Abstract
Purpose:
The present study was designed to investigate the role of ocular surface glycocalyx and mucins in graft versus host disease (GVHD)-associated dry eye. The ameliorative effect of topical rebamipide, a mucin secretagogue, on GVHD-associated dry eye was also tested.
Methods:
A mouse model of allogeneic transplantation was used to induce ocular GVHD with C57BL/6 as donors and B6D2F1 as recipient mice. Phenol red thread method and fluorescein staining was used to quantify tear secretion and corneal keratopathy. At 8 weeks after the allogeneic transplantation, corneas were harvested to perform glycocalyx staining and confocal microscopy. Goblet cell staining was performed using periodic acid Schiff's staining. Corneal and tear film levels of Mucin 1, 4, 16, 19, and 5AC were quantified using ELISA and real-time PCR. Rebamipide was applied topically twice daily to mice eyes.
Results:
Allogeneic transplantation resulted in ocular GVHD-associated dry eye characterized by a significant decrease in tear film volume and the onset of corneal keratopathy. Ocular GVHD caused a significant decrease in the area and thickness of corneal glycocalyx. A significant decrease in the goblet cells was also noted. A significant decrease in mucin 4 and 5AC levels was also observed. Topical treatment with rebamipide partially attenuated ocular GVHD-mediated decrease in tear film volume and significantly reduced the severity of corneal keratopathy.
Conclusions:
Ocular GVHD has detrimental impact on ocular surface glycocalyx and mucins. Rebamipide, a mucin secretagogue, partially prevents ocular GVHD-associated decrease in tear film and reduces the severity of corneal keratopathy.
Allogeneic hematopoietic stem cell transplantation is a successful treatment option for hematologic malignancies. However, graft versus host disease (GVHD) is a serious complication of hematopoietic stem cell transplantation and its incidence remains high in spite of the advances in human leukocytes antigens (HLA) matching. Depending upon the time of onset and clinical manifestations, GVHD is divided into acute and chronic phases. Acute GVHD primarily affects liver, skin, and intestine.
1–3 On the other hand, chronic GVHD has been shown to cause a high incidence of ocular complications.
4,5 As high as 60% to 90% of chronic GVHD patients suffer from ocular manifestations.
4,5 Ocular signs in chronic GVHD patients may be noticeable even before the other systemic symptoms.
6–8 Dry eye disease is one of the most frequent complications of ocular GVHD.
9,10 The dry eye disease in ocular GVHD patients is severe, resulting in symptoms of blurred vision, photophobia, redness, gritty sensation, and pain.
9,10 These symptoms cause significant visual discomfort, and reduce the overall quality of life of GVHD patients.
9,10 In absence of timely and appropriate treatment, dry eye disease in GVHD patients may progress to corneal keratopathy, ulceration, and visual impairment.
8,11
The lacrimal functional unit, including ocular surface nerves, apical surface glycocalyx, lacrimal glands, meibomian glands, and a normal blinking response, all collectively contribute to the secretion and maintenance of a healthy tear film.
12 Tear film is critical for keeping the ocular surface hydrated and lubricated, thus preventing desiccation-induced damage to the ocular surface. Tear film comprises the mucoaqueous gel layer, which underlies but partially integrates into lipid layer.
13
Mucins are high molecular weight glycoproteins made up of a protein core with extensive glycan N-acetyl galactosamine side chains. The heavy glycosylation imparts the mucins with hydrophilicity and a negative charge.
14–18 These structural features account for the two key physiologic functions of ocular surface mucins, which include repelling pathogens and keeping the ocular surface hydrated.
14–18 The mucins present on the ocular surface and in the tear film include membrane-bound mucins MUC1, MUC4, MUC16, and gel-forming secreted mucins, MUC19 and MUC5AC, respectively.
14–18 The membrane-bound mucins are expressed on the apical surface of corneal and conjunctival epithelial cells. The gel-forming mucin MUC5AC in the tear film is primarily secreted by the goblet cells.
19 Lacrimal gland acinar cells have been shown to express MUC7 transcript but the glycoprotein has not been detected in the tear film.
20 Tear film mucins keep the eye surface lubricated and entrap allergens and pathogens.
14–18 Patients with dry eye disease show reduced levels of mucins or an alteration in the degree of their glycosylation.
21–23 A significant aqueous deficit has been observed in the tears of GVHD patients suffering from dry eye.
24 Multiple studies have shown that GVHD causes lacrimal gland fibrosis.
25–28 The effect of GVHD on ocular surface mucins has not been investigated. Therefore, the aim of the present study was to investigate the role of ocular surface mucins in GVHD-associated dry eye and to test the ameliorative effect of rebamipide, a mucin secretagogue, on GVHD-associated dry eye.
The eyes along with eyelids were harvested from euthanized animals at 8 weeks after the allogeneic transplantation and processed for paraffin embedding. The 7-μM thin paraffin sections were cut and Periodic Acid Schiff's (PAS) staining was performed for goblet cells using a commercially available kit (Polysciences, Inc., Warrington, PA, USA). The stained sections were imaged at ×100 magnification using a brightfield microscope (Keyence corporation of America, Itasca, IL, USA).
At 8 weeks after the allogeneic transplantation, animals were euthanized by CO2 administration. The eyeballs were collected and the corneas were separated. The corneas were homogenized in RIPA buffer containing protease inhibitor (Pierce, Thermo Fisher Scientific, Hanover Park, IL, USA). The total protein in the corneal homogenates was quantified by BCA method using a commercially available kit (Pierce, Thermo Fisher Scientific). The Muc1, 4, and 16 levels were quantified in the corneal protein lysates using commercially available ELISA kits (LSBio, Seattle, WA, USA). The mucin levels were normalized for the milligram of total protein in the corneal lysates.
Corneas were harvested from animals at 8 weeks after transplantation as described above. The mRNA was extracted from the corneas using the RNeasy Mini kit (RNeasy kit; Qiagen Inc., Valencia, CA, USA). The mRNA was immediately reverse transcribed to cDNA using a commercially available kit (Superscript III First-strand synthesis; Thermo Fisher Scientific) for complementary (c)DNA synthesis. The cDNA was used to quantify Muc1, Muc4, Muc16, and Muc19 gene expressions using real-time PCR. A 20-μL reaction mixture containing 2 μL of cDNA, 2 μL of forward primer (200 nM), 2 μL of reverse primer (200 nM), and 10 μL of 2× SYBR green super mix was run at a universal cycle (95°C for 10 minutes, 40 cycles at 95°C for 15 seconds, and 55°C for 60 seconds) in a thermocycler (Biorad CFX thermocycler; Bio-Rad Laboratories, Hercules, CA, USA). β-actin was used as the housekeeping gene. The relative change in gene expression was calculated using ΔΔCt method.
Effect of Topical Rebamipide on Ocular GVHD-Mediated Changes in Tear Film and Corneal Keratopathy
Supported by a new investigator grant from American Association of Colleges of Pharmacy (AACP; Arlington, VA, USA) to Ajay Sharma and Chapman University School of Pharmacy Start up fund.
Disclosure: K. Shamloo, None; A. Barbarino, None; S. Alfuraih, None; A. Sharma, None