Differences in vulnerability to desiccation stress between corneal and conjunctival epithelium in rabbit models of short-term ocular surface exposure

Rabbit model of short-term exposure keratopathy

New Zealand white rabbits (weight, 2.0‒2.2 kg) were used in this study. The study was conducted in accordance with ARRIVE guidelines. All procedures adhered to the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmology and vision research (ARVO Animal Policy). Approval for this study was obtained from the Korea University Guro Hospital Institutional Review Board, Seoul, South Korea (KOREA-2021–0122).

All in vivo experimental procedures (including ocular surface staining, tear sample collection, and impression cytology) were performed under general anesthesia induced by intramuscular injection of ketamine hydrochloride (35 mg/ kg) and xylazine hydrochloride (5 mg/kg)50. The rabbits’ right eyes were used for all experiments, and the left eyes were untreated and used as controls. The interpalpebral fissures were held wide open for 30 minutes to ensure exposure of the central cornea, limbus, and perilimbial conjunctiva, with manual blinking performed once per minute. The eyes were evaluated at each instant: 3 min, 10 min, 20 min and 30 min.

Ocular surface staining and conjunctival hyperemia

The cornea and conjunctiva of rabbits were examined under a portable slit lamp microscope with cobalt blue filter, under general anesthesia, after instillation of 1 drop of 1% fluorescein solution. A yellow filter was used to assess conjunctiva staining in detail. For corneal fluorescein staining (CFS) scores, strips of paper impregnated with sodium fluorescein (Haag-Streit, Bern, Switzerland) were applied to the superior bulbar surface after retracting the upper eyelid and after wetting the eyelid. end of the strip with 5 μL of saline solution.

Ocular staining score was assessed by a single experienced ophthalmologist according to the standard National Eye Institute (NEI) scoring system. Briefly, the cornea was divided into five zones (central, superior, nasal, inferior and temporal); punctate fluorescein staining in each area was scored on a scale of 0 to 3, and the total score (0 to 15) is the sum of the scores for the five areas. The conjunctiva was divided into six areas and the total score (0-18) is the sum of the scores for the six areas4. Conjunctival hyperemia was graded using the Efron scale for conjunctival redness, which consists of five grades (0–4)51.

Collection of tear samples and enzyme immunoassay

Tear samples were collected from rabbit eyes and after 3, 10, 20 and 30 min exposure of the conjunctiva and cornea. Sixty microliters of normal saline solution was gently applied to the inferior fornix using a micropipette, twice. Subsequently, a total volume of 120 μl of a tear sample was gently collected52. Tear samples were used to measure MUC5AC levels, to study mucin secretion from conjunctival goblet cells in tears. The level of MUC5AC in the tear sample was assessed using an enzyme immunoassay kit for rabbit MUC5AC (MyBiosource, San Diego, CA, USA). All measurements were performed according to the manufacturer’s protocol, using a microplate spectrophotometer (Spectramax Plus® 384; Molecular Devices, Sunnyvale, CA, USA).

Print cytology and Western blot analysis

After the last tear sample was collected after 30 minutes of eye exposure, corneal and conjunctival impression cytology was performed. After instillation of 0.5% proparacaine hydrochloride eye drops (Alcaine®, Bausch & Lomb, Rochester, NY, USA). An 8 mm diameter nitrocellulose membrane (Millipore, Bedford, MA, USA) was applied to the cornea and conjunctiva. The membrane was then gently peeled off with smooth forceps. The membrane was immediately immersed in a well which was filled with fixation solution. Hematoxylin and eosin (H&E) and periodic acid Schiff (PAS) staining were used for histological staining of the membrane in the specimen53.

