuorescein staining were employed for investigation of a scopolamine induced dry eye mouse model. Acquired data was used to evaluate common and complementary ﬁndings of the two modalities. Central corneal thickness as measured by UHR-OCT increased signiﬁcantly over the study period of 24 hours, from 89.0 ± 3.57 µm to 92.2 ± 4.07 µm. Both techniques were able to show corneal lesions with a large range of severity. Localized ﬂuorescein staining was detected in 5% and diﬀuse staining in 45% of cases where no epithelial damage was visible with OCT. However, OCT revealed stromal defects in 6% and endothelial defects in 18% of the cases, which could not be visualized via ﬂuorescein staining. Thus, while ﬂuorescein staining widely detected defects of the corneal surface in a mouse model of dry eye disease, OCT non-invasively revealed additional information about defect depth and involvement of particular layers. and non-invasive in vivo applicability, OCT allows for thickness quantiﬁcation of corneal tissue in both preclinical and clinical settings. Optical coherence tomography has previously been employed for assessing the thickness of corneal layers in patients suﬀering from DED as as for measuring CCT in an experimental dry eye model in mice The current study was performed to investigate the applicability of UHR-OCT for visualization of tissue morphology and quantitative assessment of anterior segment parameters in a DED mouse model. Furthermore, OCT data was compared to and correlated with photographs acquired after ﬂuorescein staining in order to evaluate common and complementary ﬁndings of the two modalities.


Introduction
Dry eye disease (DED) is a common disorder of the ocular surface that can impact negatively on patients' quality of life [1] and cannot yet be treated at its underlying cause [2]. The exact mechanisms of the disease are still unknown, but tear hyperosmolarity, tear film instability and a vicious cycle of ocular surface inflammations are accepted as defining causative factors [2,3].
To study DED under standardized conditions, understand the factors underlying the disease and evaluate novel therapies reliably, animal models are necessary. There are several established models for dry eye in the mouse [4,5] and many have been developed recently showing the growing interest in this topic [6][7][8]. Scopolamine (SCP) induced dry eye syndrome is a frequently used model [4,9], based on either transdermal application, subcutaneous injection or implantation of devices offering continuous delivery [9,10]. Muscarinic activity is blocked in the lacrimal glands causing tear deficiency [5]. Scopolamine is combined with a constant airflow chamber to induce desiccation. It has been classified as a combined tear film deficiency model, containing evaporation and aqueous deficiency as its causative factors [4].
Fluorescein staining is a standard method for the evaluation of corneal epithelial defects and dry eye [11]. The exact mechanism of the staining on a cellular level is uncertain, but theories include pooling in lesions, intercellular ingress due to decrease of tight junction integrity and intracellular accumulation due to a defective glycocalyx and increased cell membrane permeability shortly before a shedding event [5,12].
Optical coherence tomography (OCT) is an imaging technique that provides cross sectional images with resolutions in the micrometer range. It is well suited to assess the morphology of the anterior eye segment [13] and resolves corneal epithelium, Bowman's layer, if present in the species, corneal stroma and corneal endothelium. For measurement of the central corneal thickness (CCT) standard resolutions are sufficient, making it the most popular anterior segment parameter to be quantitatively assessed by conventional OCT systems. Advances in light source technology led to the introduction of ultrahigh-resolution OCT (UHR-OCT), which provides a superior axial resolution as compared to standard OCT machines. These systems allow for a more refined analysis of corneal morphology. This includes differentiation between Descemet membrane and corneal endothelium [14,15], visualization and quantification of the precorneal tear film [16,17] and lipid layer [18] thickness, precise quantification of the thicknesses of epithelium, Bowman's layer and stroma [19,20] and characterization of wounds and wound healing with details such as depth of the defect, involvement of surrounding structures and exact location of the lesion margins [21,22]. Due to its excellent resolution and its non-contact and non-invasive in vivo applicability, OCT allows for thickness quantification of corneal tissue in both preclinical and clinical settings. Optical coherence tomography has previously been employed for assessing the thickness of corneal layers in patients suffering from DED [23][24][25][26] as well as for measuring CCT in an experimental dry eye model in mice [27].
The current study was performed to investigate the applicability of UHR-OCT for visualization of tissue morphology and quantitative assessment of anterior segment parameters in a DED mouse model. Furthermore, OCT data was compared to and correlated with photographs acquired after fluorescein staining in order to evaluate common and complementary findings of the two modalities.

