TECHNIQUES AND UPDATES



Techniques AND Updates

John Mould

Eye Veterinary Clinic

Leominster HR6 0PH

The Direct Ophthalmoscope

Standard features

On/off switch This incorporates a rheostat allowing the light intensity to be varied. With rechargeable units the intensity may be over-bright for the patient.

Beam selector This provides a choice of beam types for different applications. The following are the most useful:

← Small diameter beam is used for small diameter pupils but consequently gives a narrower field of view than the larger beam. Try to avoid.

← Large diameter beam is the routine beam for use with a pupil of adequate size.

← Cobalt blue light can be useful for observing fluorescein on the cornea.

← Slit beam may be useful as a simple slit lamp substitute

Lens magazine Instruments contain a magazine of interchangeable lenses mounted on a disc. These start at 0 (i.e. no lens) and then move in plus dioptres by moving the wheel clockwise and minus dioptres by moving the wheel anti-clockwise. Initially they move in 1 diopter intervals but after about ±8 dioptres the intervals usually widen. An illuminated window indicates which lens is in the viewing path.

The lens magazine is important and serves 3 purposes:

1. It allows the examiner to compensate for the refractive error in his/her eye. Myopic (short-sighted) examiners require minus lenses and hypermetropic (long-sighted) examiners require plus lenses according to their prescription. If this is not known it can be established by trial and error. Contact lens wearers and clinicians with normal refraction need not be concerned with this.

2. It allows the examiner to compensate for the refractive error in the patient's eye. This is important in man but animal eyes are usually near normal.

3. It allows the examiner to focus on structures at different depths in the eye. Progressively plus lenses are needed to focus through to the anterior eye. Typical values would be 0 dioptres (1-2 for the fundus up to 20 dioptres for the anterior and surface structures.

Some ophthalmoscopes have a rapid lens change switch so that the examiner can change the level of focus rapidly (usually from the plane of the retina to the anterior segment) without going through the lenses in the magazine one by one. This is a convenience but make absolutely sure this switch is not wrongly set or a large focussing error will be present.

It pays to become fully familiar with the instrument before use. Spectacle wearers must remove their spectacles to use a direct ophthalmoscope especially with horses. This brings the instrument closer to the eye enlarging the field and the spectacles will not be damaged if the animal moves suddenly. The necessary dioptre correction should be established, by trial and error if necessary. When you pick up the instrument, check the lens setting first and set it to your own requirement.

There are essentially three ways in which you can use a direct ophthalmoscope:

1. From a distance at arm’s length. This retro-illuminates the media by light reflected from the tapetum and is a good way to identify opacities. It is much easier to do and to demonstrate than to describe. Make this part of your technique.

2. From close to the patient to examine the fundus and intraocular structures.

In most species the two golden rules are:

“close to your eye – close to the patient’s eye” (to maximise the field of view)

“left eye for the left eye and right eye for the left eye” (for best access)

Neither of these is applicable to the horse. Contact with the long vibrissae around the eye will lead to excessive blinking and so a distance of 7-10cm is usual in the horse although the ophthalmoscope should still be touching your own orbital margin. Because of the flat sides to the equine head access is not a problem and the same eye can be used to examine both sides.

3. As a simple magnification device with illumination for the ocular surface. The magnification achievable, however, is low (much lower than with the fundus) and inferior to a slitlamp.

There is little tradition in equine practice of dilating pupils for fundus examination and it is not a requirement of pre-purchase examination. It does, however, make fundus examination easier and enables the examiner to cover a larger area. Most clinicians would be surprised how small a proportion of the fundus they cover with a direct ophthalmoscope. Use tropicamide eye drops only for examination and never atropine.

Although the basic design does not change the modern instruments are lighter, have good illumination and optics and are well made. Do not persist with a museum piece.

It is essential that you can use a direct ophthalmoscope with confidence.

Do not hesitate to seek help in the practical sessions.

Indirect ophthalmoscopy

This is underrated and underused in equine work but enables a wider field of view to be surveyed than with a direct ophthalmoscope. Headset units are expensive and not necessary for the non-specialist as it can be performed with an appropriate lens and a pen torch. 20D acrylic lenses are adequate for a start and can be purchased for approx. £40. Glass lenses come in a range of dioptres and cost around £120. Lenses will be available for use in the practical sessions.

The technique will be demonstrated but the most important points are:

• The working distance must be correct. This varies between lenses but at the right distance the image should fill the lens from side to side.

• A pupil of adequate size is essential.

• The lens must be parallel to the iris and the centre of the lens must be on the same axis as the pupil.

There is a knack to all this but it is not more difficult to master than a direct ophthalmoscope and arguably easier.

Lavage apparatus

There may be problems with medication of a painful eye in the horse. Eyelid closure is very powerful. More importantly the horse may become resentful of frequent handling and a vicious circle of inadequate treatment and deteriorating behaviour can result. Few medications are actually irritant and so this is really a handling problem. A lavage apparatus can solve the problem by delivering the medication locally but administering it at a distance.

The possible routes are upper or lower lid fornix or delivery retrogradely up the nasolacrimal duct. The other choice is between home-made and commercial kits. It sounds logical to deliver the medication to the upper lid so that gravity allows it to run over the eye. In effect the site of delivery is unimportant since the action of the lids distributes the fluid. Personal preference may play some part but many now use the lower lid fornix as the site of choice with commercial kits and complication rates have been shown to be lower. The footplate is placed between the lower lid and the third eyelid. It cannot migrate towards the eye under gravity and cannot irritate the cornea since it is between two conjunctival surfaces.

Under sedation plus topical and infiltrated anaesthesia the trochar is used to place the tubing which is then sutured to the skin and taken back to the withers area. The technique of placement will be illustrated in the lecture and available for practice in a cadaver station. With grateful thanks to Brian Patterson MRCVS. The procedure illustrated is by Brian as are some of the illustrative slides used.

Equine Ophthalmology books

Every equine practice should own an ophthalmology textbook. These three books will be illustrated and evaluated.

Brooks D. E. (2002) Ophthalmology for the Equine Practitioner Teton NewMedia, Jackson, Wyoming $55

Barnett K. C., Crispin S. M., Lavach J. D. and Matthews A. G. (2004) Equine Ophthalmology An Atlas and Text (2nd. Ed.) Saunders, London £85

Gilger B. C. ed. (2005) Equine Ophthalmology Elsevier Saunders, St. Louis, Missouri $145/£85

Intraocular melanoma

Melanoma is a tumour of melanocytes, the pigmented cells which give rise to the colour of the skin and parts of the eye. It is an important skin tumour of man and of the horse where there is a marked predisposition in old grey horses. Melanoma is also the most common intraocular tumour in man, the dog, the cat and the horse. They are said to be rare in the horse and few descriptions have been published but this may be a false picture.

A wide mix of breeds and ages may be represented but the great majority of cases occur in greys. The most common clinical sign is a visible mass but in a minority of cases the horse may also be presented because of secondary signs including glaucoma, uveitis and corneal opacity. Pigmentary proliferations may not be readily noticed in dark eyes. Tumours may occur in the iris and/or ciliary body and less commonly in the choroid. In marked contrast to other species tumours may be found at two separate locations in the same eye and in both eyes simultaneously.

