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 Table of Contents  
Year : 2023  |  Volume : 13  |  Issue : 1  |  Page : 34-42

Dry eye syndrome model established in rabbits via mitomycin C injection in the lacrimal gland

1 Department of Ophthalmology, Taipei Medical University Shuang Ho Hospital, New Taipei City; Department of Ophthalmology, School of Medicine, College of Medicine, Taipei Medical University, Taipei City, Taiwan
2 School of Medicine, College of Medicine, Taipei Medical University, Taipei City, Taiwan; Department of General Medicine, MacKay Memorial Hospital, New Taipei City, Taiwan
3 Graduate Institute of Biomedical Materials and Tissue Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei City, Taiwan
4 Department of Ophthalmology, School of Medicine, College of Medicine, Taipei Medical University; Department of Ophthalmology, Taipei Veterans General Hospital, Taipei City, Taiwan
5 Graduate Institute of Biomedical Materials and Tissue Engineering; International Ph.D. Program in Biomedical Engineering, College of Biomedical Engineering, Taipei Medical University; International Ph.D. Program in Cell Therapy and Regenerative Medicine, College of Medicine, Taipei Medical University, Taipei City, Taiwan; Research Center of Biomedical Device, College of Biomedical Engineering, Taipei Medical University, Taipei City, Taiwan

Date of Submission02-Sep-2021
Date of Acceptance14-Feb-2022
Date of Web Publication04-May-2022

Correspondence Address:
Prof. Ching-Li Tseng
Graduate Institute of Biomedical Materials and Tissue Engineering, Taipei Medical University, No. 250 Wu-Hsing Street, Taipei City 110
Prof. Ko-Hua Chen
Department of Ophthalmology, Taipei Veterans General, Hospital, No. 201, Sec. 2, Shipai Rd., Beitou District, Taipei City
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/tjo.tjo_11_22

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PURPOSE: To develop a new dry eye syndrome (DES) animal model by injecting mitomycin C (MMC) into the lacrimal glands (LGs) of rabbits evaluated by clinical examinations.
MATERIALS AND METHODS: A volume of 0.1 mL of MMC solution was injected in the LG and the infraorbital lobe of the accessory LG of rabbits for DES induction. Twenty male rabbits were separated into three groups, the control group, and different concentration of MMC, (MMC 0.25: 0.25 mg/mL or MMC 0.50: 0.5 mg/mL) were tested. Both MMC-treated groups received MMC twice injection on day 0 and day 7. Assessment of DES included changes in tear production (Schirmer's test), fluorescein staining pattern, conjunctival impression cytology, and corneal histological examination.
RESULTS: After MMC injection, no obvious changes in the rabbit's eyes were noted by slit-lamp examination. Both the MMC 0.25 and the MMC 0.5 groups revealed decreased tear secretion after injection, and the MMC 0.25 group showed a continuous decrease in tear secretion up to 14 days. Fluorescent staining showed punctate keratopathy in both MMC-treated groups. In addition, both MMC-treated groups demonstrated decreased numbers of conjunctival goblet cells after injection.
CONCLUSION: This model induced decreased tear production, punctate keratopathy, and decreased numbers of goblet cells, which are consistent with the current understanding of DES. Therefore, injecting MMC (0.25 mg/mL) into the LGs is an easy and reliable method to establish a rabbit DES model which can apply in new drug screening.

Keywords: Animal model, dry eye syndrome, goblet cells, lacrimal gland, mitomycin C, rabbit, tear production

How to cite this article:
Lin IC, Wang YC, Chen YZ, Tang YJ, Chen KH, Tseng CL. Dry eye syndrome model established in rabbits via mitomycin C injection in the lacrimal gland. Taiwan J Ophthalmol 2023;13:34-42

How to cite this URL:
Lin IC, Wang YC, Chen YZ, Tang YJ, Chen KH, Tseng CL. Dry eye syndrome model established in rabbits via mitomycin C injection in the lacrimal gland. Taiwan J Ophthalmol [serial online] 2023 [cited 2023 Apr 2];13:34-42. Available from: https://www.e-tjo.org/text.asp?2023/13/1/34/344835