Animals
Osmotic pumps (ALZET Osmotic Pumps, 10260 Bubb Road, Cupertino, CA, USA) delivering 0.1 mg SCP per day were implanted subcutaneously in 57 female C57BL/6 mice (Charles River, Sulzfeld, Germany) under general ketamine/xylazine anesthesia (Ketavet 90 mg/kg and Rompun 8 mg/kg, i.p.) in the Department of Functional and Clinical Anatomy of the Friedrich-Alexander University Erlangen-Nürnberg (FAU), Germany. Eyes were moistened during surgery (Oleovital Augensalbe, Fresenius Kabi Austria GmbH, Austria). After implantation, the animals were kept in a hood with a continuous airflow blower and a defined humidity of 30% for ten days. The animals were then delivered to the Center for Biomedical Research, Medical University of Vienna, Austria. Mice were fed a commercial pelleted diet (ssniff K-H, Soest, Germany) and tap water was supplied, both ad libitum. The light/dark cycle was 12 hours/12 hours. All animal procedures were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the local Animal Welfare Committee and the Federal Ministry of Science, Research and Economy (GZ BMWFW-66.009/0009-WF/V/3b/2015, Az 54-2532.1-4/13).

UHR-OCT system
Corneal anatomy of the mice was assessed with a custom-built UHR-OCT system as described previously [15,16], after adapting the optics to imaging of the anterior segment of mice. As compared to the previously presented system, a superluminescent diode (SLD) with a central wavelength of 850 nm and a full width at half maximum (FWHM) bandwidth of 165 nm was used. The theoretical axial resolution given by the bandwidth of this light source is 1.38 µm in corneal tissue, while the lateral resolution given by the focusing optics is approximately 18 µm. Using a spectrum-centered Hanning window to decrease the side lobe level of the coherence function, an axial resolution of 1.69 µm was measured. The incident power of the probe beam onto the cornea was set to 2.0 mW, well below the maximum permissible exposure limits as specified by ANSI [28] and IEC 60825-1 [29].

Measurement protocol
Optical coherence tomography measurements were performed under isoflurane (2%) anesthesia applied via face mask. Animals were placed in a custom-built holder allowing for precise positioning of the animal with respect to the imaging system. In all animals, the left eye was used for investigation. A video camera directed towards the eye was used to assure correct positioning of the probe beam centrally on the cornea. Fifty-seven animals were measured on the day after arrival at the research facility (day 0, D0; two days after removal from the climate chamber), and on the following day at the same time (day 1, D1). In a subgroup of ten animals, 10 µl phosphate buffered saline (PBS, Sigma-Aldrich Handels Gmbh, Vienna, Austria) were administered onto the ocular surface of the contralateral eye after the first measurement. In this group, anterior segment imaging of the right eye was additionally performed, including time points 10 min, 60 min, 120 min, 240 min and 480 min after PBS application in an effort to assess a short-term effect of lubrication of the dry ocular surface. The right eyes of ten animals with no instillation of any substance were measured at the same time points serving as control. At each measurement session, a three-dimensional OCT data set of the central and paracentral cornea comprising 512 × 128 × 1024 voxel corresponding to 1.6 × 1.1 × 1.5 mm 3 (lateral x vertical x depth) was acquired within two minutes after induction of the anesthesia. The recording of one data set lasted about two seconds.

Data acquisition, processing and evaluation
The OCT data acquisition and visualization was done in Labview (Labview 2013, National Instruments, Austin, TX, USA), while the processing was carried out in MATLAB (MATLAB R2013b, The MathWorks Inc., Natick, MA, USA). Processed OCT images were evaluated with ImageJ (National Institutes of Health, Bethesda, MD; available in the public domain at http://rsbweb.nih.gov/ij/). Thickness measurements of the epithelium and stroma were performed at the horizontal meridian on the central slices of each volume in the center of the B-scan. For calculation of absolute thickness values, due to the lack of consistent data for the murine cornea, group refractive indices of the human stroma (1.380 anterior, 1.373 posterior) and epithelium (1.401) [30] were considered. Scans containing central corneal defects were excluded from thickness measurements (n = 9), as were measurements in which the OCT beam did not aim centrally at the cornea (n = 10). Epithelial surface maps were created by fitting the cornea to a parabola and subsequent flattening and segmentation of the outer half of the epithelium. Maximum intensity projections of the cornea were produced, and spots revealing a higher reflectivity were counted manually.
After OCT measurements on D1, one drop of 0.1% sodium fluorescein was instilled into the left eye of 39 animals and photographs were taken under illumination with cobalt blue light using a digital camera (Canon Powershot G15, Canon Austria GmbH). Corneal thickness could be measured in the OCT data of those 34 animals on D1. For the investigation of the