Equine uveal melanomas tend to be heavily pigmented and with well defined margins. Invasion beyond the uvea and ring spread are rare or unknown. Tumours consist predominantly of a mixture of spindle cells and large heavily pigmented round or polygonal cells. Less well differentiated cells may be seen but uncommonly. Melanophages comprise a large proportion of the tumour. Mitoses are very uncommon. In general, therefore, the appearance is benign in most cases and the metastatic risk is considered low.

Management

Possible treatment modalities include local excision, laser and enucleation. In view of the benign pathology and minimal secondary change, however, it is arguable that some of these tumours could be managed by serial observation initially.

summary

← All parts of the uvea may give rise to melanomas in the horse.

← Bilateral cases and multiple tumours in the same eye may occur.

← Most appear as a visible pigmented mass.

← Grey horses are heavily over-represented.

← A high proportion of phagocytes, multifocal distribution and a predisposition in grey horses are all features of cutaneous melanoma in the horse.

← Most of these tumours were well defined, relatively benign masses and are interpreted as being unlikely to lead to metastasis. Radical surgery may not be justified in many cases.

← The true incidence of ocular melanoma is unknown.

←

Exotic? Rare? Emerging? – Some UK examples

There are various diseases which were once regarded as exotic (or at least more common outside the UK) and/or uncommon in the horse in general but which are now increasingly recognised in this country. Full details are given in Dr Brooks’ notes and lectures and in the textbooks so only brief summaries are given here but the main purpose of this section will be to illustrate these problems with examples from the UK.

Glaucoma

Glaucoma is not often diagnosed by non-specialists. The reasons may be:

• Low index of suspicion (beware Appaloosas, but not exclusively)

• Loss of vision slow and insidious

• Clinical signs which are not dramatic (corneal and optic disc changes)

• Lack of facilities for tonometry

• Equine eyes more readily become phthisical than glaucomatous after severe disease

“The horse’s eye seems to tolerate elevations in IOP for months to years that would blind a dog: however, blindness is always the end result”. Brooks, 2001.

There are two generalisations which apply to nearly all glaucomas:

• The problem does not go away (without surgery)

• Vision that is lost is lost for ever

The newer anti-glaucoma drugs fall into three categories:

• Prostaglandin analogues (Xalatan, Travatan, Lumigan)

• Topical carbonic anhydrase inhibitors (Trusopt, Azopt)

• Beta-blockers (Timolol)

Not surprisingly perhaps, horses are said not to tolerate prostaglandins because of the intense miosis (constriction of the pupil) mimicking one of the complex of signs seen in uveitis. The other two classes, however, are effective and CoSopt, which combines a carbonic anhydrase inhibitor and a beta-blocker in the same bottle, is probably the preparation of choice. Atropine is contraindicated in glaucoma in most species but was regarded at one time as useful in the horse. This has now been revised and atropine is not recommended. Laser is an additional option.

Deep stromal abscess

Even minor trauma can inoculate the cornea with pathogens. If these were then to form an intrastromal abscess you would expect the epithelium to heal poorly over it and/or the abscess to break out externally. That is what happens in most species and hence stromal abscesses are rare in small animals. Deep stromal abscess (DSA) can certainly occur in the horse, however, and is potentially serious because:

• Confirmation of the pathogens involved (bacteria vs fungi) can be difficult

• Fungi > bacteria according to the literature

• Penetration of medication is poor

• Severe uveitis can accompany the cornea problem especially if the abscess ruptures internally

• Advanced microsurgery is usually required

Fungal keratitis/keratomycosis

• May begin with a minor disruption to the surface, Rose Bengal +ve rather than fluorescein

• Specific diagnosis (vs bacteria) important in therapy – use smears and culture readily

• Antibiotics and steroids likely to promote fungal infection

• In established cases the density of organisms in the tissue is very high

• Vascularisation may be slow

• Fungi readily invade deeply into the cornea (and then into the eye)

• Anterior uveitis common

• Usually a surgical problem - excision of the affected tissue and reconstruction

Advances in diagnosis & treatment of corneal disease

Dennis E Brooks DVM PhD Diplomate ACVO

Professor of Ophthalmology

University of Florida

Gainesville, Florida USA

dbrooks@ufl.edu

Equine Corneal Microanatomy/Physiology

The cornea and precorneal tear film combine to function as a strong refractive lens. The precorneal tear film (PTF) is a lipid-bilayered, aqueous-mucin dominated gel that aids lubrication, transfers oxygen to the cornea, and smoothes out small surface irregularities in the anterior corneal epithelium in order to maintain a uniform optical surface. The tear film also removes exfoliating corneal epithelial cells and provides a means for inflammatory cells to reach the cornea. Tear film proteins serve to control infectious agents, are species specific, and include albumin, IgA, IgG, IgD and IgE, plasminogen activator, complement, interferon, prostaglandins, the antimicrobial proteins lysozyme, ß-lysin and lactoferrin, and the tear film proteinases.

In order to produce such an optically powerful structure, the corneal microanatomy consists of an epithelium and thin epithelial basement membrane, a thick relatively acellular stroma, Descemet’s membrane, and a monolayered endothelium. The total thickness of the normal equine cornea is 0.8 to1.0 mm. The thickness of the sclera at the horse equator is 0.53 ± 0.01 microns. The normal equine corneal epithelium consists of 8-10 cell layers and is ~60 µm thick. It has a thin PAS positive basement membrane that attaches the basal epithelial cells, via hemidesmosomes, collagen fibrils and laminin, to the stroma. The thickest corneal layer (~700 µm thick in the horse), the stroma, is composed of a three-dimensional mosaic of interconnected keratocytes in an extracellular matrix (ECM) of proteoglycans, and small diameter collagen fibrils. These fibrils are combined into highly ordered, sheet-like lamellae that lie essentially parallel to the corneal surface. The horse stroma has regional biochemical differences of the proteoglycans chondroitin-6-sulfate (C6S) and chondroitin-4-sulfate (C4S) with higher levels of C6S in the superficial and superficial central layers, and more C4S in the deeper areas and deeper peripheral layers. The C6S has more water holding capacity than the C4S causing corneal edema to become more prominent in the anterior stroma of the horse under corneal disease conditions. The endothelium (7 µm) of the horse cornea is a cellular monolayer with a thick collagenous basement membrane (Descemet’s membrane; 14-21 µm thick). The PAS positive Descemet’s membrane of the horse is quite elastic and the thickness increases with age. The density of horse endothelial cells is ~3,155 cells/mm2. The mitotic potential of the horse corneal endothelium is probably limited as the number of equine endothelial cells decreases with age.

The normal equine corneal epithelium increases to 10 to 15 cell layers with hypertrophy of the basal epithelial cells after corneal trephination injury. Epithelial cells migrate at a rate of 0.6 to 1.2 mm/day in noninfected horse corneas. Healing of superficial, noninfected corneal ulcers in horses is generally rapid and linear for 5-7 days, and then slows. The equine epithelial basement membrane does not completely adhere to the stroma for six weeks despite the epithelium completely covering the ulcer site. Corneal vascularization occurs at a rate of ~ 1mm/day in the horse. Neutrophils migrate in the cornea at ~ 8mm/day.

Corneal Healing-General Concepts

Healing of corneal is a complicated process that depends on the centripetal migration of healthy corneal and conjunctival epithelial cells to cover the ulcer site.

