Apical Corneal Barrier Disruption in Experimental Murine Dry Eye Is Abrogated by Methylprednisolone and Doxycycline

Abstract

purpose. To evaluate the mechanism of apical corneal epithelial barrier disruption in response to experimental ocular surface desiccation and the effects of two anti-inflammatory agents (methylprednisolone and doxycycline) on this process.

methods. Experimental dry eye (EDE) was created in C57BL/6 mice, without or with topical therapy, 1% methylprednisolone, 0.025% doxycycline, or physiologic saline solution (PSS) four times per day. Corneal smoothness and Oregon green dextran (OGD) permeability were assessed. Desquamation of and cornified envelope protein (involucrin and small proline-rich protein [SPRR]-2) expression by the corneal epithelium was evaluated by laser scanning confocal microscopy. Levels of cornified envelope proteins mRNA were measured by real-time PCR.

results. Corneal OGD permeability, surface irregularity, and the number of desquamating apical corneal epithelia significantly increased in EDE. Desiccating stress significantly increased expression of involucrin and SPRR-2 in the corneal epithelia. Treatment of EDE with methylprednisolone or doxycycline reduced corneal permeability to OGD, improved corneal smoothness, and decreased involucrin and SPRR-2 immunoreactivity compared with EDE+PSS.

conclusions. Disruption of apical corneal epithelial barrier function in dry eye is accompanied by increased apical desquamation and increased expression of cornified envelope proteins. Topical treatment of EDE with the anti-inflammatory agents methylprednisolone or doxycycline preserves apical corneal barrier function.

The ocular surface is the most environmentally exposed mucosal surface of the body. The corneal epithelial barrier function, in conjunction with the tear film, plays an essential role in preventing pathogens, allergens and irritants from entering the eye. The unstable tear film and the altered corneal permeability in keratitis sicca, the ocular surface disease of dry eye, leave the cornea vulnerable to sterile ulceration ¹ and microbial infection. ² Next to contact lens wear, dry eye is the second highest risk factor for microbial keratitis. ³ Corneal ulceration resulting from keratoconjunctivitis sicca (KCS) may lead to reduced vision, blindness, and even loss of the eye. ^{4 5}  

An altered corneal epithelial barrier is identified clinically by staining and fluorometrically by increased permeability to sodium fluorescein dye. ^{6 7 8} Corneal epithelial permeability to fluorescein dye in patients with untreated dry eye was reported to be 2.7- to 3 times greater than in eyes with normal tear function. ^{9 10} This increased permeability leads to a poorly lubricated and irregular corneal surface that causes visual morbidity (blurred and fluctuating vision) and decreases contrast sensitivity. ^{11 12 13}  

Inflammation plays a critical role in the development of dry eye. ¹⁴ Significant clinical improvement in corneal epithelial disease has been reported with anti-inflammatory therapies such as topical corticosteroids and cyclosporine. ^{13 15}  

The purpose of this study was to investigate the effects of desiccating stress on corneal epithelial smoothness and apical corneal barrier function and the effects of two anti-inflammatory agents, the corticosteroid methylprednisolone and the tetracycline doxycycline on this process. 

Methods

Mouse Model of Dry Eye

This research protocol was approved by the Baylor College of Medicine Center for Comparative Medicine, and it conformed to the standards in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Experimental dry eye (EDE) was induced in C57BL/6 mice, aged 6 to 8 weeks, by subcutaneous injection of 0.5 mg/0.2 mL scopolamine hydrobromide (Sigma-Aldrich, St. Louis, MO) in alternating hindquarters administered four times a day (8 AM, 11 AM, 2 PM, and 5 PM) and by exposure to an air draft and <40% ambient humidity for 18 hours per day, as previously reported. ¹⁶  

Five experimental groups were evaluated: normal untreated control subjects (UT, no dry eye treatment and no topical treatment); EDE control (received no eye drops); EDE+physiologic saline solution (EDE+PSS) (BSS; Alcon, Forth Worth, TX), received 1 μL/eye bilaterally of PSS four times a day; EDE+steroid (ST), received 1 μL/eye bilaterally of 1% methylprednisolone preservative free (Leiter’s, San Jose, CA), four times a day; and EDE+doxycycline (DOXY), received 1 μL/eye bilaterally of 0.025% doxycycline preservative free (Leiter’s) four times a day. 

