- Top of page
- Material and Methods
Following any corneal insult, numerous cellular interactions and alterations occur. Those are mediated by leukocytes, fibroblasts, endothelial cells as well as by combined actions of various substances such as proteinases, growth factors, and cytokines, aiming at corneal regeneration.
Proteinases can be classified as matrix metalloproteinases (MMPs), serine proteinases, aspartic proteinases, and cysteine proteinases.[2, 3] MMPs constitute an enzymatic family responsible for the remodeling and degradation of extracellular matrix and basal membrane components.[4, 5] MMP-2 and MMP-9 are the enzymes of major importance in the remodeling and degradation of the corneal stromal collagen in rats, humans,[7, 8] mice, rabbits, dogs,[11, 12] and horses.[13, 14]
Matrix metalloproteinase-2 is mainly produced by stromal keratocytes and epithelial cells and is found in its inactive form on the intact corneal epithelium and stroma. It performs a surveillance function, becoming locally activated to degrade collagen molecules that occasionally become damaged as a result of normal aging.[6, 14-16] Matrix metalloproteinase-9 is produced by corneal epithelial cells and keratinocytes, as well as by inflammatory cells following corneal wounding.[1, 6, 14, 16-18] This MMP plays an important role as it is able to destroy the adhesive structure, such as collagens, laminin, and proteoglycans of the epithelial basement membrane.[7, 16]
The activity of MMPs is tightly coordinated by tissue inhibitors of matrix metalloproteinases (TIMPs); an imbalance between MMPs and TIMPs in favor of proteinases may cause excessive degradation of normal healthy tissue impairing wound healing and leading to corneal melting.[19-22] Thus, the agents capable of inhibiting MMPs, such as N-acetylcysteine, ethylenediaminetetraacetic acid (EDTA), tetracyclines, and blood serum are recommended for topical use in cases of ulcerative keratitis.[2, 3, 21, 22]
EDTA, tetracyclines, and N-acetylcysteine chelate calcium and zinc, essential minerals for the proteinases activity.[22-24] By calcium chelation, EDTA interferes with the stability of MMPs and decreases the stimulation of the migration of polymorphonuclear cells to the corneal wound.[22, 24]
N-acetylcysteine is an MMP inhibitor commonly used in humans as well as veterinary ophthalmology.[23-26] In one study performed with tears of horses with ulcerative keratitis, 10% N-acetylcysteine decreased proteolytic activity by 98.9%. Results of studies performed in living animals with this agent are based only on direct observation of corneal re-epithelization and are described as being beneficial or detrimental to the healing process in accordance with the concentration used.[27, 28]
Glycosaminoglycans such as sodium hyaluronate and chondroitin sulfate have been used for medical treatment of ulcerative keratitis.[29-33] Their antiproteolytic properties are observed mainly in cartilage and synovial fluid in humans, and it is reported that the chondroitin sulfate is able to inhibit proteolytic enzymes, such as collagenases and elastases, as well as to protect cellular membranes from the action of free radicals.
To date, only one in vitro experiment tested the effects of commonly used substances in veterinary ophthalmology able to decrease the MMPs activity. In another study, several agents were evaluated to determine the gelatinase activity present in the tear film of normal dogs. To the author's knowledge, there are no studies performed in living animals with ulcerative keratitis, in which the effects of commonly used substances to inhibit MMPs were evaluated. Therefore, this study aimed to compare the effects of 1% EDTA, 3% chondroitin sulfate, and 10% N-acetylcysteine in alkali-burned rat corneas and to evaluate the effects of this treatment on the re-epithelization time, the expression of MMP-2 and MMP-9, and the density of corneal microvilli.
- Top of page
- Material and Methods
The benefits of EDTA in inhibiting MMPs have been previously reported.[3, 22, 26, 32] However, this substance commonly used as a preservative and corneal penetrating enhancer in ophthalmic solutions induces oxidative DNA damage in the corneal cells at high concentrations.[37, 38] The high concentration used herein (1%) may have caused the significant larger ulcerated area observed at the 6-h time point following corneal injury, in association with a higher area under the curve in the EDTA treatment group. Although concentrations at 0.05–0.2% have been recommended in the veterinary literature, 1% EDTA was chosen to be studied based on reports of its potent antiproteinase action in dogs.