Corneal ulcer healing involves transformation of fibroblasts; intercellular signaling between epithelial and stromal cells; action of matrix metalloproteases; and protection of tissue from free radical damage.

Polymorphonuclear leucocytes (PMN) are the first cells to migrate into the injured cornea where they release cytokines, growth factors and proteinases to initiate the cellular interactions necessary to heal the corneal wound. Early in the repair process, fibronectin and fibrin are deposited on the epithelial basement membrane and stroma, and then disappear as the epithelial cells cover the defect. Fibronectin is a plasma glycoprotein that functions as an attachment factor and promoter of epithelial cell migration. Excessive numbers of PMNs are associated with catastrophic tear and corneal proteinase levels. Uninjured epithelial cells at the margin of the wound loosen their intercellular and basal attachments and begin to migrate en masse to cover the ulcerated region. The extent and speed of corneal epithelial migration is controlled at least in part by contact inhibition of migrating corneal cells. The new epithelium in healing ulcers will be much thinner than normal until mitotic division reestablishes the normal corneal thickness. Re-formation of epithelial basement membrane will not be complete for several weeks after injury.

Corneal injuries involving the anterior third of the stroma are initially covered by proliferating epithelium. Exposure of the relatively dehydrated corneal stroma to tears will cause swelling of the corneal stroma. As the stroma heals, new collagen is produced by keratocytes or transformed monocytes. The new collagen is different from the native collagen in size and in orientation and causes the healed wound to be clinically opaque. In ulcers with stromal involvement, the epithelium is hyperplastic and the corneal stroma thinned resulting in a stromal scar characterized by an increased number and haphazard arrangement of keratocytes.

Full-thickness ulcers of the cornea are associated with retraction of Descemet's membrane and separation of the posterior aspect of the wound. Fibrin filled aqueous humor may help seal the wound posteriorly until healthy surrounding endothelial cells can spread and migrate into the injured area. Migrated endothelial cells will produce new Descemet's membrane. The portions of Descemet's membrane displaced into the stroma are not resorbed, but remain as the histologic marker of the site of injury. The anterior cellular surface in full thickness ulcers is repaired by the migration and mitosis of epithelial cells as for more superficial corneal wounds.

Tear film Proteinases-General Concepts and Applicability to Horse Ulcers

Tear film and corneal proteinases provide a mechanism for the detection, repair and removal of damaged corneal epithelial cells, altered corneal stromal collagen and abnormal components of the extracellular matrix (ECM) caused by normal wear and tear of the cornea. The maintenance and the repair of the corneal ECM involve a tightly coordinated balance of collagen and ECM synthesis, degradation and remodeling. Proteinases exist in inactive latent forms, become activated during inflammation, and can be produced by both corneal and inflammatory cells. They are involved in leukocyte chemotaxis, pathogen destruction following corneal infection, corneal epithelial cell migration in corneal ulceration, and corneal angiogenesis.

The tear film proteinases that predominate in the disease processes and wound healing of the cornea of equine ulcerative keratitis are the matrix metalloproteases (MMP), and the serine proteases such as neutrophil elastase (NE). Theses proteases normally exist in balance with protease inhibitors or antiproteases. In corneal ulcers, the combination of over-expression of certain destructive proteinases and reduction in antiproteinase activity can lead to rapid degradation of collagen and other components of the corneal ECM. Normal controls of routine degradative activity appear to be lost leading to pathologic destruction of the ulcerated cornea. Corneal ulceration can thus be considered a disorder of protease homeostasis.

The matrix metalloproteases are a multigene family of tightly regulated, zinc-dependent enzymes. All MMPs degrade at least one component of the ECM, share genetic homology to that of the collagenase enzyme, are secreted in a latent form, possess zinc at the active catalytic site, and are inhibited by naturally occurring tissue inhibitors of metalloproteases (TIMPs). Matrix metalloproteases are present at relatively low levels in normal horse corneal tissues as very little is constitutively expressed. MMP-2 exists in an inactive form in the normal, non-injured horse cornea. The synthesis of MMP-9 is induced during corneal injury. Matrix metalloproteases that contribute to corneal ulceration in the early stages of infection could be of bacterial or corneal cell origin. In the later stages as PMNs and monoctyes invade and accumulate, leukocyte-derived proteinases predominate as the main factor in corneal tissue destruction. MMP-2 and MMP-9 are overexpressed at the healing edge of reepithelializing corneal ulcers in horses. MMP-9 is expressed in migrating basal epithelial cells after tissue wounding. Some evidence indicates that TIMP levels are low in nonhealing chronic ulcers.

Neutrophil elastase (NE) is the most abundant serine protease in horse tears, and is synthesized by PMN leucocytes and macrophages. The clinical significance of NE and the serine proteases remains to be determined, as significant inherent antiprotease activity against NE is present in horses despite high tear film levels of NE.

MMPs and serine proteases are elevated in the tears of both eyes of horses with an ulcer in one eye!! The ulcerated eye has higher tear proteinase levels. MMP-2 increased 83%, MMP-9 increased 232%, and neutrophil elastase (NE) increased by 172% in the PTF of equine eyes with ulcers. MMP tear levels decrease with treatment. MMP tear levels decline as an ulcer resolves/heals. Infected ulcer tear MMP levels must decline more to heal. Serine proteases (neutrophil elastase, NE) are inhibited by serum and alpha 1 protease (A1P; pre-albumin inhibitor in the horse). Serum contains alpha-2-macroglobulins and platelet derived growth factors. Something in the horse tears attracts PMNs to the injured corneal site, and some presently unknown substance in the horse tears inhibits NE’s direct collagenase and protease effects

MMPs are inhibited by tissue inhibitors of metalloproteases (TIMPS), serum, EDTA, tetracyclines, ilomostat, and acetylcysteine. NE inhibits the activity of TIMPS!! MMP inhibits pre-albumin inhibitors!! The large amount of NE may thus cause indirect breakdown of collagen by increasing the MMP activity.

ULCERATIVE KERATITIS IN THE HORSE

Ulcers in horses can range from simple, superficial ulcers to full-thickness corneal perforations with iris prolapse. Epithelial cell layer disruption may so slight as to be limited to only a few superficial epithelial cell layers and is termed an abrasion. Abrasions stain very weakly with fluorescein dye. Erosions are abrasions with loss of all epithelial layers but an intact epithelial basement membrane. The term erosion is generally used to refer to persistent or recurrent abrasions with an underlying disorder of epithelial adherence to the epithelial basement membrane. Complete disruption of the epithelial cell layers and varying amounts of stromal damage is called an ulcer. Ulcers stain brightly with fluorescein dye. Absence of the mucin dominated precorneal tear film will also result in Rose Bengal retention of erosions and ulcers.

Healing of the avascular cornea of horses and other animals is dependent on interrelationships with the tear film, conjunctiva, limbus, stroma, endothelium, and aqueous humor for nutrients, humoral molecules, cytokines, growth factors and leucocytes. Tear film proteinases are elevated by 2-4 times normal levels, and must be reduced to baseline levels before healing is complete. Tear film MMP levels parallel the severity of the corneal disease. These levels diminish when treatment is initiated and as the ulcer heals.