Immunofluorescent Staining and Laser Scanning Confocal Microscopy

Occludin, involucrin, and small proline-rich protein 2 (SPRR-2) were evaluated by laser scanning confocal microscopy in wholemounted corneas and in tissue sections. 

The eyes and adnexa of mice from each group were excised, embedded in optimal cutting temperature (OCT compound; VWR, Suwanee, GA), and flash frozen in liquid nitrogen. Sagittal 8-μm sections were cut with a cryostat (HM 500; Micron, Waldorf, Germany) and placed on glass slides that were stored at −80°C. Tissue sections were used for immunofluorescent staining, as previously described. ^{17 18} Whole, freshly harvested murine corneas from UT and EDE control subjects and the three treatment groups (two corneas/group per experiment, in three different sets of experiments, for each protein evaluated) were used for laser scanning confocal microscopy. 

Tissue sections and whole corneas were processed by the same protocol. They were fixed with either cold methanol 4°C for 10 minutes (for occludin) or acetone at −20°C for 5 minutes (for involucrin and SPRR-2). After fixation, they were permeabilized with PBS containing 0.1% Triton-X for 10 minutes. After blocking with 20% normal goat serum in PBS for 45 to 60 minutes (occludin and SPRR-2) or 20% normal horse serum (involucrin), primary polyclonal rabbit antibody against occludin (1:50 dilution, 5 μg/mL, Zymed, San Francisco, CA), polyclonal rabbit serum against SPRR-2 (1:100 dilution of neat serum; Alexis Biochemicals, San Diego, CA) or polyclonal goat anti-involucrin (1:20 dilution, 2 μg/mL; Santa Cruz Biotechnology, Santa Cruz, CA) were applied and incubated for 1 hour at RT. Secondary antibodies, Alexa-Fluor 488–conjugated goat anti-rabbit IgG or Alexa-Fluor 488–conjugated donkey anti-goat IgG (1:300) were then applied and incubated in a dark chamber for 1 hour, followed by counterstaining with propidium iodide (PI; 2 μg/mL in PBS) for 30 minutes. Whole corneas were flattened on microscope slides; covered with antifade mounting medium (Gel/Mount; Fisher, Atlanta, GA), and coverslips were applied. Cryosections and wholemounted digital images (512 × 512 pixels) were captured with a laser-scanning confocal microscope (LSM 510; Zeiss with krypton-argon and He-Ne laser; Carl Zeiss Meditec, Ltd. Thornwood, NY) with 488-excitation and 543-nm emission filters, LP505 and LP560, respectively. They were acquired with a 40/1.3× oil-immersion objective. Images from treatment and control corneas were captured with identical photomultiplier tube gain settings and processed (LSM-PC software; Carl Zeiss Meditec, Inc.; and Photoshop ver. 6.0; Adobe Inc., Mountain View, CA). The number of desquamating superficial epithelial cells was counted in five different 40× microscopic fields in each wholemounted cornea, stained for occludin in the UT and EDE controls and the three treatment groups in three different sets of experiments, and the results were averaged. 

Corneal Permeability

Corneal epithelial permeability to Oregon green dextran (OGD; 70,000 molecular weight [MW]; Invitrogen, Eugene, OR) was assessed in the UT and EDE controls and the three treatment groups (10 eyes/group/experiment, in three different sets of experiments). In our preliminary experiments (data not shown), corneal permeability to two different fluorescent molecules, OGD, and 0.3% carboxyfluorescein (376 MW; Sigma-Aldrich) was evaluated. OGD was chosen because it gave the same staining pattern as carboxyfluorescein, with a much lower green fluorescent background. Because of its larger size, we found OGD to be more discriminating than carboxyfluorescein. To verify any possible damage of the corneal epithelium by dextran, hematoxylin-eosin–stained corneas of UT mice were evaluated; no damage was observed (data not shown). 