Research Article
Vol. 10, No. 9 / 1 September 2019 / Biomedical Optics Express 4887 correlation between findings from fluorescein staining and corneal thickness values obtained via OCT, fluorescein images were assessed by the Oxford grading schema [11], which was slightly adapted to the mouse eye, as in the mouse only the cornea and not the conjunctiva can be seen. In addition, using the green channel of the acquired photographs and subtracting the red and blue channel to filter white light reflexes, fluorescein mean intensity images were created, allowing for a quantitative evaluation of staining by grey value after manually selecting the region of interest.

Statistics
A paired t-test was used for comparison of values in the same mouse between D0 and D1. For comparison of thicknesses of the corneal layers between D0 and D1, nineteen mice were excluded from the analyses because one or both thickness measurements could not be performed. To compare the thicknesses over time after instillation of PBS versus control, a mixed model including random effects was used. A Spearman correlation was calculated for the epithelial and stromal thickness and fluorescein mean intensity after observing a non-linear correlation in the scatter plot. A Pearson correlation was calculated for all other correlations. The two-tailed significance level was set to 0.05 for all statistical tests. P-values were Bonferroni-corrected to account for multiple testing.

Results
Ultrahigh-resolution OCT revealed all major layers of the mouse cornea, namely the epithelium, stroma and endothelium (Fig. 1). In addition, the anterior lens with its capsule, epithelium and cortex as well as the iris were visualized.
After OCT measurements on D1, one drop of 0.1% sodium fluorescein was instilled into the study eye of 39 animals and photographs were taken under illumination with cobalt blue light using a digital camera (Canon Powershot G15, Canon Austria GmbH). Of these 39 animals corneal thickness could be measured in the OCT data of 34 animals on D1. For the investigation of the correlation between findings from fluorescein staining and corneal thickness values obtained via OCT, fluorescein images were assessed by the Oxford grading schema [11], which was slightly adapted to the mouse eye, as in the mouse only the cornea and not the conjunctiva can be seen. In addition, using the green channel of the acquired photographs and subtracting the red and blue channel to filter white light reflexes, fluorescein mean intensity images were created, allowing for a quantitative evaluation of staining by grey value after manually selecting the region of interest.

Statistics
A paired t-test was used for comparison of values in the same mouse between D0 and D1. For comparison of thicknesses of the corneal layers between D0 and D1, nineteen mice were excluded from the analysis because one or both thickness measurements could not be performed. To compare the thicknesses over time after instillation of PBS versus control, a mixed model including random effects was used. A Spearman correlation was calculated for the epithelial and stromal thickness and fluorescein mean intensity after observing a nonlinear correlation in the scatter plot. A Pearson correlation was calculated for all other correlations. The two-tailed significance level was set to 0.05 for all statistical tests. P-values were Bonferroni-corrected to account for multiple testing.

Results
Ultrahigh-resolution OCT revealed all major layers of the mouse cornea, namely the epithelium, stroma and endothelium (Fig. 1). In addition, the anterior lens with its capsule, epithelium and cortex as well as the iris were visualized.  In Fig. 2, exemplary photographs after fluorescein staining and their green channel images are shown in an eye without apparent lesions and an eye exhibiting increased staining.

Thickness of corneal layers
Of the 57 mice included in the study, the quality of OCT image data of 38 animals was adequate on both study days for analysis of the CCT, as well as the central epithelial and stromal thickness. Nine animals had to be excluded due to central defects and ten due to insufficient centering of the OCT recording with respect to cornea. The results of the measurements are summarized in

Thickness of corneal layers
Of the 57 mice included in the study, the quality of OCT image data of 38 animals was adequate on both study days for analysis of the CCT, as well as central epithelial and stromal thickness. Nine animals had to be excluded due to central defects and ten due to insufficient centering of the OCT recording with respect to cornea. The results of the measurements are summarized in Table 1. The mean thickness of all corneal layers increased significantly from day 0 (D0) to day 1 (D1) (p = 0.001 for epithelium and stroma, p < 0.001 for CCT; paired t-test). In 26 % of animals, a decrease in stromal thickness could be observed. While the thickness of the epithelium could not be correlated to the thickness of the stroma for the measurements performed on D0 (r = 0.280; p = 0.069; Pearson correlation), a weak correlation was found for measurements performed on D1 (r = 0.339; p = 0.018). On the first study day, no difference in epithelial thickness between inferior and superior cornea was found.

