Clinical Commentary Calcium Degeneration and Ocular Surface Failure in the Horse D

8 EQUINE VETERINARY EDUCATION / AE / JANUARY 2012

Clinical Commentary Calcium degeneration and ocular surface failure in the horse D. E. Brooks College of Veterinary Medicine, University of Florida, 2015 SW 16 Ave, Gainesville, Florida 32608, USA.

Introduction horse tears is 8.33 Ϯ 0.15 (Ollivier 2004) and tears in man 6.5–7.6 (Abelson et al. 1981). The ocular surface and its associated adnexal structures The precorneal tear film (PTF) lubricates the rough form an integrated functional optical unit of over corneal surface, prevents premature evaporation of the 2000 mm2 in area in the horse (Rauz and Saw 2010). The PTF, provides oxygen and nutrition to the avascular cornea, equine ocular surface consists of a thick mucin dominated enables epithelial cell proliferation, maturation and precorneal tear film, externalised lymph tissue of the movement over the ocular surface and participates in conjunctiva, transitional limbus and refractive cornea. ocular immunological defences. Normal eyelid blinking function of 14 blinks/min in the horse The total thickness of the normal trilayered equine is also a vital component of the equine ocular surface. cornea is 0.8–1.0 mm (Plummer et al. 2003; Brooks and Matthews 2007). The avascular cornea derives nutrition Ocular surface anteriorly from the tear film and internally from the aqueous humour. Disease affecting the aqueous humour The architecture of the precorneal tear film (PTF) is a or tear film can alter corneal metabolism and physiology layered, mucoaqueous gradient gel (Mishima 1965; Smolek (Rauz and Saw 2010). The normal equine corneal and Klyce 2005; Rauz and Saw 2010). The anterior-most layer epithelium consists of 8–10 cell layers and has a thin is the lipid or oily layer derived from secretions of the eyelid basement membrane that attaches the basal cuboidal margin meibomian glands. The thick middle aqueous tear epithelial cells via haemidesmosomes, collagen fibrils film layer is produced by the lacrimal and third eyelid and laminin to the stroma. Microvilli of the superficial glands. The posterior mucin layer of the PTF is derived from apical corneal epithelial cells are coated with a secretions of conjunctival goblet cells. mucopolysaccharide glycocalyx that provides a scaffold Lysozyme, lactoferrin, albumin, immunoglobulins, for the attachment of the precorneal tear film to the epidermal growth factor, transforming growth factors, cornea in order produce an optically smooth corneal interleukin cytokines, proteases, glucose, antioxidants and surface. The thick corneal stroma is composed of a electrolytes such as sodium, chloride and calcium are 3-dimensional mosaic of interconnected keratocytes in an present in human tears (Davson 1990; Rauz and Saw 2010). extracellular matrix (ECM) of proteoglycans and highly The PTF of the horse contains IgA, IgM, IgG and IgT (Martin ordered, small diameter collagen fibrils parallel to the et al. 1997; Schlegel et al. 2003), urea (Zapata et al. 2005), corneal surface (Brooks and Matthews 2007). The inner connective tissue growth factor (Ollivier et al. 2004) and endothelium of the horse cornea is a cellular monolayer various classes of proteases (Strubbe et al. 2000; Ollivier with a thick collagenous basement membrane 2004; Ollivier et al. 2004). Calcium is important to mucin (Descemet’s membrane) (Brooks and Matthews 2007). formation in the goblet cells (Paz et al. 2003). The corneoscleral limbus is the transition zone between The PTF thickness averages ~93 mm in the horse and the cornea and conjunctival epithelium. Limbal stem cells 7 mm in man, tear flow rate is ~34 ml/min (horse) and 1.2 ml/ amplify, proliferate and differentiate into the corneal min (man), tear volume is ~234 ml (horse) and 7 mlinman epithelium and are critical for corneal ulcer healing. Limbal and tear replacement rate is ~7 min (horse) and ~4 min stem cell deficiency following severe corneal inflammation (man) (Mishima 1965; Chen and Ward 2010). The pH of leads to invasion of conjunctival epithelial cells and blood vessels onto the corneal surface to result in a slightly opaque, irregular and unstable ‘corneal’ epithelium prone Corresponding author email: [email protected] to damage following minor injury (Rauz and Saw 2010).