Briefly, 0.5 μL of 50 μg/mL OGD was instilled onto the ocular surface 1 minute before euthanasia. Corneas were rinsed with 2 mL of PBS and photographed with a stereoscopic zoom microscope (model SMZ 1500; Nikon, Melville, NY), under fluorescence excitation at 470 nm. Images were obtained 2 hours after instillation of the last treatment drop and were processed (Metavue 6.24r software; Universal Imaging Corp., West Chester, PA). The severity of corneal OGD staining was graded in digital images by two masked observers, who used the Baylor grading scheme for corneal fluorescent staining, ¹⁹ . Briefly, the number of dots of fluorescein staining was graded in the 1 mm central cornea zone of each eye, on a standardized 5-point scale (0 dots, 0; 1–5 dots, 1; 6–15 dots, 2; 16–30 dots, 3; and >30 dots, 4). One point was added to the score if there was one area of confluent staining and 2 points were added for two or more areas of confluence. 

Oregon Green Dextran Uptake

OGD uptake in the corneal epithelium was evaluated using a technique reported by Ubels et al., ²⁰ with modification. Briefly, 1.0 μL of 50 μg/mL OGD was instilled bilaterally onto the ocular surface 15 minutes before euthanasia. Freshly harvested whole murine corneas from each experimental group (two corneas/group per experiment, in two different sets of experiments) were fixed with cold methanol 4°C for 10 minutes. After fixation, they were permeabilized with PBS containing 0.1% Triton-X for 10 minutes, followed by counterstaining with PI (2 μg/mL in PBS) for 30 minutes. Whole corneas were flattened on microscope slides and covered with antifade medium (Gel/Mount; Fisher), and coverslips were applied. Wholemount digital images (512 × 512 pixels) were captured with a laser-scanning confocal microscope (LSM 510 with krypton-argon and He-Ne laser; Carl Zeiss Meditec) with 488-nm excitation and 543-nm emission filters (LP505 and LP560, respectively; Carl Zeiss Meditec). The images were acquired with a 63/1.4× oil-immersion objective and a zoom setting of 0.7. Images from the treatment and the control groups were captured with identical photomultiplier tube gain settings and processed (LSM-PC; Carl Zeiss Meditec, Inc.; the software and Photoshop ver. 6.0; Adobe Inc., San Jose), using the Z-stack option. This technique has the advantage of allowing multiple scans from the surface to the basal layer of the epithelium. The software can then combine these pictures into a three-dimensional (3-D) configuration, generating a cross section that is perpendicular to the apical plane. By this method, penetration of OGD into the apical–subapical–basal layer can be evaluated. 

Evaluation of Corneal Smoothness

Corneal smoothness was assessed in 10 eyes of the UT and EDE controls and in 10 eyes of mice in each of the three treatment groups, in three different sets of experiments. Reflected images of a white ring from the fiber-optic ring illuminator of the stereoscopic zoom microscope (SMZ 1500; Nikon) were taken immediately after euthanasia, 2 hours after instillation of the last treatment drop. This ring light is firmly attached and surrounds the bottom of the microscope objective. Because the illumination path is nearly coincident with the optical axis of the microscope, the viewing area is evenly illuminated and nearly shadowless. The projected ring light will reflect off a wet surface and the regularity of the reflected ring depends on the surface’s smoothness. The smoothness of the reflected rings was graded in digital images by two masked observers. The projected ring was divided into four quarters, of 3 clock hours each. The corneal irregularity severity score was calculated using a 5-point scale based on the number of distorted quarters in the reflected ring: 0, no distortion; 1, distortion in one quarter of the ring (3 clock hours); 2, distortion in two quarters (6 clock hours); 3, distortion in three quarters (9 clock hours); 4, distortion in all four quarters (12 clock hours); and 5, severe distortion, in which no ring could be recognized. 

To determine whether this method was valid to evaluate corneal smoothness, two untreated mice were euthanatized, and sequential images were taken immediately after and at 10 and 15 minutes after death. During this time course, the eyes remained open, and the corneas desiccated. Ring smoothness in these control images was evaluated by the two masked observers. 

