MELTING CORNEAL ULCERS IN HORSES: DIAGNOSIS AND TREATMENT METHODS

Key words

keratomalacia, melting cornea, stroma, proteinase, horse

Most corneal ulcers heal very quickly with the appropriate treatment, but some can be complicated by corneal melting or keratomalacia. Melting ulcers can be very severe, progress rapidly and are potentially sightthreatening, therefore, they should be identified and managed promptly.

The cornea is traditionally divided into four layers. From the outside to the inside, these layers are the epithelium, stroma, Descemet’s membrane and endothelium. The thickest corneal layer is the stroma, which is composed of a three-dimensional mosaic of interconnected keratocytes in an extracellular matrix of proteoglycans and collagen fibres¹.

During normal corneal healing following injury, proteinases are produced that aid in removal of debris and remodelling of the corneal stroma². These proteinases are produced by microorganisms, inflammatory cells, corneal epithelial cells and fibroblasts, and can be divided in two main groups – matrix metalloproteinases (MMPs, such as MMP-2 and MMP-9) and serine proteinases (such as neutrophil elastase, NE).

The cornea contains naturally occurring inhibitors of MMPs and serine proteinases – corneal destruction occurs when there is an imbalance between proteinases and their inhibitors²,³.

Diagnosis

In early stages, melting ulcers may look very innocuous. Later, they tend to appear as a grey, mucoid or gelatinous (liquefaction) opacity (^{Figure 1}). A complete eye examination must be carried out, including staining with fluorescein to detect corneal epithelial defects, tear film deficiencies or leakage from the anterior chamber (the Seidel test).

Cytologic examination and microbiologic cultures of melting ulcers are imperative². Samples for cytology can be obtained with a previously soaked microbiology swab, cytology brushes or the blunt end of the scalpel blade. The sample is then rolled onto a glass slide. Common stains include Gram and DiffQuik.

Although results of the culture are often more sensitive than cytology, examination of Gram-stained slides can give immediate information about the causative agent, allowing prompt, more focused therapy².

Cultures should be obtained before application of any topical solution – such as fluorescein or local anaesthetics – as they contain preservatives that may impair culture results.

Medical therapy for melting ulcers

• Appropriate antimicrobial therapy

Aggressive antimicrobial therapy should be immediately implemented, ideally based on the results of cytology and culture. Gram staining will allow a rapid, more adequate selection of antimicrobial while waiting for the results of culture and sensitivity. If this is not possible, the choice can be made empirically, based on the normal components of the conjunctival flora in horses⁴.

Although uncommon in the UK, fungal keratitis can also occur and detection of fungal elements on cytology and/or culture should prompt rapid antifungal treatment.

Further discussion on antimicrobial choice is beyond the scope of this article and the reader is directed to the excellent review by Matthews (2009)⁵.

Control of uveitis

Uveitis is a common consequence of corneal disease and should be appropriately managed to avoid damage to the internal structures of the globe. Symptomatic treatment is achieved with the topical use of cycloplegics (atropine one per cent) and systemic anti-inflammatories.

Flunixin meglumine is anecdotally believed to be the most effective systemic NSAID and analgesic for ocular conditions, but its long-term use can delay the development of corneal vascularisation, which is important for corneal healing. One should aim to change to a different compound, such as phenylbutazone, as soon as the eye is comfortable enough, or flunixin and phenylbutazone can be used on alternate days.

A study found the concentration of firocoxib in the aqueous humour of horses is superior to that of flunixin⁶ and, therefore, may provide another option for the treatment of uveitis. However, to the author’s knowledge, firocoxib is only licensed in the UK for the treatment of musculoskeletal conditions in horses.

Topical NSAIDs, such as diclofenac and flurbiprofen, can be used in cases of uveitis, secondary to corneal ulceration. However, their application can cause some degree of ocular discomfort, delayed corneal neovascularisation and their efficacy is questionable. Again, more detailed treatment of uveitis is beyond the scope of this article.

• Inhibition of proteinases

Proteinase inhibitors have been recommended for treatment of corneal ulcers to reduce the progression of the stromal melting, accelerate epithelial healing and minimise corneal scarring¹,³.