  Introduction Top

Dry eye syndrome (DES) is an ocular disease that commonly affects people older than 50 years of age.[1] DES occurs due to the inflammatory disruption of tear formation mechanisms in the lacrimal glands (LGs), and this may cause tear instability and changes in tear composition.[2] The current diagnostic criteria for DES are mainly based on patient symptoms, including burning or pruritic sensation in the eyes, similar to the presence of foreign bodies, excessive tearing, redness, and photophobia.[3] However, DES must be distinguished from infection or allergy because incorrect treatments may lead to the progression of DES.[4] The tests to distinguish them include a comprehensive symptom-oriented questionnaire,[5] tear film breakup time,[6] ocular surface staining of corneal injury,[7] Schirmer's test,[3] and clinical examination of the eyelid and conjunctiva.[8],[9] The development of DES therapeutic agents/regimens relies on the results from animal studies. Therefore, it is impotent to develop a suitable animal model for DES.

Several DES animals' models have been established, including a model of surgical removal of LGs,[10],[11] or a model of exposure to low humidity environments.[12] The former requires high surgical skills, while the latter requires modification of breeding facilities or conditions. Moreover, these methods cannot fully reflect the pathogenesis of chronic DES, in which the inflammatory response usually involves both the LGs and conjunctiva.[13] A commonly used DES animal model is induction with 0.1%–0.2% benzalkonium chloride (BAC) in mice, rats, rabbits, and dogs.[14],[15] However, BAC reduces corneal cell proliferation and viability, impairs corneal healing, and disrupts epithelial barrier function.[16] With the differences of BAC concentration (0.05-0.2%) for DES induction and the use of anesthesia during eye examination, these factors resulted in the variation of DES development process. This may increase in each animal group because each drop of BAC may be on the conjunctival or corneal surfaces.[17] In contrast, a low concentration (0.1% BAC) requires 4–8 weeks to induce DES in rabbits,[18] which takes a long time to induct a DES model. Therefore, a quick, feasible, and stable DES model is needed to facilitate the development of DES innovative therapies.

Mitomycin C (MMC) has been applied for various ocular diseases treatment, including glaucoma, pterygium, corneal refractive surgery, corneal and conjunctival dysplasia and neoplasia, and allergic eye disease.[19],[20] It is an alkylating chemotherapy drug that causes DNA crosslinking resulting in transcription arrest and apoptosis.[21] Apoptosis has been proposed as a possible mechanism responsible for the impairment of LG secretory function associated with Sjogren syndrome.[22]

In this study, rabbits were tested for new DES model establishment. There are three groups: the control group, the MMC 0.25 group, and the MMC 0.5 group. The MMC 0.25, and the MMC 0.5 group received injection with 0.1 mL of MMC at 0.25 mg/mL or 0.5 mg/mL in both the LG and the infraorbital lobe of the accessory LG for DES induction. Then, the tear production, fluorescent staining pattern, and histological changes of the cornea were examined to find optimal concentration of MMC to develop a new DES animal model.

  Materials and Methods Top

Chemicals and reagent

MMC was purchased from Sigma-Aldrich (St. Louis, MO, USA). Zoletil 50 and 2% Rompun solution were obtained from Virbac Animal Health (Vauvert, Nice, France) and Bayer Korea Ltd. (Ansan-City, Kyonggi-do, Korea), respectively. Schirmer strips (Tear Touch; Madhu Instruments, New Delhi, India), topical anesthesia solution (0.5% Alcaine®; Alcon-Couvreur N. V., Puurs, Belgium), and fluorescein (FL) paper strips (Haag-Streit AG, Koniz/Bern, Switzerland) were also obtained. The remaining chemicals were purchased as reagent grade from Sigma-Aldrich.