RNA Isolation and Real-Time PCR

Total RNA from the corneal epithelia collected and pooled from each of the five experimental groups (10 eyes/group/experiment, in three different sets of experiments) was extracted using an acid guanidium thiocyanate-phenol-chloroform method. ²¹ The RNA concentration was measured by its absorption at 260 nm and stored at −80°C before use. 

First-strand cDNA was synthesized from 1 μg of total RNA using random hexamers and M-MuLV reverse transcriptase (Ready-To-Go You-Prime First-Strand Beads; GE Healthcare, Inc., Piscataway, NJ), as previously described. ^{22 23} Real-time PCR was performed using gene expression assay primers and MGB probes specific for GAPDH, involucrin, transglutaminase 1, and SPRR2-a (assay IDs: MM9999915-g1, Mm00515219-s1, Mm00498375-m1, Mm 0084512-m1, respectively; and a Taqman Universal PCR Master Mix AmpErase UNG; Applied Biosystems [ABI], Foster City, CA), in a thermal cycler (Smart Cycler; Cepheid, Sunnyvale, CA), according to the manufacturer’s protocol. Assays were performed in duplicate and repeated three times using different samples from different experiments. A nontemplate control was included in all the experiments to evaluate DNA contamination of the reagents used. The GAPDH gene was used as an endogenous reference for each reaction to correct for differences in the amount of total RNA added. The results of quantitative PCR were analyzed by the comparative C_t method (User Bulletin, No. 2, P/N 4303859; ABI). ²³ The cycle threshold (C_t) was determined using the primary (fluorescent) signal as the cycle at which the signal crossed a user-defined threshold. The results were normalized by the C_t of GAPDH and the relative mRNA level in the untreated group was used as the calibrator. 

Statistical Analysis

The Mann-Whitney test was used to compare the controls (UT versus EDE) or the treatment groups (EDE+PSS versus EDE+ST or EDE+DOXY). P ≤ 0.05 was considered statistically significant. Analyses were performed on computer (Prism 3.0 software; GraphPad Software, San Diego, CA). 

Results

EDE and Corneal Permeability

A hallmark of human KCS is increased corneal epithelial permeability to the diagnostic dye sodium fluorescein. Corneal epithelial permeability to high molecular weight fluorescent OGD (70,000) was assessed in the UT and EDE controls and the three treatment groups (Fig. 1 , left column). Compared with the UT group, corneal uptake of OGD was significantly increased in EDE, with punctate and confluent dye staining, mimicking human KCS (OGD staining score [mean ± SD] 0.95 ± 0.95 vs. 4.18 ± 1.89; P < 0.0001). Treatment with methylprednisolone and doxycycline had a protective effect. The OGD staining score in eyes treated with these agents was significantly less than with EDE+PSS (OGD score 2.11 ± 1.33 and 1.67 ± 1.23; P > 0.05 vs. 4.72 ± 2.02, P = 0.0005 and P = 0.0002, respectively; Table 1 ). 

OGD uptake was evaluated by laser scanning confocal microscopy of wholemounted corneas. The dye was added to the ocular surface (1 μL/eye, bilaterally) 15 minutes before euthanasia, to allow the dye to diffuse passively through the cornea. If an altered corneal barrier is present, the dye is expected to penetrate the apical and subepithelial layers. Z-stack images of 21.45 ± 3.376-μm thickness of all experimental groups were obtained. Figure 1(central and right columns) shows series of composite images from two different eyes in each experimental group. In each group, the surface of the corneal epithelium is shown in the lower image and the generated cross sections (CS) in the top image. The white line in the bottom image demonstrates where the software (LMS 510; Nikon) generated the cross sections, perpendicular to the apical layer. Compared with the UT control, both the EDE+PSS and EDE groups showed similar increased uptake of OGD by some apical epithelial cells (arrows) and diffusion of the dye into the subepithelial layers (increased background of green particulates). OGD uptake in UT, EDE+ST, and EDE+DOXY groups was rarely noted. 