Topical 0.2 per cent ethylenediamine tetra-acetic acid (EDTA) has been shown to cause a 99.4 per cent reduction in proteinase activity in the ocular surface³,⁷,⁸. EDTA is a calcium and zinc chelator that inhibits MMP activity and can be easily prepared by adding sterile water to a commercial blood collection tube.

Autologous serum can inhibit both MMPs and serine proteases, due to the presence of α1-antitrypsin and α2-macroglobulins³,⁷,⁹. Serum has been shown to decrease proteinase activity by 90 per cent. A long clotting time (120 minutes), a sharp centrifugation (3,000g for 15 minutes) and dilution with balanced salt solution improve the ability of serum eye drops to support the metabolism of corneal epithelial cells⁸.

Serum contamination is unlikely, but it should, nevertheless, be closely monitored for changes in its turbidity that may indicate contamination. Plasma can also be used topically, but it lacks the beneficial platelet-derived growth factors found in serum.

N-acetylcysteine is another MMP inhibitor, with an efficacy of 98.9 per cent³,⁷. However, its use should be minimised because it can disturb the stability of the tear film.

Tetracyclines (doxycycline and oxytetracycline) also have an inhibitory effect on the MMPs on the ocular surface. Doxycycline was shown to reduce proteinase activity by 98.8 per cent³. Unfortunately, the availability of a topical ophthalmic preparation containing doxycycline is limited, however, a study by Baker et al (2008)¹⁰ showed detectable levels of doxycycline on the preocular tear film, following oral administration.

Topically applied tetanus antitoxin (120units/ml) has also shown some anti-proteinase activity, compared in efficacy to serum and N-acetylcysteine¹¹.

Because these compounds use different mechanisms to inhibit various families of proteinases, combining several protease inhibitors (for example, alternating administration of serum with EDTA) may be beneficial³,⁷.

Horses with corneal ulcers are often in pain and administration of topical treatment is usually difficult. In addition, keratomalacia cases require very frequent medication. Placement of a subpalpebral lavage system is therefore recommended (^{Figure 2}).

Various techniques and systems have been described. The author likes to use 8FR silicone tubing with a single hole and footplate. The subpalpebral lavage system can be placed in the superior or the ventral palpebral fornix.

Higher concentration of medication into the cornea and anterior chamber is achieved when the system is placed in the superior fornix⁸, although the rate of complications is considered lower with a ventral placement.

In severe cases, a constant delivery device can be attached to the subpalpebral lavage system, to provide constant administration of medication to the corneal surface.

Surgical therapy for melting ulcers

• Keratectomy

Removing necrotic infected tissue by keratectomy speeds healing, encourages neovascularisation, minimises scarring and reduces the stimulus for uveitis¹,²,⁸. This can be achieved with a cellulose swab, Colibri forceps and microsurgical corneal scissors or corneal dissector.

• Conjunctival autografts

Conjunctival autografts or flaps are frequently used in equine ophthalmology for the treatment of deep, large or melting ulcers²,⁸,¹². The ulcer should be stabilised with medical therapy, if possible, before the placement of the flap, to prevent digestion of the sutures.

The conjunctival graft is best mobilised from the bulbar conjunctiva and then can be attached on to the cornea with absorbable 5/0 to 7/0 sutures. Two additional tension-relieving sutures are placed at the base of the graft. These grafts provide structural strength to the damaged cornea and, in addition, offer significant antibacterial, antifungal, antiviral, antiprotease and anticollagenase effects²,⁸,¹².

There are several types of conjunctival grafts. The rotational conjunctival pedicle graft is the most commonly used and is best harvested from the dorsal or temporal quadrants. The pedicle graft should be oriented vertically on the cornea, so as to minimise eyelid trauma to the corneal sutures and conjunctival graft¹².

The bridge pedicle graft is similar to the pedicle graft, but the conjunctiva is attached to both ends rather than just one. The hood or 180° graft can be used for peripheral lesions and the complete or 360° graft can be used when the whole cornea is affected¹².

• Amniotic membrane graft

Equine amniotic membrane has been used in equine ophthalmology for the treatment of melting ulcers. Amnion consists of an epithelium, a thick basement membrane and an avascular stroma.

Amniotic membrane has the ability to facilitate the migration and differentiation of the epithelial cells and the reinforcement of cellular adhesion. It also has the ability to modulate stromal scarring and decrease ocular surface inflammation, as well as having antiangiogenic and antimicrobial effects. It contains growth factors and several proteinase inhibitors¹³,¹⁴,¹⁵.