Induction of dry eye syndrome in rabbits

Twenty male New Zealand white rabbits (weighing 2.5–3.5 kg) with no signs of ocular inflammation or gross abnormalities were used in this study and separated into two batches. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Taipei Medical University (approval no. LAC-2017-0395, March 19, 2018). The rabbits were housed in standard cages in a light-controlled room at 23°C ± 2°C, relative humidity of 60% ± 10%, and alternating 12-h light-dark cycles (6 AM to 6 PM). Each rabbit was given food and water ad libitum. All examinations procedures on rabbits were performed under general anesthesia, administered through an intramuscular injection of a mixture of Zoletil 50 and 2% Rompun solution (1:2 ratio, 1 mL/kg).

MMC was dissolved in phosphate-buffered saline (PBS) and adjusted to designated concentrations of 0.25 and 0.5 mg/mL. The rabbits were randomly separated into three groups. Rabbits in the MMC 0.25 group received injection with 0.1 mL of MMC (at 0.25 mg/mL) using a 31-G insulin needle in both the LG and the infraorbital lobe of the accessory LG, the same us MMC 0.5 group by injected with 0.1 mL of MMC at 0.5 mg/mL. The rabbits which did not receive MMC injection were categorized as the control group. The injection site is shown in [Figure 1]. To increase the duration of this DES model, MMC was injected on day 0 and day 7. The assessment of DES was performed at specified time points (days 0, 3, 7, 10, and 14) and was described below.
Figure 1: The injection site of the rabbit eye. Two glands (lacrimal gland and infraorbital lobe of the accessory lacrimal gland) are depicted, along with the sites where mitomycin C is administered

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Evaluation of dry eye syndrome in rabbits

Measurement of the tear production

The tear production was measured using Schirmer strips.[18] Briefly, the rabbits were anesthetized to keep them immobile. The test was performed on scheduled dates in a standard environment by the same researcher. Schirmer strips were inserted into the space of the palpebral conjunctiva which is near the junction of the middle and outer third of the lower eyelid. The length of the moistened strip was measured and recorded in millimeter at 5 min. Each eye was tested twice at an interval more than 30 min. Due to the baseline tear volume variation between animal batches, the tear production value was recorded as a percentage that compare with the value on day 0.

Fluorescein staining

FL staining of corneal tissue was performed before and after DES induction.[23] Five FL strips were soaked in 1 mL PBS for half-hour letting dye dissolved and released. At the indicated time, 2 μL of FL solution was instilled into the conjunctival sac, and the ocular surface was examined and graded under a hand-held portable slit-lamp microscope with a cobalt blue filter (SL-17, Kowa Company, Torrance, CA, USA). The scoring criteria follow a system recommended by the National Eye Institute scale: The grading is based on a scale of 0–3 (0 = normal, 1 = mild, 2 = moderate, and 3 = severe staining) in five areas of cornea: Central, superior, inferior, nasal, and temporal quadrant. The maximum score is 15.[24]

Conjunctival impression cytology

Conjunctival impression cytology was performed after FL staining (with PBS rinsed for several times removing dye residual). A nitrocellulose filter paper (Whatman, Maidstone, UK) with a pore size of 0.45 μm was cut into small disks with a radius of 3.5 mm and then cut into semicircles. The edge of the paper was grasped with forceps, and the paper was placed with a slight pressure on the eye at the surface of the palpebral conjunctiva (near the junction of the middle and outer third of the lower eyelid), 2 mm lateral to the corneal limbus. The paper was lifted gently and immediately fixed in fresh 4% paraformaldehyde for at least 30 min; after rinsed by PBS, then the paper was treated by xylene several times turning to transparent. The papers were washed with PBS again, and then stained with periodic acid Schiff and hematoxylin, finally mounted.[25] The completed slides are examined by light microscopy (DMi8, Leica, Wetzlar, Germany). The goblet cell number was counted from the photos using ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). Three different regions of each specimen were selected and photographed randomly for quantification.