Effect of EDE on Corneal Smoothness

The regularity of a white-light ring reflecting off the mouse corneas was used to evaluate their smoothness. To evaluate the effects of desiccation on corneal smoothness, we evaluated serial reflections in normal mice immediately after euthanasia and 10 minutes and 15 minutes post mortem (Fig. 2A) . A progressive increase in corneal irregularity was observed as the corneas desiccated. Figure 2Bshows that the EDE group had greater corneal irregularity than the UT control (mean severity score, 2.6 ± 0.91 vs. 1.08 ± 0.93, respectively; P < 0.0001), which had regular reflected rings in most corneas. Eyes treated with methylprednisolone and doxycycline had more regular corneal surfaces than did those in the EDE+PSS group (1.60 ± 1.07 and 1.70 ± 1.20 vs. 2.54 ± 1.44; P = 0.0241 and P = 0.0438, respectively; Table 1 ). 

Effect of EDE on Expression of Cornified Envelope Proteins and Apical Desquamation

In an attempt to determine the cause of the altered apical corneal epithelial barrier corneal function and surface irregularity in EDE, we evaluated the expression of the occludin and the cornified envelope proteins involucrin and SPRR-2 in corneal epithelium by laser scanning confocal immunomicroscopy in tissue sections and wholemounted corneas. 

In tissue sections (Fig. 3 , left) linear occludin staining was observed in the apical layers of the corneal epithelium. Weak staining for involucrin and patchy SPRR-2 staining was noted in the apical epithelium of UT corneas (Figs. 4 and 5 , left columns). After 5 days of EDE, increased cytoplasmic staining was observed in sections stained for occludin, and there was a marked increase in involucrin and SPRR-2 staining in the apical and subapical layers. 

The number of desquamating cells was evaluated by laser scanning confocal microscopy of wholemounted corneas stained for occludin. Detached apical epithelial cells (Fig. 3 , arrows) were occasionally observed in the central area of UT corneas, and there was a significant increase in the number of detached cells after 5 days of EDE (2.18 ± 1.33 vs. 8.54 ± 3.27 cells, P = 0.0001). Numerous “holes” were noted where cells had detached from the apical corneal epithelium (Fig. 3 , asterisks). The EDE groups that were treated with methylprednisolone and doxycycline had fewer detached apical epithelial cells than did the EDE+PSS group (1.72 ± 1.35 and 2.00 ± 1.73 vs. 8.09 ± 2.74 cells; P < 0.0001 and P = 0.0001 for each comparison). 

Minimal cytoplasmic staining for the cornified envelope proteins, involucrin (Fig. 4 , middle and right columns) and SPRR-2 (Fig. 5 ; middle and right columns) were found in confocal images of the apical corneal epithelium of UT eyes. A marked increase in staining for both of these proteins was observed in response to EDE. Strong SPRR-2 staining surrounding many apical cells was noted in the EDE group. Staining for these proteins in the EDE+ST and EDE+DOXY resembled UT corneas, and it was markedly very different from the EDE+PSS group, which showed mixed areas of intense and weak staining (similar to EDE group). 

EDE Induced Increased Expression of Involucrin, SPRR-2a, and Transglutaminase Transcripts

The levels of RNA transcripts encoding the envelope proteins (involucrin and SPRR-2a), the enzyme transglutaminase 1 (which is responsible for cross-linking involucrin and SPRR-2a), and the housekeeping gene GAPDH were evaluated by real-time PCR with pooled total RNA samples from corneal epithelia obtained from the UT and EDE controls and the three treatment groups (10 eyes/group per experiment, in three different sets of experiments). The nontemplate control showed absence of DNA contamination. Compared with the UT group, increased levels of SPRR-2a and transglutaminase 1 transcripts (both P < 0.05) were noted in the EDE group. Treatment with methylprednisolone decreased expression of SPRR2-a transcripts, compared with that in the EDE+PSS group (P < 0.05; Fig. 6 ). 