Corneal defects measured with OCT
Corneal findings were diverse and in addition to defects that presented as a locally decreased epithelial thickness, also included increases in epithelial thickness, epithelial foreign body inclusion, endothelial abnormalities and high reflectivity spots in the epithelium (Fig. 3).  While the thickness of the epithelium could not be correlated to the thickness of the stroma for the measurements performed on D0 (r = 0.280; p = 0.069; Pearson correlation), a weak correlation was found for measurements performed on D1 (r = 0.339; p = 0.018). On the first study day, no difference in epithelial thickness between inferior and superior cornea was found (p = 0.530).

Corneal defects measured with OCT
Corneal findings were diverse and in addition to defects that presented as a locally decreased epithelial thickness, also included increases in epithelial thickness, epithelial foreign body inclusion, endothelial abnormalities and high reflectivity spots in the epithelium (Fig. 3).
The occurrence of corneal defects is summarized in Table 2. In 38% of the corneas, no defect was found on either study day. The most frequently observed defects were epithelial defects (in 47% of the mice). Stromal defects were never found solitarily but always as a consequence of extensive epithelial defects. Endothelial defects were the defects most frequently found on both days.

Number of highly reflective spots and correlation to thickness measurements
Counting of highly reflective spots revealed an average number of 8.9 on D0 and 8.6 on D1. The difference between the two study days was not significant (p = 0.715, paired t-test). epithelial thickness defect (arrowheads). d: Epithelial infiltrates corresponding to high reflectivity spots in en face projections (Fig. 6). Scale bars correspond to 100 μm.
The occurrence of corneal defects is summarized in Table 2. In 38 % of the cornea, no defect was found on either study day. The most frequently observed were epithelial defects (in 47 % of the mice). Stromal defects were never found solitarily but always as a consequence of extensive epithelial defects. Endothelial defects were the defects most frequently found on both days.

Number of highly reflective spots and correlation to thickness measurements
Counting of highly reflective spots revealed an average number of 8.9 on day D0 and 8.6 on day D1. The difference between the two study days was not significant (p = 0.715, paired t-test).

Comparison of findings with fluorescein staining and OCT
Epithelial maps and fluorescein photography acquired on D1 were compared to identify features revealed by the two imaging modalities in 39 animals. In cases where no epithelial damage was visualized via OCT, fluorescein photographs showed diffuse staining in 45 % and no staining in 50 % of the cases. In five percent of the evaluated OCT-Fluorescein image pairs, fluorescein accumulated locally, but no epithelial thickness defects were found with OCT ( Fig. 4(b)). Among corneas showing an epithelial lesion in OCT on D1, each presented fluorescein markings, 67 % of which were diffuse and 33 % distinct and local ( Fig. 4(a), (c)). and epithelial thickness defect (arrowheads). d: Epithelial infiltrates corresponding to highly reflective spots in en face projections (Fig. 6). Scale bars correspond to 100 µm.

Comparison of findings with fluorescein staining and OCT
Epithelial maps and fluorescein photographs acquired on D1 were compared to identify features revealed by the two imaging modalities in 39 animals. Fluorescein photographs showed diffuse staining in 45% and no staining in 50% of the cases where no epithelial damage was visualized via OCT. In five percent of the evaluated OCT-Fluorescein image pairs, fluorescein accumulated locally, but no epithelial thickness defects were found with OCT ( Fig. 4(b)). Among corneas showing an epithelial lesion in OCT on D1, each presented fluorescein markings, 67% of which were diffuse and 33% distinct and local ( Fig. 4(a), (c)). Optical coherence tomography additionally revealed stromal defects in 6% and endothelial defects in 18% of cases on D1. Fluorescein grading using an adapted Oxford schema was highly correlated to the calculated fluorescein mean intensity (r = 0.711; p < 0.004, Pearson correlation) and showed a weak correlation to epithelial thickness on D1 (r = 0.495; p = 0.018, Spearman correlation), but no correlation to stromal thickness on D1 (p = 1.000). As for structural changes, the number of highly reflective spots on D1 did not correlate with fluorescein grading (p = 1.000, Pearson correlation).
Optical coherence tomography additionally revealed stromal defects in 6 % and endothelial defects in 18 % of cases on D1. Fluorescein grading using an adapted Oxford schema was highly correlated to the calculated fluorescein mean intensity (r = 0.711; p < 0.004, Pearson correlation) and showed a weak correlation to epithelial thickness on D1 (r = 0.495; p = 0.018, Spearman correlation), but no correlation to stromal thickness on D1 (p = 1.000). As for structural changes, the number of highly reflective spots at D1 did not correlate with fluorescein grading (p = 1.000, Pearson correlation).

