Histopathological Changes of the Anterior Surface Structures of Rabbit Eye after Subconjunctival Injection of Different Doses of Mitomycin C

INTRODUCTION

The front of the human eye consists of clear cornea centrally and white sclera peripherally. Cornea and sclera, respectively, are covered by the corneal and conjunctival epithelia. These epithelia are non-keratinized and stratified structures. The corneal epithelium is 5–7 cell layers thick, while the conjunctival epithelium is 3–5 cell layers thick. These two epithelial structures are bathed in a tear film, and together they form the front surface of the eye (the ocular surface).¹ Mitomycin C (MMC) is a chemotherapeutic antibiotic isolated from Streptomyces caespitosus and is a cell-cycle non-specific alkylating agent. MMC covalently binds to and results in DNA cross-linking; thus, it has antimetabolite activity as it inhibits cellular replication in a dose-dependent manner.² The use of MMC was first reported in the eye to treat pterygia in

1963. It has become increasingly popular in ophthalmology because of its properties as a modulator for wound healing as well as for the treatment of pterygia, it is also used in glaucoma drainage, corneal and conjunctival dysplasia, and neoplasia, allergic eye disease, and as a measure to prevent and treat haze formation in corneal refractive surgery.³ MMC is an alkalizing agent and a potent fibroblast inhibitor that causes irreversible damage to the cell's DNA structure. It inhibits both fibroblast migration and the synthesis of new collagen and thus affects wound healing. Subconjunctival MMC is known to reduce the number of stromal fibroblast cells. There is also morphological and structural damage to vascular endothelial cells.⁴

Because of its anti-fibroblast effect, MMC is used in a variety of ocular surgical procedures. The optimal dose of MMC is not known but is usually used at a concentration of 0.1 mg/ml (0.01%) to 0.5 mg/ml (0.05%) in various clinical setups.⁵ Despite the important function of MMC in the treatment of eye diseases such as pterygium and glaucoma, complications may occur, including corneal edema, corneal melting and perforation, corneal blood staining, pigmentation, scleral calcification and melting, secondary glaucoma, prolonged hyperemia, late hemorrhage, chemosis, lid oedema, excessive tearing, ptosis, photophobia, and endophthalmitis.⁶ Preoperative use of MMC as a subconjunctival injection into the body of pterygium before surgical excision is another modality in which the recurrence rate is similar to intraoperative use of MMC. Subconjunctival injection of MMC avoids direct MMC contact with corneal and conjunctival epithelium, which can cause a permanent corneal or conjunctival defect.⁷ Compared to intraoperative use of MMC, the preoperative subconjunctival injection into the body of pterygium is preferable because its concentration is not diluted in the tear film, there is less intraocular penetration as the corneal epithelium is intact, and as a result, there is more prolonged contact time with the subconjunctival tissue.⁸

This study had evaluated the changes in the normal histology of the anterior surface structures of the rabbit eye “conjunctiva, cornea and sclera” after subconjunctival injection of different doses of Mitomycin C (0.1, 0.2 and 0.4 mg/ml) after different periods (1, 7 and 14 days).

MATERIAL AND METHODS

A comparative prospective case-control animal study included eighteen eyes of eighteen healthy adult New Zealand white male rabbits, weighing approximately 2.0-4.0 kg. It was carried out from April 2019 to October 2019 at Pathology Department, Faculty of Medicine, Al-Azhar University.

Eighteen rabbits were divided into 3 groups each group contains six rabbits. The three groups (group A, group B, and group C) were given a subconjunctival injection of 0.2 ml MMC with different concentrations (0.1, 0.2, and 0.4 mg/ml respectively) 2 mm from the limbus at 3 O’clock position in the left eye of every rabbit. Another control group was formed from six eyes from the right eyes that had not been treated with MMC. The rabbits were anesthetized with ketamine hydrochloride (50 mg/kg) and xylazine hydrochloride (5 mg/kg) before subconjunctival injections.

Histopathology: Six rabbits (two from each group) were sacrificed at 1 day, another six rabbits (two from each group) at 7 days, and the last six rabbits (two from each group) at 14 days. Eyes were enucleated, fixed immediately in formaldehyde 10%. The globes have then bisected vertically. The specimens had embedded in paraffin and sectioned with a microtome. Sections (5-7 µm) were stained with hematoxylin-eosin (H&E) for cellularity including fibroblast, Periodic acid-Schiff (PAS) for goblet cell identification, Masson's Trichrome (MT) for collagen density and architecture and Orcein stain for elastic fiber detection. The grading system to assess cellularity, collagen deposition and goblet cell number will be calculated using the average cell number.