Discussion

This study provides insight into the cause of disruption of the apical corneal barrier and surface irregularity in an experimental murine model of dry eye that develops corneal epithelial disease that is similar to human keratitis sicca. We observed increased staining of certain apical epithelial cells as well as increased diffusion of the fluorescent dye OGD into deeper layers of the corneal epithelium in this model (although we could not demonstrate its penetration into the stroma), findings that indicate apical disruption of the corneal epithelial permeability barrier. This was accompanied by loss of cell–cell adhesion with microscopic observation of increased apical cell desquamation and numerous desquamation holes. It is likely that the punctate epithelial erosions and increase fluorescein uptake that are seen in human keratitis sicca are due to these same epithelial changes. We also observed increased expression of skinlike cornified envelope proteins (involucrin, SPRR-2). These proteins may be an attempt to reestablish an alternate apical barrier. Treatment of EDE with topical steroid or doxycycline attenuated these pathologic changes. 

The mechanism(s) by which these two anti-inflammatory agents preserve apical corneal barrier function in response to experimental desiccation remains to be fully determined, although our previously reported studies provide clues. We have found that this experimental model of dry eye activates mitogen-activated protein kinases (MAPK) signaling pathways in the ocular surface epithelia, increases production of the proinflammatory cytokines IL-1 and TNF-α, and stimulates production and activation of matrix metalloproteinase (MMP)-9. ²³  

We have previously reported that MMP-9 plays a key role in acute disruption of apical corneal epithelial barrier function in dry eye. MMP-9 knockout mice showed significantly less alteration of apical corneal epithelial barrier function in response to EDE than did wild-type mice. This protective effect was abrogated by topical application of MMP-9 to the ocular surface. ¹⁶ A significant increase in the concentration and activity of MMP-9 in the tear film has been reported in human patients with KCS, ^{24 25 26} as well as in the corneal epithelium and tear fluid of mice with EDE. ¹⁶ Both hyperosmolar stress and the inflammatory cytokines that are elevated in keratitis sicca (i.e., IL-1, TNF-α, and TGF-β) have been found to increase expression of MMP-9 by the corneal epithelium (Li D-Q, et al., IOVS 2002;43:ARVO E-Abstract 1981). 

Activation of MAPK intracellular signaling pathways by the desiccating and hyperosmolar stress of dry eye is another possible cause of the observed pathologic corneal epithelium. Certain MAPKs such as ERK have been reported to modulate barrier function, increasing transepithelial electrical resistance in human cultured epithelial cells. ²⁷ Other MAPKs, such as JNK, have been reported to stimulate production of cornified envelope proteins, such as involucrin in the corneal epithelium. ²⁸ These kinase signaling pathways also regulate the expression of cytokines and MMPs (MMP-9, -1, -3, and -13) (Li D-Q, et al., IOVS 2002;43:ARVO E-Abstract 1981). ^{29 30 31 32 33}  

Corticosteroids and tetracyclines have the capacity to inhibit directly or indirectly one or more of these pathways or mediators of corneal epithelial disease in dry eye. Corticosteroids are global inhibitors of inflammation and have been reported to decrease the production of a number of inflammatory cytokines (IL-1, IL-6, IL-8, TNF-α, and GM-CSF) and MMP-9 production by the corneal epithelium (Djalilian A, et al., IOVS 2001;42:ARVO Abstract 3086). They may secondarily inhibit MAPKs by decreasing production of IL-1 and TNF-α, which activates MAPKs. They also induce MAPK phosphatase 1, which dephosphorylates and inactivates all members of the MAPK family of proteins. ^{34 35 36} Tetracyclines are a potent inhibitor of MMPs and they have recently been shown to inhibit p38 and JNK MAPK pathways. ^{37 38} In our study, topical treatment with 1% methylprednisolone and 0.025% doxycycline significantly preserved apical corneal barrier function and corneal smoothness. It also inhibited the production of the cornified envelope proteins involucrin and SPRR-2 in response to EDE. This is an important finding, because increased apical desquamation and increased production of these cornified proteins in focal areas may promote corneal surface irregularity, which is a key sight-threatening feature of human KCS. ^{16 39} Clinical improvement in corneal epithelial disease in dry eye has been reported with topical corticosteroids, and these findings in our murine EDE model shed new light on their mechanism of action in this condition. ^{15 40}  

 Presented at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2005.