Central corneal thickness and intraocular pressure examination

Central corneal thickness was determined using an ultrasonic pachymeter (iPac pachymeter, Reichert Technologies, NY, USA) with a hand-held solid probe, with the probe tip held perpendicular to the central cornea. Averages of 10 readings were recorded for each eye. The intraocular pressure (IOP) of the rabbits was measured using Icare TONOVET tonometer (TV01, Vantaa, Finland) in compliance with the manufacturer's instructions. To determine IOP, five readings were taken for each eye alternating between the left and right eyes, and the average IOP value was calculated.[26]

Histological examination of the cornea

After the examination on day 14, the rabbits were sacrificed, the eyeballs were excised, and the corneas were isolated. The corneas were fixed in 3.7% buffered formaldehyde solution for at least 24 h. The fixed corneas were then embedded in paraffin and sectioned into 5 μm thick slide. The sections were stained with hematoxylin and eosin (H and E) for histological examination. The stained slides were examined under light microscopy and photographed. To measure corneal epithelial thickness, the Image J software was used. At least five points were used to measure the thickness of the corneal epithelial layers from the H and E images.

Statistical analysis

Data are presented as the mean ± standard deviation of 2–3 independent experiments from 4 to 8 rabbits in each group. Statistical analyses were performed using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). A one-way ANOVA followed by Tukey's post hoc test was used to compare the groups. Statistical significance was set at P < 0.05.

  Results Top

Ocular observation after mitomycin C injection

The changes in the cornea using slit-lamp microscopy and FL staining were presented [Figure 2] and [Figure 3]. No redness, discomfort, or discharge was noted in MMC-injected eyes. However, slight corneal opacity and roughness were observed after day 7 in both the MMC 0.5 and the MMC 0.25 groups [Figure 2].
Figure 2: Representative slit-lamp examination photographs of the rabbit eyes from control, mitomycin C (MMC) 0.25, and MMC 0.5 groups

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Figure 3: Photographs of fluorescein staining examination of the rabbit eyes. (a) Slit-lamp photography of the rabbit eyes in each group. (b) Cornea grading according to the National Eye Institute scale. Data were analysed using the one-way ANOVA and are expressed as the mean ± standard deviation; n = 3, (*P < 0.05; **P < 0.001 compared with the control group)

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Corneal FL staining is used to evaluate the condition of the cornea in rabbits. In the control group, there was no punctate staining on the cornea throughout the experimental period. Punctate keratopathy in the MMC-treated groups was noted from day 3 to day 14 [Figure 3]a. [Figure 3]b shows the grading score after treatment with MMC at different days. The control group had almost no staining during the experimental period. Both in the MMC 0.25 and MMC 0.5 groups, staining began on day 3 (4.67 ± 4.16 and 4.00 ± 1.73), reach the highest peak on day 7 (2.67 ± 0.58 and 6.33 ± 3.51), and remained constant through day 14 (2.00 ± 1.73 and 3.67 ± 2.08).

The central corneal thickness of the control group was 370–403 μm [Table 1]. There was no significant difference among the control group, the MMC 0.25 group, and the MMC 0.25 group during the study periods. The IOP of the MMC 0.25 group and the MMC 0.5 group was also not significantly different from that in the control group [Table 1]. These findings indicated no changes in central corneal thickness and IOP related to the MMC injection.
Table 1: Central corneal thickness and intraocular pressure results

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Tear secretion after mitomycin C injection

The tear secretion was evaluated by using Schirmer's test on days 0, 3, 7, and 14. The value of tear production on day 0 was considered as 100%. In the MMC 0.25 group, the tear secretion volume slightly decreased on day 3 (89.2% ± 14.5%) and then significantly decreased on day 14 (72.0% ±6.8%; *P < 0.05, compared with control group). In the MMC 0.5 group, the tear secretion volume decreased during the first 3 days; however, it rebounded after day 3 and peaked at 140.6% ± 6.9% on day 14 [Figure 4].
Figure 4: Results of Schirmer's test of all groups. Data are expressed as mean ± standard deviation (n = 4). *P < 0.05 compared with the value of the control group (one-way ANOVA and Tukey's post hoc test)