Pathogenesis of Equine Infectious Corneal Disease in Horses

The healthy corneal epithelium and tear film of the horse are formidable barriers to the stromal invasion of bacteria or fungi. Bacteria and fungi must first adhere to the corneal surface in order to resist the natural corneal defense mechanisms that suppress bacterial and fungal growth. The pathogenicity of a bacterial or fungal species for the cornea may thus relate to its ability to adhere to injured corneal epithelium. Bacteria do not generally adhere well to intact corneal epithelium or to stroma completely denuded of epithelium, whereas they readily adhere to injured or diseased epithelium at the edge of an epithelial defect. Fungi in horses appear, however, to be able to adhere to healthy corneal epithelial cells in the absence of a normal precorneal tear film layer. An organism’s virulence (i.e., ability to rapidly cause serious disease) thus relates to its ability to adhere, and induce inflammation and activation of destructive enzymes. It may also be possible that these microbes are capable of producing biofilms. Biofilms would protect the microbes from antibacterials and prevent our ability to culture them. Pathogenic fungi adhere more easily to cornea, chemotactically attract more leukocytes, are associated with higher levels of MMP-9, and penetrate deeper vertically into the cornea. Fungal organisms have a unique propensity to move or “tunnel” deep in the stroma towards Descemet’s membrane of horses.

A vicious cycle may be initiated after relatively minor injury to the cornea, with the "second injury to the cornea" occurring because of the release and action of massive amounts of powerful inflammatory cytokines. These cytokines trigger a rapid and extensive infiltration of the corneal stroma by PMNs and a few T-lymphocytes that are chemotactically attracted from the limbal circulation and the tear film.

Corneal ulceration develops when epithelial wound healing fails. The formation of plasmin from plasminogen by plasminogen activator (PA) is an early step in ulceration. Plasmin produced in the cornea causes production of MMP-1. Prostaglandins, leucotrienes, and cytokines are released from epithelial cells and subepithelial keratocytes in response to injury. Interleukin-1 enhances MMP synthesis and activity but is not elevated in the tears of horses with ulcers. Interleukin-6 increases TIMP activity. Leucocytes in the stroma appear clinically as white infiltrates. Ulcers are relatively clear without leucocyte infiltration, and turbid white if present. Leucocytes can eliminate bacteria but not fungi by phagocytosis, and also release massive quantities of degradative enzymes to cause stromal “melting”. Leucocyte infiltration of the cornea is highly undesirable when excessive. Leucocytes release lysosomal enzymes, superoxide radicals and peroxides to damage the predominantly cell free stroma. In the present era of very effective antibiotics, microbes are rapidly eliminated leaving the remaining polymorphonuclear (PMN) leucocytes to destroy the cornea.

Pathogenesis of Infectious Equine Corneal Stromal Diseases

Healing of infected stroma in horses is typically manifested by the massive infiltration of polymorphonuclear (PMN) leucocytes, delayed epithelialization, and slight to severe neovascularization. PMNs chemotactically attracted to the horse cornea from the limbal blood vessels and the precorneal tear film then rapidly infiltrate the corneal stromal lamellae. The destruction of corneal stroma is due to the liberation of proteolytic enzymes by both the microbial organisms and the PMNs. After the PMNs enter the cornea, a destructive process involving necrosis of stromal keratocytes and phagocytosis of these dead cells by PMNs then occurs. As PMNs migrate peripherally in the anterior stroma, the overlying epithelial basement membrane is destroyed, thus leading to sloughing of the epithelium. The enlarged epithelial defect further enhances the migration of PMNs into the corneal stroma. Stromal destruction and loss of collagen fibrils increases with the migration and degranulation of PMNs. If the replication and spread of bacteria is not halted by the immune response of the horse eye, or the instillation of antibiotics, the process of stromal collagen and ECM degradation ultimately leads to total loss of stromal tissue and corneal perforation. Ulcers can however continue to deteriorate despite sterilization of the ulcer site due to excessive proteinase activity. This is generally followed by extensive fibroblastic collagen deposition and corneal scarring. The horse cornea does have the often unrecognized amazing ability to slowly remodel itself to diminish the size of large corneal scars.

Equine Tear Film Proteinases and Stromal “Melting” or Keratomalacia

Keratomalacia is liquefactive necrosis of the stroma with collagen fragmentation and loss of keratocytes. The clinical term for keratomalacia is “melting”. The matrix metalloproteases (MMP-2 and MMP-9) and the serine proteases (neutrophil elastase, NE) are important highly destructive mediators of corneal stromal keratomalacia in horses. MMP-2, MMP-9, and NE levels are significantly increased in the tear film of ulcerated horse eyes. Tear film proteinases including collagenases and elastases are produced by bacteria, fungi, epithelial cells, keratocytes, and PMNs. Neutrophils, the most abundant phagocytes in blood, contain a variety of antimicrobial proteinases and proteinase inhibitors. Numerous roles for these corneal proteinases have been hypothesized including the routine maintenance of corneal stromal collagen and extracellular matrix components, leukocyte chemotaxis and activation during inflammatory keratopathies, pathogen destruction following corneal infection, corneal epithelial migration following corneal ulceration, and corneal angiogenesis. These enzymes normally exist in balance with proteinase inhibitors. In the normal cornea and in some cases of ulcerative keratitis, antiproteinases may serve to prevent excessive tissue destruction. Degradative proteolytic activity appears to overwhelm the normal inhibitory control mechanisms in the “melting” corneal ulcer leading to rapid stromal tissue destruction and ulcer progression.

THERAPY FOR KERATOMALACIA IN THE HORSE

Treatment of keratomalacia or corneal melting consists of three concurrent components. The first is sterilization of the cornea with topical antimicrobials. I do not believe that this is a difficult goal to attain in most cases, but this sterilization does not necessarily equate with rapid healing of the epithelium or resolution of the inflammation. The initial topical antibiotic therapy in the inflamed and ulcerative cornea should be intensive with frequent application of broad spectrum or fortified antibiotics to “marinade” the cornea and produce very high stromal drug concentrations. Two to three days of sustained high concentrations of antibiotic are usually enough to eliminate most sensitive and partially sensitive bacterial and fungal populations. The second component is to speed healing of the cornea by reducing the tear film proteinase levels as rapidly as possible. MMP and neutrophil elastase levels were significantly higher in the tear film of horse eyes with “sterile” ulcers than eyes with fungal ulcers. Corneal perforation caused by direct microbial extension or indirect stromal proteolysis must be averted. A third problem in horse eyes with keratomalacia is controlling the often severe secondary anterior uveitis.

Antimicrobials

Empirical antibacterial therapy in keratitis is based on the likely pathogen, the available drugs, and the severity of the condition. Medical therapy for infected stromal keratopathies is most effective when the infectious organism is identified. It is often still contentious as to whether there is a need for microbial culturing and sensitivity testing in all horses, but the antimicrobial sensitivity results can be invaluable in difficult cases. Antibiotics can be used alone, or in combination, depending on the results of sensitivity testing. Increasing the frequency of application and the concentration of topical antibiotics increases the corneal concentration levels and improves the therapeutic response. There are emerging resistance and gaps in the spectrum of activity for microbes commonly causing infectious keratitis in horses.