Topical application of 10% N-acetylcysteine every 1–4 h has been recommended to treat corneal ulcers of dogs and horses. In this study, 10% N-acetylcysteine was not able to accelerate corneal re-epithelization and enhanced the diameter of the ulcerated area 6 h after corneal wounding. At concentrations of 10 and 20%, N-acetylcysteine did not accelerate corneal re-epithelization in rats and in dogs. However, ulcerated corneas of dogs and rabbits treated with 3% N-acetylcysteine re-epithelized faster than controls. Thermes et al. reported that at concentration of 20%, N-acetylcysteine promoted focal necrosis of corneal and conjunctival epithelium cells of rabbits. Ramaesh et al. demonstrated in vitro that human corneal cell migration is inhibited by N-acetylcysteine and that such inhibition is concentration-dependent. It has been postulated that lysis of disulfide linkages between mucopolysaccharides and tissue proteins by acetylcysteine is responsible for the apparent tissue destruction. A possible alternative explanation is that the hyperosmotic solution draws fluid into the corneal stroma causing mechanical damage. The concentration of 10% used herein may have impaired corneal migration at early stages of healing.
Similarly to the other tested substances, 3% chondroitin sulfate did not show any benefit on corneal re-epithelization. Similar results were reported in corneal cultures of rabbits, as well as in dogs with corneal ulcers treated with commercially ophthalmic solutions of chondroitin sulfate associated with antibiotics. One experiment demonstrated that polysulfated glycosaminoglycan causes a concentration-dependent effect on morphologic characteristics and migration of canine corneal epithelial cells in tissue culture.
In this experiment, latent and active forms of MMP-2 were found in healthy and ulcerated rat corneas, while MMP-9 was only found in ulcerated corneas in its inactive form. In rabbits, the latent form of MMP-9 was only activated 3 days following keratectomy. Shi et al. have demonstrated by immunohistochemistry that MMP-9 can be found in the corneal epithelium and stromal cells of rats 24 h following alkali injury and that such expression increases seven days later in parallel with corneal neovascularization. However, once antibodies used in that study for MMP-2 and MMP-9 were able to recognize both latent and active forms of such enzymes, it is not possible to make comparisons with our findings in regard of activation of MMP-9.
In vitro studies reported the efficacy of 10% N-acetylcysteine on reducing the proteolytic activity by up to 98% in equine tears affected by keratomalacia. However, 10% N-acetylcysteine did not reduce enzyme activity in the tears of healthy dogs after 48 h of treatment. In our study, when compared with saline group, the levels of the latent form of MMP-2, and MMP-9 were significantly increased in corneas treated with 10% N-acetylcysteine by the end of the experiment. The aforementioned information in regard to the corneal toxicity of the 10% N-acetylcysteine may explain the elevated levels of MMPs found in our study.[41-43]
Forty-two hours after corneal injury, significantly higher levels of the active form of MMP-2 was only observed in 3% chondroitin sulfate group. Based on the results of the present research, it can be postulated that the solution containing chondroitin sulfate might have acted as a substrate to the MMP-2, elevating the quantitative of this enzyme at the end of the experiment. Actually, it has been demonstrated in melanoma cell culture that the latent form of MMP-2 directly binds to chondroitin sulfate through the C-domain, facilitating the generation of the active form of MMP-2. However, it has been demonstrated that MMP-2 participation in stromal remodeling may not depend on coexisting epithelial repair.
In vitro, EDTA was able to reduce the gelatinolytic activity in the tears of horses with keratomalacia by up to 99.4% and by up to 68% in the tears of healthy dogs following 48 h of treatment. After re-epithelization has been completed, 1% EDTA was able to significantly reduce latent forms of MMP-2 and MMP-9, when compared with the eyes that receive chondroitin and N-acetylcysteine. Nevertheless, such reduction was not significant, when compared with the saline group.
The evaluation of corneal microvilli density by scanning electron microscopy has been shown to be a value method in assessing the corneal surface integrity after alkali injury and experimentally induced dry eye in rabbits.
Our experiment failed to show benefits of any tested agent in decreasing the levels of MMP-2 and MMP-9 after corneal alkali injury in rats. Despite the possible toxic corneal effects of EDTA[41-43] and N-acetylcysteine[37, 38] have been previously discussed herein, they may have acted only in acute phases of the corneal healing, once the corneal epithelium surface tended to appear restored at the end of the study.