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Variation in conjunctival goblet cells after mitomycin C injection

Conjunctival goblet cells are responsible for the mucous layer of the tear film, so they are considered a critical factor in the evaluation of DES. The impression cytology result obtained from representative eyes is shown in [Figure 5]a. In the control group, there was no substantial change in the cell number during the experimental period. On days 0, 7, and 14, the average number of goblet cells in the control group was 169 ± 19, 185 ± 31, and 170 ± 16 cells/photo, respectively. For the MMC 0.25 and the MMC 0.5 groups, the number of conjunctival goblet cells on day 7, especially on day 14, were significantly less than those on day 0 and the control group [Figure 5]a. In the MMC 0.25 group, the average number of goblet cells was decreased to 92 ± 25 cells/photo on day 7, and it was continuously decreased to 63 ± 10 cells/photo on day 14. In this group, the number of goblet cells on day 14 showed a significant difference compared to that on day 0 (#P < 0.05, compared with day 0) [Figure 5]b. In the MMC 0.5 group, the average number of goblet cells was 74 ± 15 cells/photo on day 7, and 61 ± 6 cells/photo on day 14. The difference between the MMC 0.25 and the MMC 0.5 groups showed no statistical significance on day 7 and day 14. In addition, the number of goblet cells in both groups revealed statistical significance compared with that in the control group [*P < 0.05, [Figure 5]b]. The results demonstrated that the reduction of conjunctiva goblet cells after MMC injection.
Figure 5: (a) Photographs of conjunctival impression cytology stained with periodic acid Schiff and hematoxylin (scale bar = 100 μm). (b) Quantification data were acquired from those images. Data are expressed as mean ± standard deviation (n = 5). *P < 0.05 compared with the value of the control group, #P < 0.05 compared with the value on day 0

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Histological examination of the cornea

The corneal tissue section stain with H and E was observed under a light microscope. The cornea structure of the control group is composed of multiple layers of epithelial cells and dense collagen fibrils in the stroma [Figure 6]a. However, slightly loose stroma with space between collagen parts was noted in both MMC-treated groups [Figure 6]b and [Figure 6]c. The corneal epithelial thickness (measured by corneal section photos) was presented on [Table 2]. The corneal epithelium thickness was 39.5 ± 1.9 μm in the control group on day 14, and the thickness of the MMC 0.25 and MMC 0.5 groups were 40.0 ± 3.5 μm and 37.3 ± 8.5 μm, respectively. The histological results indicated that no remarkable changes in corneal epithelium thickness after MMC injection.
Figure 6: Representative images of H and E stained corneal sections from cornea tissue acquired on day 14. (a) Control group, (b) mitomycin C (MMC) 0.25 group, and (c) MMC 0.5 group (Scale bar = 250 μm). The right panel showed an enlarge image of the square in the whole corneal section

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Table 2: Corneal epithelium thickness

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  Discussion Top

In this study, we demonstrated a new method to established DES model in rabbits through MMC injection into the LG. The manifestations of this model included decreased tear secretion, punctate epitheliopathy, and decreased numbers of goblet cells, are consistent with those clinical features of DES. This new DES model can be induced within 2 weeks, and it can be applied in the DES drug screening and evaluation.

MMC has been widely used in ophthalmology, including in the surgical treatment of pterygia, glaucoma, corneal and conjunctival dysplasia, and neoplasia. In addition, it has been used in prevent haze formation in corneal refractive surgery such as laser-assisted in situ keratomileusis, it is used in concentrations ranging from 0.2 to 0.4 mg/mL.[27] However, Sauder and Jonas reported limbal stem cell deficiency after subconjunctival injection of 0.2 mg/mL MMC to patients.[28] Another study from Mietz et al. found rabbit sclera treat with 0.1–0.2 mg/mL MMC showed pathologic changes in the ciliary.[29] Our preliminary data have tried the concentration of MMC at 0.125 mg/mL, but the tear production and FL stain results showed no obvious difference with the control group. Therefore, the concentration of MMC which we selected to test thereafter was 0.25 and 0.5 mg/mL.