Effect of topical PBS application
For the experiments investigating the effect of a single topical instillation of PBS on the thickness of the corneal layers, quantitative analysis could be performed in eight mice. In the control group were no substance was applied, thickness data could be assessed in seven mice. Two and three animals had to be excluded from the analysis due to non-central measurements, in the PBS and control groups, respectively. In corneas treated with one drop of PBS, the stromal thickness increased more than in those that received no treatment over a period of eight hours (Fig. 5). A significant interaction between time and treatment group was found (p = 0.004, mixed model). The increase in stromal thickness between measurements taken at 0 min and 480 min (p = 0.004), 10 min and 480 min (p < 0.001) and 60 min and 480 min (p = 0.012) was significantly different between the two treatment groups. Differences between 120 min and 480 min (p = 0.168) and 240 min and 480 min (p = 0.267) were not significant.

Effect of topical PBS application
For the experiments investigating the effect of a single topical instillation of PBS on the thickness of the corneal layers, quantitative analysis could be performed in eight mice. In the control group where no substance was applied, thickness data could be assessed in seven mice. Two and three animals in the PBS and control group, respectively, had to be excluded from the analysis due to non-central measurements. In corneas treated with one drop of PBS, the stromal thickness increased more than in those that received no treatment over a period of eight hours (Fig. 5). A significant interaction between time and treatment group was found (p = 0.004, mixed model).
The increase in stromal thickness between measurements taken at 0 min and 480 min (p = 0.004), 10 min and 480 min (p < 0.001) and 60 min and 480 min (p = 0.012) was significantly different between the two treatment groups. Differences between 120 min and 480 min (p = 0.168) and 240 min and 480 min (p = 0.267) were not significant. The thickness of the epithelium was dependent on time (p = 0.001), but no interaction between the observed timeline and treatment group was found (p = 0.550). The baseline values were significant determinants of subsequent epithelial and stromal thicknesses (p < 0.001 for both).
In Fig. 6, en face projections generated from the acquired UHR-OCT data sets from five different mice visualize the range and extension of epithelial defects observed in the study. The thickness of the epithelium was dependent on the time (p = 0.001), but no interaction between observed timeline and the treatment group was found (p = 0.550). The baseline values were significant determinants of subsequent epithelial and stromal thicknesses (p < 0.001 for both). In Fig. 6, en face projections generated from the acquired UHR-OCT data sets from five different mice visualize the range and extension of epithelial defects observed in the study.

Discussion
Corneal damages with a large range of severity were found in both OCT and in fluorescein photographs of mice with SCP-induced DED. While fluorescein staining could be observed very frequently, the cause is often not clear. OCT offered the possibility to investigate the layers of the cornea and evaluate the severity and extension of the defect in more detail.  The thickness of the epithelium was dependent on the time (p = 0.001), but no interaction between observed timeline and the treatment group was found (p = 0.550). The baseline values were significant determinants of subsequent epithelial and stromal thicknesses (p < 0.001 for both). In Fig. 6, en face projections generated from the acquired UHR-OCT data sets from five different mice visualize the range and extension of epithelial defects observed in the study.

Discussion
Corneal damages with a large range of severity were found in both OCT and in fluorescein photographs of mice with SCP-induced DED. While fluorescein staining could be observed very frequently, the cause is often not clear. OCT offered the possibility to investigate the layers of the cornea and evaluate the severity and extension of the defect in more detail.