 Supported by National Eye Institute Grants EY11915 (SCP), EY014553 (D-QL), EY016928-01, and EY07001-29 (CSDP); an unrestricted grant from Research to Prevent Blindness; the Oshman Foundation; The William Stamps Farish Fund; and an unrestricted grant from Allergan, Inc.

 Submitted for publication September 27, 2005; revised January 4 and March 2, 2006; accepted May 17, 2006.

 Disclosure: C.S. De Paiva, Allergan, Inc. (F); R.M. Corrales, Allergan, Inc. (F); A.L. Villarreal, Allergan (F); W. Farley, Allergan (F); D.-Q. Li, Allergan, Inc. (F); M.E. Stern, Allergan, Inc. (E, F); S.C. Pflugfelder, Allergan, Inc. (F)

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Corneal permeability was measured in two ways: as the severity of clinically graded staining with OGD (left) and by laser scanning confocal microscopy of wholemounted corneas (middle, right) stained with OGD (green) and the nuclear counterstain PI (red) in UT and EDE controls and the three treatment groups (PSS, ST, DOXY). (Middle, right) Series of composite images from two different eyes for each experimental group. For each group, the surface of the corneal epithelium is shown in the bottom image and the generated cross-sections (CS) in the top image. White line in the bottom image demonstrates where the software generated the cross sections, perpendicular to the apical layer. Arrows: areas of increased OGD dye uptake.

Table 1.

 

Corneal Staining and Smoothness Scores and Number of Desquamating Cells in the Study Groups

	UT	EDE	PSS	ST	DOXY
OGD staining score	0.95 ± 0.95	4.18 ± 1.89	4.72 ± 2.02	2.11 ± 1.33	1.67 ± 1.23
		P < 0.0001		P = 0.0005	P = 0.0002
Corneal smoothness score	1.08 ± 0.93	2.60 ± 0.91	2.54 ± 1.44	1.60 ± 1.07	1.70 ± 1.20
		P < 0.0001		P = 0.0241	P = 0.0438
Desquamating cells (n)	2.18 ± 1.33	8.54 ± 3.27	8.09 ± 2.74	1.72 ± 1.35	2.0 ± 1.73
		P = 0.0001		P < 0.0001	P = 0.0001

 Data are shown as mean ± SD. Probabilities compare UT versus EDE control and EDE + PSS versus EDE + ST or EDE + DOXY. OGD, Oregon green dextran.

Figure 2.

 

(A) Corneal smoothness evaluated by reflection of white ring light in two different untreated animals (UT1 and UT2), 30 seconds and 10 and 15 minutes after euthanasia. The animals had their eyes opened and the corneas were allowed to desiccate.(B) Corneal smoothness evaluated by reflection of light ring in three different mice (subjects 1, 2, 3) in the five experimental groups 30 seconds after euthanasia. Scale bar, 500 μm.

Figure 3.

 

Immunofluorescent staining in tissue sections (left, merged image) and laser scanning confocal microscopy of wholemounted corneas (middle, right) stained for occludin (green) with PI (red) nucleus counterstaining, in UT and EDE controls and the three treatment groups. Arrows: desquamating apical epithelial cells; asterisks: holes resulting from the detached cells.

Figure 4.

 

Immunofluorescent staining in tissue sections (left, merged image) and laser scanning confocal microscopy of wholemounted corneas (middle, right) stained for involucrin (green) with PI (red) nucleus counterstaining, in UT and EDE controls and the three treatment groups.

Figure 5.

 

Immunofluorescent staining in tissue sections (left, merged image) and laser scanning confocal microscopy of wholemounted corneas (middle, right) stained for SPRR-2 (green) with PI (red) nucleus counterstaining in UT and EDE controls and the three treatment groups.

Figure 6.

 

Real-time PCR showing mRNA levels of expression for involucrin, SPRR-2a, and transglutaminase 1 in cornea epithelia of the UT and EDE controls and the three treatment groups. *P ≤ 0.05.