Due to the high prevalence of DES, an effective therapeutic agent for DES treatment is required. Several animal DES model establishment methods have been reported, such as local administration of BAC,[18],[30],[31] or raising animals under low-humidity (19% ±5%) and high-airflow (flow rate: 15 L/min) environmental conditions,[32] or complete dacryoadenectomy in rabbits.[33] BAC is a common preservative in topical eye drops. The BAC-induced DES rabbit model developed by Xiong et al. showed obvious corneal surface damage, decreased aqueous tear secretion, loss of goblet cells, and corneal epithelial changes.[18] However, this BAC-induced model was associated with the risk of reduced corneal cell proliferation and viability, impaired corneal wound healing, and disrupted epithelial barrier function.[30] The topical application of BAC causes ocular surface discomfort and inflammation,[31],[34] similar to the clinical manifestations of keratitis. The corneal epithelial layer became thinner in BAC-treated cornea compared to the control groups.[30],[35] In this study, H and E staining revealed that the morphology of the corneal epithelial layer [Figure 6] and the measured thickness [Table 2] in both MMC 0.25 and MMC 0.5 groups was not significantly different compared to those in the normal cornea. In addition, there were no significant differences in central corneal thickness between the control group and the MMC-treated group. No redness and discharges were noted in the MMC-treated groups. We concluded that this model does not change the corneal thickness, and it does not appear to be toxic to the corneal epithelial cells as BAC method.

A DES model induced by excising the exorbital and intraorbital LGs (ELG and ILG, respectively) of mice has been reported. After ELG and ILG excision, the tear production significantly decreased, and the corneal FL infiltration score also significantly increased in those dacryoadenectomized mice compared with normal eyes.[11] Honkanen et al. used complete dacryoadenectomy to develop a rabbit DES model, which was claimed to present a stable, chronic, and predominantly aqueous-deficient DES that recapitulated the key clinical and histological changes of human DES. Complete dacryoadenectomy resulted in suppression of the Schirmer's test values on week 1, which remained stable throughout the 8 weeks of observation. However, complete dacryoadenectomy requires precise surgical skill and prior experience in orbital surgery, and the surgical time is approximately 1–2 h.[33],[36] Moreover, it is failed to completely reduce the Schirmer's test value due to other possible sources of residual tear fluid, including the largely untouched accessory LGs, other conjunctival sources, and plasma leakage from conjunctival vessels.[36]

A reciprocating phenomenon of tear secretion was noted on day 7 in the MMC 0.5 group [Figure 4]. Although the main LG is considered to be the major source of tear production, it has been reported that rabbit conjunctival epithelium has the capacity to become the primary source of the tear film.[37],[38] Gilbard et al. demonstrated that cauterization of the main LG excretory duct and removal of the nictitating membrane and Harderian gland did not reduce tear secretion as compared with control eyes, which is thought to be due to the presence of the accessory LGs, cornea, and conjunctiva of tear secretion unit.[39] Moreover, previous studies have provided evidence that conjunctival and/or corneal tissues can secrete or transport fluid into the tear film.[40],[41],[42] Honkanen et al. also reported that incomplete reduction of Schirmer's test values in dacryoadenectomized rabbits. They speculated that the remaining tear fluids come from the accessory LGs, conjunctiva, and plasma leakage from conjunctival blood vessels.[36] Taken together, it is plausible to speculate that the robust tear production on day 7 in the MMC 0.5 group may be due to compensatory tear secretion from the conjunctiva and cornea.