Statistical analysis: Data from different groups along the three sacrifices were collected, revised, encoded, and entered in the GraphPad Prism Version 7.0 (Two Way ANOVA Test) statistical package. Quantitative data were presented as mean, standard deviations, and ranges when their distribution was parametric. Qualitative variables have also been presented as numbers and percentages. The p-value was thus found to be significant as follows: P > 0.05: non-significant, p < 0.05: significant, P < 0.01: highly significant.

RESULTS

Immediately after injection: All of the treated rabbit eyes showed in the injected area a bubble-like elevation appeared immediately after injection, which gradually vanished within the next 1-2 days. There was no evidence of corneal stromal edema. The sclera appeared normal, specifically, without inflammatory infiltrates or scarring     

During the follow up: Group A: Rabbits treated with a low dose of MMC (0.1 mg/ml) showed non-significant findings except that one rabbit showed slight conjunctival congestion after one day of injection and disappeared three days later. Group B: This group showed more apparent findings, all injected rabbits showed conjunctival congestion from the second day of injection. Two rabbits of them showed blepharospasm with excessive tearing in the treated eyes for about 48 hours  (Figure 1).

Group C: Compared to the other groups, there were more significant clinical findings, all rabbits showed severe congestion, blepharospasm, and excessive tearing. Furthermore, two rabbits showed severe chemosis, lid edema, and lacerations. Injected eye of one of them showed scleral Dellen (Figure 2).

Corneal thickness: There were non-significant changes in the total corneal thickness within the different sacrifices unless the 3^rd sacrifice after two weeks, administration of high doses of MMC showed a marked decrease in the corneal thickness (Table 1) (Figure 3).

Keratocytes count: There were no significant changes in the count of keratocytes in different sacrifices unless there was a marked decrease in high doses of MMC (0.4 mg/ml) during the third sacrifice after two weeks of injection.

Scleral thickness: Comparing the changes of the Scleral thickness in the three study groups and the control group with variable doses of MMC along with the three different sacrifices. Scleral thickness was markedly decreased at the 3^rd sacrifice in comparison with the 1^st and 2^nd sacrifices  (P < 0.01) in all study groups. Group A results showed a non-significant decrease in the thickness of the sclera except in the 3^rd sacrifice (P < 0.01) (Figure 4).

Conjunctival goblet cells counting: Administration of MMC at different doses even the low dose significantly decreased the goblet cells in time and dose-dependent manner (P < 0.01) (Figure 5).

Hematoxylin and Eosin (H&E) stain: 1^st sacrifice showed non-significant findings except within group C conjunctival sections. 2^nd and 3^rd sacrifices showed significant changes mainly in conjunctival and scleral sections of groups B and C (Figure 6).

Periodic acid-Schiff (PAS) stain: Goblet cells stained with PAS showed a significant decrease with MMC administration at different doses even the low dose from the 1^st to the 3^rd sacrifice in the dose and time-dependent manner (Figure 7).

Masson's Trichrome (MT) stain: The intensity of collagen staining within corneal stroma showed non-significant decreased in all groups of the three sacrifices.

While episcleral and scleral collagen content showed a significant decrease of the in the dose and time-dependent manner in all treated groups from 1^st to 3^rd sacrifices (Figure 8).

Orcein stain: The elastic tissues within the scleral portion were markedly decreased from the 1^st to the 3^rd sacrifice and within all treated groups in a dose and time-dependent manner (Figure 9).

Two-way ANOVA test

Data of corneal thickness are expressed as mean ± standard deviation.

Table 1:Comparison of the corneal thickness in the three study groups and control group with variable doses of MMC along with the three different sacrifices.

Fig. 3: A chart showing corneal thickness within the different sacrifices at different doses of mitomycin, data were expressed as mean±SE.

Fig. 4: A chart showing scleral thickness within the different sacrifices at different doses of mitomycin, data were expressed as mean±SE. a indicates significance in comparing with the control group, b indicates significance in comparing with group A, c indicates significance in comparing with group B.

Fig. 5: A chart showing goblet cell number within the different sacrifices at different doses of mitomycin, data were expressed as mean±SE. ND means non-detectable cells. a indicates significance in comparing with the control group, b indicates significance in comparing with group A, c indicates significance in comparing with group B.