The balance between bacterial and fungal growth on the horse cornea is normally quite precarious. Bacterial production of antifungal compounds released into the tear film may normally suppress fungal growth. Use of antifungals in therapy of keratomycosis should mimic the natural state. Streptomyces natalensis produces natamycin and is found on the conjunctiva of many horses suggesting natamycin may be the most physiologically appropriate drug to utilize in equine keratomycosis. It is possible that keratomycosis is so common in horses as the horse eye possesses immunodeficiencies of the tear film and/or cornea that predispose them to fungal infection. The reasons for the aggressive nature of corneal fungal infections in some horse breeds, and the propensity of fungi to grow deep in the equine cornea are unknown.

Killing the fungi infecting the horse cornea is not as difficult as I once thought. The problem is that the dead hyphae incite a dramatic PMN inflammatory response. The inflammatory response is out of control. Treatment must be directed against the fungi, as well as against the corneal and intraocular inflammatory responses that occur following fungal replication and hyphal death. Long duration of antifungal drug exposure is required for complete fungal destruction and resolution of the clinical signs. Prophylactic use of antifungal medications is debatable as some antifungals have strong immunosuppressive properties. These drugs should only be used when there is convincing evidence of fungal infection.

Topical Antiproteinase Therapy and Collagenolysis Prevention.

It is important to realize that healing of stromal disease in horses occurs when the tear film levels of the elevated proteinases are reduced. The therapeutic use of proteinase inhibitors is therefore critical, and yet unfortunately underutilized, in horses with keratomalacia in order to reduce the progression of stromal ulceration, speed epithelial healing, and minimize corneal scarring. Specific antiproteinases for ophthalmic use include N-acetylcysteine (NAC), disodium ethylene diamine tetraacetate (EDTA), tetracycline antibiotics, and autogenous serum. Serum contains alpha-2 macroglobulin which has activity against both MMP and serine proteinases, and alpha-1 antitrypsin which inhibits serine proteases. N-acetylcysteine, disodium EDTA, and tetracyclines are metal-chelating agents and appear to specifically inhibit MMPs.

Autogenous serum contains a number of antiproteinases. Αlpha-2-macroglobulin is a non-specific proteinase inhibitor produced in the liver that reduces the activity of proteinases from all major proteinase classes. It is a tetrameric molecule composed of two pairs of identical, disulfide-linked subunits. Binding of the proteinase to the bait region of each subunit leads to a change in the conformation of the α-2-macroglobulin molecule and the entrapment of the proteinase within the inhibitor. This mechanism tenaciously binds 2 proteinase molecules per one α-2-macroglobulin molecule, and causes this circulating agent to be one of the strongest known inhibitors of MMPs. This multifunctional inhibitor is present at high levels in blood comprising 8-10% of the total serum proteins. For this reason, the topical application of autogenous serum (one or two drops every 1 to 2 hours) is highly recommended for the treatment of corneal ulcerations in humans, and animals. Blood drawn into sterile containers containing no anticoagulants rapidly clots and yields serum that can be separated by centrifugation. The serum can be used at room temperature or refrigerated until needed, and its inhibitory effect remains high even after several days of storage. It is important to replace it with freshly collected serum at least every 8 days, as it may provide a medium for bacterial growth if it becomes contaminated, and may show some decline in inhibitory activity. The fibronectin in serum may reduce the discomfort in corneal ulcers and is known as the “feel good factor”. Serum, rather than plasma, also contains platelet-derived growth factors that could aid corneal ulcer healing. High levels (90%) of in vitro inhibition of MMP activity by serum have been noted.

Collagenases and other proteinases can function only in the presence of cations such as calcium and zinc. When these ions are chelated, the activity of collagenase decreases. Tetracycline binds to MMPs by a calcium bridge, thus inactivating the enzyme unless additional calcium is added. Tetracycline and doxycycline can ameliorate the ravages of collagenase by affecting PMN activity, while antibiotics such as penicillin and cefazolin are ineffective.

Ethylene diamine tetraacetate, doxycycline, and NAC inhibit MMP by chelation of the zinc and the calcium that MMPs require as cofactor and stabilizing ion respectively. By chelating the calcium ion, EDTA interferes with the stability of MMPs and thus decreases the stimulation for the migration of PMNs to the corneal ulcer site. EDTA also interferes with the attachment of the MMP to the PMN cell membrane leaving the PMN in a resting, inactivated state. It seems to be well tolerated by the equine eye when used topically at 0.05% to 0.2% concentrations for corneal ulceration, and exhibits a high rate (99.4%) of in vitro anti-MMP activity.

Tetracycline type drugs exhibit MMP inhibition activity independent of their antimicrobial properties. The proposed mechanism of action of these antimicrobial agents is that tetracyclines bind to both the zinc and calcium cations necessary for MMP activation, and thus cause reduced MMP activity. Doxycycline, in particular, inhibits the synthesis of MMPs in endothelial cells and reduces the breakdown of cornea mediated by excessive collagenolytic activity. It inhibits TGF-beta induced MMP-9 production and activity in epithelial cell culture. Its topical and systemic use is recommended in equine corneal ulceration as 0.025 to 0.1% doxycycline exhibits a high rate (96.3%) of in vitro anti-MMP activity.

N-acetylcysteine is a MMP inhibitor commonly used in human as well as veterinary ophthalmology. Topical application of 5-10 % NAC every 1 to 4 hours has been recommended in the horse. NAC at 10% proved to be very effective (98.8%) in vitro in inhibiting tear film MMPs of horses.

Efforts have been made to design more powerful synthetic inhibitors of proteinases. Ilomostat, a hydroxamic acid-containing modified dipeptide also called Galardin™, appears to be a promising and powerful MMP inhibitor in the treatment of rapid corneal stromal degradation. Ilomostat (0.1%) exhibits a high rate (98.9%) of in vitro anti-MMP activity. Combinations of antiproteinases may be necessary in many horses.

Tear film cytokines and excessive corneal scarring.

Healing of corneal stromal ulcers in horses is often unfortunately associated with profound fibrosis and scar formation that can result in varying degrees of visual impairment. Transforming growth factor beta (TGF-beta) activates normally quiescent stromal keratocytes to become fibroblasts and myofibroblasts. Activated keratocytes and fibroblasts produce connective tissue growth factor (CTGF). CTGF enhances fibroblast proliferation and collagen production. CTGF is present at detectable levels in about 40% of tears from ulcerated horse eyes. Overexpression of tear film cytokines such as TGF-beta and CTGF may be responsible for the severity of corneal scarring found in stromal keratopathies of the horse.

Cyclosporine A inhibits fibroblast proliferation and increases apoptosis of fibroblasts and is now being used topically to reduce corneal scarring in horses with corneal ulcers. Studies utilizing antifibrosis inhibitors such as cyclosporine A and CTGF inhibitors are urgently needed for use in the ulcerated horse eye.

ADJUNCTIVE SURGICAL THERAPY

Not infrequently during clinical treatment of corneal ulcers in the horse, surgery is necessary to augment the medical treatments.

Keratectomy

Keratectomy may prove to be useful during the early stages of ulcerative keratitis, when infection is confined to the corneal epithelium and anterior third or half of the cornea stroma. Removing necrotic tissue by keratectomy speeds healing, minimizes scarring, and decreases the stimulus for iridocyclitis.