Prior to its use, the placenta has to be harvested during a caesarean section. The amnion is then separated from the allantois, placed on a nitrocellulose paper and stored frozen in Dulbecco’s medium – also containing antimicrobials. Most of the epithelium is lost in this process, so the graft material consists only of stroma and a basement membrane.

When it is required, the amnion is thawed and rinsed with sterile saline, cut to size and applied to the corneal surface. If the graft is placed over the cornea with the stromal side down (inlay or graft technique), the amnion will adhere and be incorporated to the corneal stroma.

If it is placed with the basement membrane facing the cornea (overlay or patch technique), epithelial cells will migrate along its surface and thus the amnion can be used as a bandage², with the amnion expected to slough in seven to 10 days.

A third technique (filling in or layering), which consists of placing multiple pieces of amnion trimmed to size and placed within the depth of the ulcer crater, is also described. In any case, the amniotic membrane is sutured to the cornea, with interrupted sutures of 5/0 to 7/0 absorbable material (^{Figure 3}).

Summary

Corneal melting or keratomalacia is a relatively common complication following corneal ulceration in horses, as a result of imbalances between proteinases and their inhibitors.

Rapid, aggressive medical or surgical treatment (or a combination of both) is often required to stop the progression of the disease, enhance the repair process and minimise scarring.

• Please note drugs mentioned within this article are appropriate to use under the cascade.

References

1. Brooks D E and Matthews A G (2007). Equine ophthalmology. In Gelatt K N (ed), Veterinary Ophthalmology, Blackwell, Ames, IA. 2. Ollivier F J (2005). Medical and surgical management of melting corneal ulcers exhibiting hyperproteinase activity in the horse, Clinical Techniques in Equine Practice 4(1): 50-71. 3. Ollivier F J et al (2007). Proteinases of the cornea and preocular tear film, Veterinary Ophthalmology 10(4): 199-206. 4. Johns I C et al (2011). Conjunctival bacterial and fungal flora in healthy horses in the UK, Veterinary Ophthalmology 14(3): 195-199. 5. Matthews A G (2009). Ophthalmic antimicrobial therapy in the horse, Equine Veterinary Education 21(5): 271-280. 6. Hilton H G et al (2011). Distribution of flunixin meglumine and firo- Figure 3. Amnion membrane sutured to the corneal surface. coxib into aqueous humor of horses, J Vet Intern Med 25(5):1,127-1,133. 7. Ollivier F J et al (2003). Evaluation of various compounds to inhibit activity of matrix metalloproteinases in the tear film of horses with ulcerative keratitis, American Journal of Veterinary Research 64(9): 1,081-1,087. 8. Brooks D E (2010). Catastrophic ocular surface failure in the horse. In Annual Convention of the AAEP, Baltimore. 9. Brooks D E (2004). Inflammatory stromal keratopathies: medical management of stromal keratomalacia, stromal abscesses, eosinophilic keratitis, and band keratopathy in the horse, Veterinary Clinics of North America: Equine Practice 20(2): 345-360. 10. Baker A et al (2008). Doxycycline levels in preocular tear film of horses following oral administration, Veterinary Ophthalmology 11(6): 381-385. 11. Haffner J C, Fecteau K A and Eiler H (2003). Inhibition of collagenase breakdown of equine corneas by tetanus antitoxin, equine serum and acetylcysteine, Veterinary Ophthalmology 6(1): 67-72. 12. Denis H M (2004). Equine corneal surgery and transplantation, Veterinary Clinics of North America: Equine Practice 20(2): 361-380. 13. Ollivier F J et al (2006). Amniotic membrane transplantation for corneal surface reconstruction after excision of corneolimbal squamous cell carcinomas in nine horses, Veterinary Ophthalmology 9(6): 404-413. 14. Plummer C E (2009). The use of amniotic membrane transplantation for ocular surface reconstruction: a review and series of 58 equine clinical cases (2002-2008), Veterinary Ophthalmology 12(Suppl 1): 17-24. 15. Lassaline M E et al (2005). Equine amniotic membrane transplantation for corneal ulceration and keratomalacia in three horses, Veterinary Ophthalmology 8(5): 311-317.