The conjunctiva has a nonkeratinised, vascularised is also reported secondary to topical steroid therapy mucosal epithelium interspersed with mucin-producing (Taravella et al. 1994; Dubielzig et al. 2010). goblet cells and lymphoid follicles and a connective tissue There appear to be 2 primary forms of calcium stroma that extends from the eyelid margin to the associated ocular surface failure in the horse (Rebhun corneo-scleral limbus (Rauz and Saw 2010). Conjunctival 1992; Rebhun et al. 1993; Håkanson and Dubielzig 1994; epithelial cells are continuous with the corneal epithelium Brooks 2002; Matthews 2004; Andrew and Willis 2005). and secrete a glycocalyx of mucopolysaccharides that Calcific band keratopathy (CBK) is a superficial type of coat the conjunctival and corneal surfaces to evenly calcific corneal degeneration. Calcareous degeneration distribute the precorneal tear film. Conjunctiva-associated is a second type of calcific degenerative process of lymphoid tissue is critical for ocular surface defence. The diseased eyes that affects the deeper posterior stroma conjunctival goblet cells produce the hydrophobic inner (Goodfriend and Ching 1995). layer of the tear film, the mucin layer, responsible for precorneal tear film adherence and stability (Rauz and Calcific band keratopathy Saw 2010). Calcific band keratopathy (CBK) is a complication of Ocular surface failure chronic uveitis in the horse and consists of deposition of dystrophic calcium in the superficial corneal epithelium and stroma (Figs 1–5). Calcific band keratopathy refers Failure of the ocular surface is precipitated by trauma, specifically to the deposition of calcium salts at the level of inflammation and infection (Rauz and Saw 2010). Eyelid the epithelial basement membrane and anterior stroma abnormalities, tear film problems and altered corneal that is found in about 6% of horses with chronic equine sensation exacerbate the ocular surface failure. Ocular recurrent uveitis (Brooks 2002). It may also occur in surface failure is most often manifested as corneal ulceration in the horse, but would also include precorneal tear film and conjunctival disease. The core principal for managing ocular surface failure in the horse is promoting and preserving the integrity of the corneal epithelium. The therapeutic approach for ocular surface failure is to eliminate infection, reduce PTF protease activity and suppress iridocyclitis (Rauz and Saw 2010).

Calcium and ocular surface failure

Calcium (Ca2+) plays an important role in the physiology and biochemistry of the normal eye and is closely regulated to maintain specific levels to ensure adequate calcium homeostasis. It is involved in aqueous humour outflow, lens metabolism, retinal phototransduction, neurotransmitter release, transmembrane potential maintenance and contraction of muscles. Many enzymes Fig 1: Calcific band keratopathy in the central cornea is dense and associated with fluorescein staining corneal ulcers. require calcium ions as a cofactor. Calcium also acts as one of the primary regulators of osmotic stress. Recent reports hypothesise that calcium plays an important role in providing cationic shielding to keep negatively charged mucins condensed and tightly packed within conjunctival goblet cells (Paz et al. 2003). Derangement of calcium homeostasis can lead to systemic or local hypercalcaemia or hypocalcaemia, both of which can have important consequences for the health of the ocular surface. Calcium precipitation of the cornea, conjunctiva and sclera occurs in regions of high pH and increased evaporation to cause high local calcium levels (Krachmer and Palay 1995). Calcification of dead or dying tissue and previously undamaged tissue may occur. Calcium deposition in the cornea and conjunctiva may be secondary to inflammation or be associated with a primary Fig 2: Histology of Case 1 found calcium deposited in the dystrophic condition (Peiffer 1983). Corneal calcification subepithelial region (Von Kossa stain).

Fig 3: Miniature horse mare with ERU is being treated with topical Fig 5: Same horse in Figures 3 and 4 two weeks later with near prednisolone acetate. resolution of the CBK.

horses not treated medically. The specific cause of the mineralisation is unknown although local alterations in pH may predispose to calcification. Treatment is topically administered calcium chelaters such as dipotassium ethylene diamine tetra-acetate (EDTA, 0.4–1.5%) to decrease tear film and tissue free calcium levels (Brooks 2002; Matthews 2004; Andrew and Willis 2005). Topical antibiotics, atropine and systemic nonsteroidal anti-inflammatory drugs are also beneficial for ulcers. Superficial keratectomy may be necessary to remove the painful calcium deposits. The healing of keratectomy sites in eyes with CBK can occur with severe scarring (Brooks 2002; Matthews 2004; Andrew and Willis 2005). Recurrence of calcium band keratopathy is possible with continued episodes of uveitis. The prognosis for vision is guarded because of subsequent corneal scarring and further uveitis episodes.

Fig 4: Ulcers in the interpalpebral fissure from calcific band Corneal and conjunctival mineralisation keratopathy are present 3 weeks later in the case from Figure 3. association with corneal ulcers in horses (Brooks 2002; Corneal calcification is found in a variety of ocular Matthews 2004; Andrew and Willis 2005). White bands of conditions in animals. Local mineralisation of superficial calcium of varying density are noted in the interpalpebral corneal ulcers are occasionally seen in the horse region of the central cornea. Small lucent areas may be (Håkanson and Dubielzig 1994) and, in most cases, will present within the lesion, possibly corresponding to the resolve as the lesion heals. Mineralisation of the anterior points of penetration of corneal nerves through the corneal stroma can also occur as an uncommon sequel to basement membrane (Brooks 2002; Matthews 2004; chronic or recurrent keratitis arising from various causes Andrew and Willis 2005). Thicker deposits may elevate the (Rebhun 1992; Håkanson and Dubielzig 1994). Areas of corneal surface, resulting in excoriation of the epithelium stromal mineralisation are generally nonprogressive, dense, and shallow scattered areas of painful, fluorescein retaining semi-refractile opacities and are usually prominently corneal ulcers (Brooks 2002; Matthews 2004; Andrew and vascularised. There is no specific treatment and the cornea Willis 2005). A gritty sensation is often found during scraping may clear spontaneously following resolution of the for corneal cytology. These thicker lesions may provoke their primary problem. Excision by lamellar keratectomy of own inflammatory response (Brooks 2002; Matthews 2004; severe corneal stromal calcification could be attempted Andrew and Willis 2005). Calcific band keratopathy but the probability of post surgical corneal fibrosis and appears to develop in the eyes of horses most aggressively infection presents a serious drawback (Brooks 2002; treated with topical corticosteroids for ERU and is rare in ERU Matthews 2004; Andrew and Willis 2005).