Conjunctival Grafts  

Conjunctival grafts or flaps are frequently used in equine ophthalmology for clinical treatment of deep, melting, and large corneal ulcers, descemetoceles, and perforated corneal ulcers both with and without iris prolapse. Conjunctival flaps are best mobilized from the bulbar conjunctiva. I do not recommend using the conjunctiva near the nictitans, however, because postoperative nictitans movement can put tension on the graft and, thereby, result in premature graft release. Conjunctival flaps can be transposed and sutured onto the cornea to provide sufficient tissue to strengthen most weakened corneas, but they are not as strong as corneal grafts. Conjunctival autografts contain limbal stem cells, blood vessels, and lymphatics, thus offering significant antibacterial, antifungal, antiviral, antiproteinase, and anticollagenase effects. With conjunctival grafts, PMNs, antibodies, serum, and alpha-2-macroglobulins are immediately placed in the corneal ulcer bed. Systemic antibiotics can enter the ulcer site at higher levels through leakage from the conjunctival graft vasculature. The fibrovascular, or deeper, layer of the conjunctival transplant offers immediate fibroblasts and collagen with which to begin rebuilding the corneal stroma.

Conjunctival grafts, particularly for large-diameter ulcers, cause significant scarring and may impair vision, and do not always provide sufficient tectonic support. There may be a short term increase in PTF proteinase activity postoperatively due to tissue manipulation and corneal suturing. Scarring can be minimized, however, by removal of necrotic cornea with keratectomy before graft placement. Postoperative topical corticosteroids can reduce this postoperative scar tissue formation to a minimum, but corneal scarring after conjunctival grafts should be anticipated.

Amniotic Membrane Transplants

Amniotic membrane, which has been used to facilitate nonocular wound healing, as well as to reconstruct diseased cornea, has been suggested as a material useful in meeting both goals of strength and clarity. The amnion is the layer of the fetal membrane that envelops the fetus, and is attached to the rest of the placenta only at the umbilicus. Amnion is composed of an epithelium, a thick basement membrane, and an avascular stroma. It is avascular and strong, contains anti-angiogenic and anti-inflammatory factors, contains growth factors, and is antifibrotic. Basement membranes in general facilitate epithelial migration, reinforce epithelial adhesion, and prevent epithelial apoptosis. The mechanism of action by which amnion exerts its antiangiogenic and anti-inflammatory properties is unknown. The principles of application of amnion are dictated by its composition of a single layer of cuboidal epithelial cells, a basement membrane, which is a network of reticular fibers, and a stroma. Amnion can be applied as a single sheet, or multiple sheets in a blanket-fold. Orientation of application of amniotic membrane on the cornea, epithelium up (i.e. basement membrane if frozen amnion is used, as epithelium is lost during freezing), or stroma up determines whether the AMT will be incorporated into the recipient cornea or sloughed. This is because regenerating corneal epithelium grows along the AMT basement membrane.

I have used AMT to successfully treat corneal melting of the entire cornea which progressed in the face of aggressive medical therapy. These total corneal “melts” have previously had no reasonable therapy till the AMT. The AMT appears to have provided adequate support and been stable in the face of proteinases. AMT appeared to have anti-angiogenic properties (as evidenced by the progression of corneal vascularization only at the periphery of the AMT in each case), and appeared to have been permeable to topical medications (as evidenced by control of corneal infection). I have also used AMT following keratectomy and beta radiation of corneal SCC in the horse. There was much less corneal scarring and a better visual outcome postoperatively with the AMT than with conjunctival grafts used in similar cases.

When to stop therapy for melting ulcers?

It is easier to start therapy than to know when to stop therapy! It is often difficult to perceive when the infectious agents are still active. Closure of the epithelium over an ulcer and a lack of fluorescein dye staining are correctly used as significant end-points in defining a cure. The signs of resolution are also a diminution of the clinical signs of uveitis. The circulation to a healing ulcer will change from intense redness during active stages of the infection to pale pink/white when the inciting stimulus for the stromal lesion is absent.

|Antiproteinase Drugs |Dose |Proteinase-types inhibited and mechanism of action |

|Serum: (α-2-macroglobulin, α-1-proteinase inhibitor, and |Nondilute; q2h |MMP and serine proteinase inhibitor; Chelates Ca and|

|platelet-derived growth factors) | |Zn |

|Plasma: (α-2-macroglobulin, and pre-albumin inhibitor) |Nondilute; q2h |MMP and serine proteinase inhibitor; Chelates Ca and|

| | |Zn |

|EDTA 0.2% |q2h |MMP and serine proteinase inhibitor; Chelates Ca and|

| | |Zn |

|Ilomostat 0.1% |q2h |MMP and serine proteinase inhibitor; Chelates Ca and|

| | |Zn |

|Acetylcysteine 10% |q4h |MMP and serine proteinase inhibitor; Chelates Ca and|

| | |Zn |

|Pre-albumin inhibitor 0.1% |q2h |Serine proteinase inhibitor; proteinase entrapment |

|Doxycycline 0.1% |q8h |MMP and serine proteinase inhibitor; Chelates Ca and|

| | |Zn |

Suggested Reading:

Ollivier FJ, Brooks DE, Kallberg ME, Komaromy AM, Lassaline ME, Andrew SE, Gelatt KN, Stevens SR, Blalock TD, van Setten G, Schultz GS: 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): 1081-1087, 2003.

Ollivier FJ, Brooks DE, Van Setten GB, Schultz GS, Gelatt KN, Stevens GR, Blalock TD, Andrew SE, Komaromy AM, Lassaline ME, Kallberg ME, Cutler TJ: Profiles of matrix metalloproteinase activity in equine tear fluid during corneal healing in 10 horses with ulcerative keratitis. Veterinary Ophthalmology 7(6): 397-406, 2004.

Brooks DE: Inflammatory stromal keratopathies: medical management of stromal keratomalacia, stromal abscesses, eosinophilic keratitis, and band keratopathy in the horse. Veterinary Clinics Equine Practice 20(2):345-360, 2004.

Lassaline ME, Brooks DE, Ollivier FJ, Komaromy AM, Kallberg ME, Gelatt KN: Equine amniotic membrane transplantation for corneal ulceration and keratomalacia in three horses. Veterinary Ophthalmology 8(5): 311-318, 2005.

Ollivier FJ, Kallberg ME, Plummer CE, Barrie KP, O'Reilly S, Taylor DF, Gelatt KN, Brooks DE: Amniotic membrane transplantation for corneal surface reconstruction after excision of corneolimbal squamous cell carcinoma in nine horses. Veterinary Ophthalmology 9(6): 404-413, 2006.

Brooks, D.E., Matthews A.G.: Chapter 25, Equine Ophthalmology. In Gelatt, K.N. (ed.), Veterinary Ophthalmology, 4th ed., Blackwell Pub, Ames, IA,, pp 1165-1274, 2007.

Equine Recurrent Uveitis

Brian Patterson

Equine Eye Clinic

Rowe Veterinary Group

01454 415478

brianpatterson@

Equine recurrent uveitis is one of the most common ocular diseases in horses, classically characterised by episodes of active intra-ocular inflammation followed by varying periods of 'quiescence'. During such quiescent periods, low-grade, subclinical inflammation may never-the-less be present. Regardless of the specific cause, the inflammatory events within the globe eventually lead to secondary changes. The delicate visual axis that makes precise vision possible is highly vulnerable to the destructive potential of inflammation and it is these post inflammatory complications that make this syndrome the most common cause of blindness in horses worldwide (56% of horses with ERU lose vision in the affected eye within 2 years of disease onset) .

