Introduction

An alkali burn of the cornea causes a recalcitrant keratitis characterized by frequent blister formation, recurrent epithelial breakdown, stromal cell death, inflammatory cell infiltration, and endothelial dysfunction.²⁵ Several proteinases released from the alkali-injured cornea might account for the ulcerative process. Elevated activities of proteinase and acid glycosidase have been thought to be responsible for the ulcerative process in alkali-injured corneas.⁹ 

Non-steroidal anti- inflammatory drugs (NSAIDs) are one of the most commonly prescribed classes of worldwide medications specifically in treatment of ocular disease.¹⁹ NSAIDs are potent inhibitors of COX enzymes, and thereby the synthesis of PGs. Within the eye, PGs cause vasodilatation, disruption of the blood-ocular barrier, and leukocyte migration. Consequently, their inhibition by cyclooxygenase inhibitors may have therapeutic effects.^13,14,27 Two main isoforms of COX, COX-1 and COX-2, have been identified.³⁶ COX-1, a constitutive enzyme, synthesizes PGs that regulate physiologic processes. Present in most tissues, COX-2 is an inducible enzyme that is expressed throughout the body, primary during inflammatory responses and in association with pain or fever.²⁹ Aspirin and other chemically related compounds, used systemically for many decades for their analgesic, antipyretic, and anti-inflammatory properties, have more recently been prepared in topical ophthalmic formulations; they havebeen proven to be useful in treatment of ocular inflammation, including control of miosis during surgery, postsurgical inflammation of the anterior segment, cystoid macular edema (CME) and inflammation due to noninfectious conjunctivitis such as allergic conjunctivitis. In addition, they can be used to decrease pain, neovascularization and photophobia after refractive surgery.^{12,13,14,34,35} There are some other treatments, including the steroids and anti-allergic drugs, for ocular inflammation; but NSAIDS are preferred because do not produce the classical adverse effects associated with steroids, such as elevation of intraocular pressure (IOP), cataract and aggravation of ocular infection. Although usage of topical NSAIDs is increasing in ophthalmology medicine, scientists worry about their corneal complications. An alternative medicine to suppress ocular inflammation is diclofenac that has been used in this study to evaluate the improvement rate of alkali corneal burnt in rabbit. However, the recent reports of corneal melting is surprising.¹²

Materials and Methods

15 New Zealand adult male and female rabbits with normal eyes were divided into three groups with five animals each. Prior to the study, ophthalmic examinations including, indirect ophthalmoscopy, slit lamp bio microscopy, STT, and fluorescein staining of both eyes were performed. Animals were anesthetized with xylazine (5 mg/Kg, IM) and ketamine (35 mg/Kg, IM) . The corneal alkali burn was made by placing a 9-mm diameter; circular piece of filter paper soaked in 1molar NaOH on the central cornea for 30s. Only one eye in each rabbit was treated. The cornea was rinsed with 2 ml of physiological saline immediately after alkali exposure. Then, in group A, topical diclofenac was applied every 8 hours, and in group B, 0.4 cc of diclofenac was administered through subconjunctival injection every 7 days. Duration of treatments was 21 days. Group C, as control group, remained untreated after alkali exposure (Fig.1).

The rabbits were maintained in a relatively dark room and only during the time of examination and treatments were the lights turned on. The eyes were examined daily by slit-lamp bio microscopy for 21 days. Clinical outcome was monitored by duration of blepharospasm, duration of ocular discharge, and corneal vascularization.

All rabbits were euthanized by IV injection of sodium pentobarbital in day 21 and the eyes were fixed in 10% buffered formalin and embedded in paraffin. Sections were stained with H&E, PAS, and Gram, and examined by light microscopy (Olympus BX41, Japan). To document the findings, microphotographs were obtained by digital camera (Olympus DP12, Japan). Samples were monitored by evaluating corneal thickness (9μm), numbers of epithelial rows, keratocyte density, stromal vascularization, stromal inflammation, and stromal collagen arrangement.

Statistical analysis for duration of ocular discharge, duration of blepharospasm, and corneal vascularization among the three groups was performed by Kruskal– Wallis test. Pair wise comparison between each group with control group was performed by Mann–Whitney test. P values of

Results

Freshly burned corneas became cloudy immediately after burning, and subsequently, turned opaque within 24 hours. All rabbits showed blepharospasm due to pain on the first 2 days following ulcer formation and the eyes were semi -closed. Corneal edema was most prominent during the first week after inducing ulcers in all groups. Comparison between groups showed that groups A and B, had significantly lower (P<0.05) discharge days than the control group (Fig.2).

Duration of blepharospasm in groups A and B were significantly shorter than the control group (P<0.05); however, there was no significant difference between groups A and B (Fig.3). Corneal vascularization revealed significant differences among groups. Pair wise comparison determined that group B had a significantly lower degree of corneal vascularization than the control group (Fig.4). Statistical analysis showed significantly less stromal inflammation in groups A and B than the control group (P<0.05) (Fig.5, 6 and 7).

Discussion

The lucent structure of the cornea is easily become opaque by many factors such as trauma, chemical burn, contact lens etc. The opacity blocks the light. All mentioned factors result in hypoxia, infections and finally neovascularization on the cornea.^1,4,8 Alkali burn is one of the most dangerous chemical burnt which have poor prognosis and also the most of serious chemical injuries to the anterior segment of the eye.²⁶ Many investigations have been performed to prevent and treat chemical burn complications.³³

Alkaline chemicals cause injuries via two different chemical processes: the saponification of triglycerides found in the cell membranes by the hydroxyl ions, which leads to acute lysis of these cell membranes and the denaturation and hydrolysis of proteins due to the change in PH. The hydrolysis leads to loss of the quaternary, tertiary, and secondary structures of proteins and therefore to loss of function of structural as well as transport proteins and enzymes. Consequences are cell death, reduced mechanical strength, and impaired repair mechanisms. Damage to the corneal epithelial stem cells at the limbus will particularly influence the corneal healing process and therefore the visual outcome.^10,12 Perilimbal whitening is a useful indicator in humans to judge the extent of corneal stem cell damage and indirectly the damage to the underlying ciliary body and trabecular meshwork.³² The alkaline molecules may rapidly penetrate the cornea and enter the anterior chamber. This usually destroys the keratocytes of the affected stroma and damages the corneal endothelial cells.^7,31 Damage to intraocular structures including iris, ciliary body, and lens has been reported in humans.³² A fibrinous uveitis may result from pH-induced cell lysis and release of necrotic debris into the aqueous humor. Acute clinical signs found in our rabbits included blepharospasm, chemosis-ocular discharge and conjunctival hyperemia. Furthermore, there was an extensive destruction of the corneal epithelium exposed to the alkaline substance. Saponification and denaturation of the stroma was visible as fine white opacities, and subsequent mild to moderate corneal edema was present.⁶