Discussion
Corneal damages with a large range of severity were found in both OCT and fluorescein photographs of mice with SCP induced DED. While fluorescein staining could be observed very frequently, the cause is often not clear. Optical coherence tomography offered the possibility to investigate the layers of the cornea and evaluate the severity and extension of the defect in more detail.
Factors contributing to the appearance of the OCT image include the power and incidence angle of the probe beam onto the sample, which directly influence the magnitude of the detected back-reflected and back-scattered light. Besides saturation artifacts, these factors can easily be adapted before measurement or compensated for in post-processing. In addition, the visualization of morphological changes is based on a direct detection of reflectivity changes due to the defect.
Fluorescein mean intensity images were created from the fluorescein photographs and showed a high correlation to the adapted Oxford grading, providing an automatic and objective measure of the images. However, fluorescein intensity varies with several factors that are difficult to control, including concentration of fluorescein on the ocular surface and thickness of the fluorescein layer [12]. Furthermore, it is essential to assess the staining immediately after instillation, as the pattern can be obscured otherwise due to rapid diffusion into the tissue [11]. Fluorescein imaging is an indirect method due to the need for a contrasting substance to mark the defect. Using OCT, even if the system or measurement parameters were changed, gross tissue changes such as epithelial defects would still be detectable. It has been stated that, due to the lack of understanding of mechanisms leading to fluorescein staining, interpretation of fluorescein relies solely on assumption rather than evidence-based science [12] and only provides insights into the epithelial surface. With OCT, a structural defect of the epithelium can be easily distinguished and evaluated. Furthermore, OCT delivers information about deeper lying structures and can provide quantitative thickness measurements of the layers.
Induction of experimental dry eye with SCP is a commonly used method which is easily accessible. However, antagonization of muscarinic activity does not only cause tear deficiency, but also affects the immune system, altering the physiological response and inflammation [5]. While the causative factor in the mouse model is established, DED in human patients is often multifactorial [3,5].
Over the course of 24 hours, the thicknesses of the corneal layers increased significantly. This effect could partly be explained by the subcutaneous pump reservoir set to be sufficient for SCP delivery for 14 days after implantation and measurements in the present study were started at day 12 after implantation. When SCP is discontinued, tear production was found to return to normal after two days [9]. Further, the second factor for dry eye induction, i.e. the climate chamber was not present from this day on. Therefore, recovery of normal tear production might have already started in the mice and might be the reason for the increased thickness. Preliminary UHR-OCT measurements in healthy mice revealed a mean CCT of 94.5 ± 7.37 µm, with the epithelium accounting for 34.7 ± 0.53 µm and the stroma for 59.8 ± 7.56 µm (n = 3) (unpublished data). The corneal thicknesses found in the present study are lower than these values and the literature values found for healthy corneas in the same strain by two-photon imaging [31] and histology [32]. Corneas of mice suffering from DED induced by SCP are known to be thinner than in healthy mice [27]. There are other mechanisms, however, that may influence corneal thickness, such as stromal swelling in case of integrity loss of the epithelium or endothelium. For the stroma to swell in case of epithelial integrity loss, a sufficient amount of isotonic or hypotonic fluid has to be present on the corneal surface. This may not be the case in severe dry eye. When the missing fluid was substituted with PBS, an increase in stromal thickness was observable in our study.
Furthermore, a correlation between epithelial and stromal thickness and a correlation between epithelial thickness and fluorescein staining was observed. The number of epithelial cell layers and therefore the overall thickness can increase, for example, in response to erlotinib, a drug that can also be used to induce dry eye experimentally [7]. In some cases, defects marked by fluorescein were not visible in OCT scans of the second study day, but were revealed by OCT measurements taken on the prior study day. This could indicate that fluorescein can stain newly healed tissue, for example if remnants of dead cells and debris are still present. In these cases, marking with fluorescein might coincide with a thicker epithelium. Here, OCT may provide additional information regarding the underlying cause of fluorescein mark-up.
Distinct and strongly localized appearance of fluorescein staining may be attributed to the pooling of fluorescein in the surface irregularities and accumulation in excavated cells without glycocalyx [33]. Our results indicate that in cases of distinct fluorescein staining, OCT en face maps of the epithelium mostly display the same defects. However, fluorescein usually marked a larger area with less distinct borders, confirming the finding of a study that found lesion sizes calculated from fluorescein staining to be larger than those measured in OCT [22]. Other staining methods, like rose bengal and lissamine green may offer additional insights [11] but have not been included here and could be considered for future studies.

Conclusion
A detailed insight into corneal defects and morphological changes in a SCP induced mouse model of dry eye was gained using a combination of the two imaging modalities, fluorescein staining and OCT. While fluorescein staining provided visualization of subtle changes on the ocular surface, OCT data offered both information about epithelial changes and alterations in deeper corneal layers. The complementary application of both methods might serve as a valuable tool for assessment of DED related changes in the cornea in preclinical and clinical settings.