Aetiology and Pathogenesis:

Equine recurrent uveitis represents a group of organ specific immune-mediated diseases which trigger inflammation in the uveal tract. Such triggers include trauma (both penetrating and blunt) or systemic disease. Specific conditions implicated in the pathogenesis of equine uveitis worldwide include systemic bacterial infections (Leptospira species, Brucella abortus, Streptococcus equi, Salmonellosis, Escherichia coli, Rhodococcus equi, Borelia burgdorferi), parasitic (Onchocerca cervicalis, Toxoplasma gondii, intestinal strongyles), and viral infections (Equine herpesvirus types 1 and 4, equine arteritis virus, and equine infectious anemia virus). (1, 2). The most widely investigated of these are the Leptospira spp. Although the incidence of onchocerciasis is less common due to the widespread use of ivermectin, equine recurrent uveitis is thought to be stimulated by dead or dying microfilaria that have aberrantly migrated to the eye and episodes of uveitis can therefore be seen following normal worming.

Whilst infectious agents are commonly implicated as a trigger in the disease, the development of autoimmune activity against retinal proteins and antigens is likely to be a major component in the recurrent nature of the condition.

Clinical Findings and Lesions:

The clinical signs associated with equine recurrent uveitis include both acute signs of active inflammation and chronic secondary side effects. Damage to the uveal tract leads to the release of inflammatory mediators such as leukotrienes, prostaglandins, and histamines, which in turn causes increased permeability of the anterior uveal vessels, breakdown of the blood-aqueous barrier, iris sphincter spasm, and ciliary body muscle spasm. The compromise of the blood-aqueous barrier allows for leakage of protein, fibrin, and cells into the aqueous. These responses account for the classic signs of acute uveitis: blepharospasm, epiphora, episcleral injection, corneal edema, aqueous flare, fibrin clots in the anterior chamber, and miosis. Often, the anterior segment signs restrict examination of the posterior segment. If visible, posterior segment signs of an acute episode may include an inflammatory cell infiltrate involving the retina and/or choroid, focal or diffuse retinal separation, retinal hemorrhage, and a hazy yellow / green appearance to the vitreous secondary to infiltration by inflammatory cells and proteins. Liquefaction within the vitreous may be observed as collagen molecules aggregates, separating from the fluid component of the vitreal gel. One or both eyes may be affected in cases of ERU and there may be a disparity of clinical signs between eyes.

Corneal neovascularisation, calcium band keratophies, atrophy of the corpora nigra, posterior synechia, glaucoma, cataracts, pigment clumping of the nontapetal fundus (retinal degeneration) and retinal detachment are all signs consistent with chronic equine recurrent uveitis. The presence of any of these signs should be considered significant when undertaking pre-purchase examinations.

Diagnosis:

Whilst the diagnosis of ERU is based on the presence of characteristic clinical signs, consideration should be given to identifying the underlying cause. Because an acute episode of uveitis can be the first sign of systemic disease, a thorough physical examination should always be performed in addition to the ophthalmic examination. Routine haematology, biochemistry and urinalysis (if possible) should be considered in the workup of suspected systemic disease. Serologic testing for Leptospira spp is frequently discussed in the investigation of ERU. Caution must be taken in interpreting the results of such serology as up to 25% of horse in the UK can be expected to have positive titres to at least one leptospire serovar even in the absence of clinical signs. Serology may be of use as a prognostic tool. In one study, odds-ratio analysis revealed that uveitic horses with a positive titre to leptosirosis were 4.4 times more likely to lose vision than were seronegative horses with uveitis. Conjunctival biopsies from the lateral aspect of the bulbar conjunctiva my be examined for Onchocerca microfilaria, but results must also be interpreted with caution as horses without equine recurrent uveitis may also exhibit these microfilaria. Paracentesis of either the anterior chamber or the vitreous cavity offers the possibility of identifying a causative agent either through culture or the identification of locally produced antibodies; However, the procedure may cause severe intraocular damage, and a risk-benefit assessment should be made before undertaking such a procedure.

Treatment, Prevention, and Control:

The aims of ERU therapy are to maximise visual potential, minimise pain, and to prevent or minimise recurrent attacks of uveitis. Therapy should be initiated as soon as possible once signs of the acute phase are recognised. If a specific underlying cause can be identified, it should be addressed as part of the initial treatment protocol. In addition to dealing with the causative agent, or in instances where no specific cause is found, aggressive therapy with both topical and systemic anti-inflammatory medications is started to minimise the damage associated with intraocular inflammation. Both steroidal and nonsteroidal topical medications are commonly used. Prednisolone acetate (steroid, 1% suspension - shake bottle before using!), dexamethasone (steroid, 0.1% suspension or ointment), flurbiprofen (nonsteroidal, 0.03% solution), and diclofenac (nonsteroidal, 0.1% solution) have all been successfully used. When selecting a topically applied steroid, either prednisolone or dexamethasone are preferred to hydrocortisone, which penetrates the cornea poorly and is not sufficiently potent to be an effective medication for anterior uveitis. Frequency of application depends on severity of the inflammation, but administration 4-6 times a day is not uncommon. Corneas should undergo fluorescein staining prior to initiating topical steroid therapy. Flourescein positive eyes should not be treated with steroids as a general rule due to the risk of potentiating corneal infection and melting. As the clinical signs of uveitis resolve, the frequency of medication can be slowly reduced. It is recommended that therapy be continued for one month after the signs of acute inflammation have resolved. Topical atropine (1% solution or ointment) should be used in cases of anterior uveitis as it helps stabilise the blood-ocular barrier, it reduces pain by relieving ciliary body spasm as well as reducing the likelihood of posterior synechia formation in the central visual axis. Atropine is applied every 2 hours until pupil dilatation is achieved and then, ideally, on an as needed basis to maintain pupil dilatation. Owners can be taught to assess for mydriasis and to treat with atropine accordingly. Wash hands after handling atropine to avoid self medication! Whilst available, 4% compounded atropine solutions are probably best avoided as they are unlikely to achieve mydriasis should a 1% solution fail and they are more likely to cause adverse gastrointestinal effects. Gut motility and faecal output should be monitored when using atropine with measures being taken to avoid ileus (encourage activity outside of the stable, feed a laxative diet etc).

Flunixin meglumine administered systemically, and particularly when given IV, is probably the preferred treatment for acute anterior uveitis in horses. The usual initial IV dose is 1.1 mg/kg followed by oral medication at 0.25-1.1 mg/kg, bid. Phenylbutazone (2-4 mg/kg, sid-bid) can be used as an alternative to oral flunixin. Some horses may respond to aspirin (25 mg/kg, sid-bid, PO) although this drug is more often advoated for the longterm prevention of relapses rather than treatment of an acute flare up. Its use as a preventative measure is however questionable in terms of efficacy. Systemic steroids, specifically prednisolone (0.25-1 mg/kg PO per day) and dexamethasone (2.5-10 mg/day) have also been successfully used to treat acute uveitis episodes, but their longterm use has been associated with laminitis and owners must be counseled as the risk of laminitis prior to their use. As the severity of the clinical signs lessens, the dosage and frequency of oral anti-inflammatory medications can be tapered over a 2- to 3-month treatment period. If frequent topical medication is not feasible, subconjunctival injections of triamcinolone (10-20 mg), methylprednisolone acetate (10-40 mg), or betamethasone (5-15 mg) can supply therapeutic levels of intraocular anti-inflammatories. Injections should be made into the bulbar conjunctiva (not the palpebral) However, these routes of medication should be used with caution as they cannot be easily removed once injected and can have devastating consequences should a corneal ulcer develop.