Mineralisation of the conjunctiva is mentioned with Goodfriend, A.N. and Ching, S.S.T. (1995) Corneal and conjunctival conjunctival granulomas in emus with avian tuberculosis degenerations. In: Cornea, Vol. II. Eds: J.H. Krachmer, M.J. Mannis and E.J. Holland, Mosby, St. Louis. pp 1127-1130. (Pocknell et al. 1996) and calcific mineralisation of the third Håkanson, N.E. and Dubielzig, R.R. (1994) Chronic superficial corneal eyelid conjunctiva reported in the kestrel (Kern et al. 1996), erosions with anterior stromal sequestration in three horses. Vet. but appears to be unusual in horses and other animals. The Comp. Ophthalmol. 4, 179-183. conjunctival mineralisation in this horse is also unique in that Kern, T.J., Paul-Murphy, J., Christopher, J., Murphy, C.J., Buyukmihci, N.C., it was associated with a corneal ulcer. Changes in the Burling, K., Miller, P.E., Oppenheim, Y. and Riis, R. (1996) Disorders of the third eyelid in birds: 17 cases. J. Avian Med. Surg. 10, 1218. episcleral microenvironment from the uveitis and drug Krachmer, J.H. and Palay, D.A. (1995) Cornea Color Atlas, Mosby, St. injection undoubtedly caused the mineralisation. Louis. A172 and 176. It has been hypothesised that the use of some Martin, E., Molleda, J.M., Ginel, P.J., Novales, M., Lucena, R. and Lápez, corticosteroids, but particularly steroid-phosphate R. (1997) Total protein and immunoglobulin concentrations in preparations, provide a microenvironment conducive to equine tears. Zentralbl. Veterinarmed. A 44, 461-465. the rapid precipitation of calcium phosphate in the Matthews, A.G. (2004) The cornea. In: Equine Ophthalmology. An Atlas cornea (Taravella et al. 1994). Serum and normal body and Text, 2nd edn., Eds: K.C. Barnett, S.M. Crispin, J.D. Lavach and A.G. Matthews, Saunders, Philadelphia. pp 107-148. fluids such as tears and aqueous humour contain calcium Mishima, S. (1965) Some physiologic aspects of the precorneal tear film. and phosphate concentrations that approach their Arch. Ophthalmol. 73, 233-241. solubility product. The addition of medications containing Ollivier, F.J. (2004) The precorneal tear film in horses: its importance and phosphate may push the equilibrium toward formation of disorders. Vet. Clin. N. Am.: Equine Pract. 20, 301-318. calcium phosphate. It may also be that uveitis alters Ollivier, F.J., Brooks, D.E., Schultz, G.S., Blalock, T.D., Andrew, S.E., corneal metabolism in such a way as to elevate tissue pH. Komaromy, A.M., Cutler, T.J., Lassaline, M.E., Kallberg, M.E. and Van Setten, G.B. (2004) Connective tissue growth factor in tear film of the This elevation of tissue pH then increases the likelihood of horse: detection, identification and origin. Graefes Arch. Clin. Exp. calcium deposition (Taravella et al. 1994), and may have Ophthalmol. 242, 165-171. occurred in the case report by Donaldson et al. (2012). Paz, H.B., Tisdale, A.S., Danjo, Y., Spurr-Michaud, S.J., Argüeso, P. and Alterations in the pH of the ocular surface during Gipson, I.K. (2003) The role of calcium in mucin packaging within ophthalmic disease may promote calcium deposition. goblet cells. Exp. Eye Res. 77, 69-75. Some infectious agents may also alter the ocular surface Peiffer, R.L. (1983) Comparative Ophthalmic Pathology, CC Thomas, Springfield. p 97. pH to induce pathological changes from calcium. This Plummer, C.E., Ramsey, D.T. and Hauptman, J.G. (2003) Assessment of would appear to be an interesting area of research. corneal thickness, intraocular pressure, optical corneal diameter, and axial globe dimensions in Miniature Horses. Am. J. vet. Res. 64, 661-665. Author’s declaration of interests Pocknell, A.M., Miller, B.J., Neufeld, J.L. and Grahn, B.H. (1996) Conjunctival mycobacteriosis in two emus (Dromaius No conflicts of interest have been declared. novaehollandiae). Vet. Pathol. 33, 346-348. 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