Tissue cell extracts from corneal impression cytology were subjected to Western blot analysis to measure AQP5 protein levels. Those from conjunctival impression cytology were subjected to Western blot analysis to measure protein levels of AQP5, MUC5AC and CFTR. Primary antibodies against AQP5 (1:1000; ab92320, Abcam, Cambridge, UK), MUC5AC (1:1000; ab198294, Abcam, Cambridge, UK), CFTR (1:1000; MAB3482, Sigma-Aldrich, Missouri , USA), and β-actin (1:10,000, #5125; Cell Signaling Technology, Danvers, MA, USA) were used. Impression cytology from the left eyes, which was unprocessed, was used as a control. The original images as recorded during the experiment are shown in Figure S1-S4 as Supplementary Files. We only stored images of protein bands of interest and images of full-length blots were not shown.

Scanning electron microscopy

After a 30 min exposure, for scanning electron microscopy (SEM) and immunohistochemistry, the rabbits were euthanized using a CO2 chamber under general anesthesia. Corneal and conjunctival tissues were gently excised after sacrifice and prefixed in 2% glutaraldehyde in 0.1 M phosphate buffer. Specimens were then post-fixed for 2 h in 1 % dissolved in phosphate buffered saline for SEM. Then, corneal and conjunctival tissues were treated in a graded series of ethanol and t-butyl alcohol, dried in a freeze-dryer (ES-2030; Hitachi, Tokyo, Japan), and coated with platinum using an ion coating (IB-5; Eiko, Ibaraki, Japan). The appearance of the corneal and conjunctival epithelial surface was observed by field emission-SEM (S-4700; Hitachi)54. We defined percent cell loss as the ratio of cell loss, which shows epithelial cell desquamation or morphological change relative to total epithelial cells in each of four representative digital SEM images to quantify cell loss. epithelial. First, the total area of ​​each representative image was calculated using a “defined scale” based on the known distance from the SEM using ImageJ (http://imagej.nih.gov /ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). Second, the area of ​​each corneal and conjunctival epithelial cell was calculated from the “polygon selection tool”. Third, the total number of epithelial cells in each representative image was calculated as the total area of ​​each representative image divided by the area of ​​each epithelial cell. Fourth, the number of cell losses, which shows desquamation of epithelial cells or morphological change, was calculated using a “multipoint tool” (Fig. 4C,F). Finally, percent cell loss was calculated as the ratio of cell loss to total epithelial cells in representative images.

Immunohistochemistry

The anterior segment of each eyeball was surgically removed and fixed in 10% neutral buffered formalin and then embedded in paraffin. Paraffin-embedded tissues were cut into 4 μm sections with a microtome (Leica RM 2255; Leica, Bannockburn, IL, USA) and the tissue sections were placed on microscope slides. After deparaffinization of the tissue sections with xylene, the tissue sections were immersed in a graded series of ethanol and phosphate buffered solution. Serial sections were used for immunohistochemistry of CD31 (as a vascular endothelial marker) and LYVE-1 (as a lymphatic endothelial marker). Primary antibodies were obtained commercially for CD31 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA) and LYVE-1 (1:100; Abcam Inc, Cambridge, Massachusetts). A rabbit-specific HRP/DAB (ABC) detection IHC kit (ab64261; Abcam) was used for antibody-based secondary detection according to the manufacturer’s instructions. Tissue sections were observed under an optical microscope at 400× magnification and digital images were taken with an Olympus BX51 microscope and DP72 camera (Olympus Optical Co., Ltd., Tokyo, Japan). CD31 positive cells were determined by analyzing four visual fields on a conjunctival biopsy and the results are reported in cells per square millimeter. Lymphatic space surrounded by LYVE-1 positive cells (%) was defined as the percentage of total area surrounded by LYVE-1 positive cells per total observed area. The average number of CD31 positive cells and the lymphatic space surrounded by LYVE-1 positive cells (%) were compared between the exposure model eye and the control eye.

statistical analyzes

Statistical analyzes were performed using the Mann‒Whitney U test and the Wilcoxon signed rank test in SPSS version 20.0 (IBM SPSS, Inc., Chicago, IL, USA). Values ​​are expressed as median and interquartile range. P

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