Compared with mice, rabbits are regarded as a more reliable animal model for ophthalmic examinations because they have a larger exposed ocular surface. Therefore, standard dry eye clinical tests, such as tear breakup time, tear production tests, and corneal FL staining of the ocular surface, are more reliable in rabbits than in mice.[43] DES occurs more frequently in females than in males. Sex hormone changes, especially androgen deficiency, can adversely affect ocular surface homeostasis.[44],[45] The alteration in sex hormone in female rabbits may affect tear production and lead to spontaneous dry eye formation. Therefore, we choose male rabbits to minimize the possible impact of sex hormones on the ocular effect in this study. The degree of corneal epithelial erosion in the MMC 0.25 and the MMC 0.5 groups was most prominent on day 10 [Figure 2] and [Figure 3]. In both MMC-treated groups, Schirmer's test values have decreased since day 3. In MMC 0.25 group, the Schirmer's test values decreased significantly on day 14 that may be induced by the second injection of MMC on day 7 [Figure 4]. The number of conjunctival goblet cells in the MMC 0.25 and the MMC 0.5 groups was significantly decreased compared to the control group [Figure 5]. We concluded that the optimal condition of our DES model was from day 7 to day 14, and the optimal concentration of MMC is 0.25 mg/mL. DES is also known as keratoconjunctivitis sicca, and its clinical symptoms include a decrease in the number of goblet cells leading to a decrease in tear secretion. Severe decrease number of goblet cells is related to squamous metaplasia, enlargement of the epithelial area, and occasional keratinization of the ocular surface.[46],[47] The number of conjunctival goblet cells has been found to not correspond to Schirmer's test value, but it was related to tear break-up time.[48] In the MMC 0.5 group, Schirmer's test value did not decrease significantly and did not correspond to the loss of conjunctival goblet cells.

In this study, there were no significant differences on the central corneal thickness and corneal epithelial thickness between the control groups and the MMC-treated groups. The use of MMC in ophthalmology has been increasing due to its effect on wound healing.[19] However, studies have shown that loss of endothelial cells occurs after MMC-augmented trabeculectomy[36] and reduction in endothelial cell counts occur after topical MMC use in pterygium surgery.[49] Chang found that 0.02% MMC caused corneal edema and apoptosis in a concentration- and time-dependent manner.[50] In addition, Song et al. observed increased keratocyte apoptosis by increasing the dose and exposure time of MMC. They indicated that the concentration of MMC had greater effects on the aqueous MMC concentration and keratocyte apoptosis than the exposure time of MMC.[51] Our study showed that MMC injection into the LG does not change the corneal thickness, corneal epithelium thickness and also no affect IOP. Although slight corneal opacity and roughness were observed on day 7 in both MMC-treated groups, no severe adverse effects of MMC or no complications with the procedure occurred.

  Conclusion Top

We demonstrated that the injection of 0.25 mg/mL MMC into the LG and the infraorbital lobe of the accessory LG could successfully induce a rabbit DES model with reduced tear production, corneal epithelial punctate lesions, and decreased the numbers of goblet cells. This MMC-induced DES model does not affect the corneal thickness or corneal epithelial thickness. This model is an easy, safe and reliable method to induce DES within 2 weeks. It can be applied to study the pathogenesis of DES or facilitate the development of DES innovative therapies.


This work was supported in part by a grant from the Taipei Medical University Shuang Ho Hospital (107TMU-SHH-18) and the Ministry of Science and Technology, Taiwan (MOST 107-2622-E-038-003-CC3, MOST 110-2314-B-038 -044 -MY2). And grants from the integrated research grant in health and medical sciences from National Health Research Institute, Taiwan (NHRI-EX111-10933SI). Also, thanks for the helping of research assistant, Erh- Hsuan Hsieh, for some animal tests.

Financial support and sponsorship

This work was supported in part by a grant from the Taipei Medical University Shuang Ho Hospital (107TMU-SHH-18) and the Ministry of Science and Technology, Taiwan (MOST 107-2622-E-038-003-CC3, MOST 110-2314-B-038-044-MY2). And grant from the integrated research grant in health and medical sciences from National Health Research Institute, Taiwan.

Conflicts of interest

The authors declare that there are no conflicts of interests of this paper.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

  [Table 1], [Table 2]


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