The use of systemically or topically administered antibiotics may be considered in cases of uveitis. Antibiotics should be broad spectrum, and appropriate for the geographic location of the patient. Topical antibiotics are indicated in cases of uveitis due to penetrating ocular trauma, or ulcerative keratitis. Antibiotic treatment for horses with positive titers for Leptospira remains speculative but streptomycin (11 mg/kg IM BID) may be a good choice for horses at acute and chronic stages of the disease. Penicillin G sodium (10,000 U/kg IV or IM QID) and tetracycline (6.6-11 mg/kg IV BID) at high dosages may be beneficial during acute leptospiral infections. Enrofloxacin administered at 7.5mg/ kd SID has been shown to reach levels above the reported MIC for Leptospira pomona in inflamed eyes. Intracameral injections of gentamicin can be used to chemically 'euthanase' eyes with intractable sequelae such as glaucoma.

Horses with frequent recurrences or chronic, low-grade uveitis may be managed medically with daily (or every other day) doses of oral phenylbutazone or aspirin. Although most horses tolerate this regimen well, these medications can have adverse GI and hematologic side effects and the need for daily administration might lead to problems of compliance. In addition, these regimens frequently do not eliminate recurrence. Recently, in an attempt to address the problems of medical management alone, 2 surgical procedures have been developed. Core vitrectomy removes virtually all of the vitreous through an incision ~1 cm posterior to the dorsolateral aspect of the limbus. The vitreous is then replaced with either saline or balanced salt solution. The proposed benefit of this procedure is that T lymphocytes and/or organisms in the vitreous significantly contribute to the chronic / recurrent inflammation of equine recurrent uveitis. By removing these elements, the frequency and severity of the inflammatory events can be minimized. Although the core vitrectomy procedure has been very successful in achieving this goal in Munich, postoperative formation of cataracts has led to significant vision compromise in many patients in other ophthalmic centres. An alternative procedure has recently become available in the form of suprachoroidal cyclosporine (CsA) implants. CsA, through its ability to block IL-2 transcription, leading to impaired proliferation of activated T-helper and T-cytotoxic cells among other immunologic effects is a useful drug in the prevention of reactivation of ocular inflammation. CsA may be therapeutic in known leptospiral uveitis by reducing the bacterial burden as well as treating the inflammatory component. Unfortunately topically applied cylosporin fails to penetrate into the globe effectively so the drug must be delivered adjacent to the uveal space or within the vitreous. In the procedure of suprachoroidal cyclosporine implants , a biocompatible disc containing cyclosporine is implanted under a scleral flap created 8 mm posterior to the dorsolateral aspect of the limbus. Although this procedure is still in its relative infancy, early results have been encouraging.

Good husbandry practices such as effective fly control, avoiding stable objects that eyes might be traumatised against, general stable hygiene, routine worming and vaccinations, minimising contact with cattle or wildlife, draining stagnant ponds or restricting access to soggy pastures, and maximising nutrition have all been advocated as means to reduce the effects of equine recurrent uveitis. While such measures provide overall benefits for individual horses, the extent to which they impact the clinical course of equine recurrent uveitis is unknown

Equine Glaucoma

Brian Patterson BVM&S Cert VOphthal MRCVS

Equine Eye Clinic

Rowe Veterinary Group

01454 415478

brianpatterson@

Key points

• Glaucoma represents a group of diseases that lead disturbance in aqueous outflow which results in phases of raised intraocular pressure (IOP) termed ocular hypertension.

• Blood flow within the eye is reduced and retinal ganglion cells and optic nerve axons are damaged by raised intra-ocular pressure leading to progressive loss of retinal ganglion cells and optic nerve fibres (optic neuropathy) which ultimately affects vision.

• Diagnosis is made by demonstrating raised IOP (by tonometry), history, signalement and clinical signs.

• Therapeutic considerations include addressing underlying pathology and affecting the dynamics of aqueous flow.

Intraocular pressure (IOP)

The intraocular pressure represents balance between the production of aqueous humour at the level of the ciliary body epithelia and its passage through and out of the anterior segment and globe tunic. Aqueous is a complex mixture of electrolytes, organic solutes, growth factors, and other proteins that supply nutrients to the non vascularised tissues of the anterior chamber (i.e. tubercular meshwork, lens, and corneal endothelium). It is produced by an active process of secretion through the ciliary body epithelium. This process relies on the enzyme carbonic anhydrase which splits water and carbon dioxide into bicarbonate and water. Bicarbonate enters the posterior chamber, followed by sodium, which in turn is followed by water. A small proportion of aqueous is produced through ultrafiltration of blood through the ciliary body circulation.

Aqueous flows from the posterior chamber through the pupil in to the anterior chamber where it exits the eye through the iridocorneal angle (conventional outflow) and through passage across the iris, ciliary body and sclera (unconventional outflow).

The meshwork of pecinate ligaments (observable at the the meeting point of the cornea and iris) marks the entry to the iridocorneal angle (or ciliary cleft). Aqueous journeys through the labyrinth like structures of the uveal trabecular meshwork (UTM) and the corneoscleral trabecular meshwork (CTM). These meshworks are made up of extracellular matrix (e.g. collagens) organised into a network of beams. The aqueous enters the vascular system via the angular aqueous plexus (AAP) (which serves a similar function to the Schlemm's canal of human eyes) and on to the intrascleral plexus (ISP) which in turn drains into the vortex veins. Resistance to outflow is greatest at the AAP and the ISP. The conventional outflow path shows a high capacity for handling aqueous flow when compared to other species (horse: 0.88 microliter/min/mm Hg (sd +/- 0.65), dog: 0.32 microliter/min/mm Hg (sd +/- 0.16), human: 0.28 microliter/min/mm Hg (sd +/- 0.01) (1)

In the unconventional pathway aqueous humor enters the anterior face of the iris to drain into the vortex veins as well as passing from the UTM through the ciliary body musculature to drain in to the wide and well-defined supraciliary and suprachoroidal spaces and choroid vessels.

In the horse the uveoscleral route may be just as important as the conventional pathway for the removal of aqueous humor (2,3), whereas in the dog and cat, uveoscleral outflow accounts for 15%  and 3% of aqueous exit, respectively.

Obstruction to aqueous outflow causes a rise in IOP. The reference range  for normal IOP in the horse as measured by the Tonopen applanation tonometer is reported as 7 to 37mmHg, with a mean of 23.3 +/- 6.9mmHg (4). Values obtained with a rebound tonometer have been reported as 22.1 +/- 5.9 mm Hg (range, 10 to 34 mm Hg)  (5) Head position has a significant effect when it comes to measuring IOP  with 87% of horses showing an increase in IOP of 8.20 +/- 1.01 mm Hg when their heads were positioned below heart level following sedation (6). Failure to use a APNB may result in over-estimation of IOP. Intravenous administration of xylazine resulted in a statistically significant (P < 0.05) decrease in IOP of between 23 and 27%(7)

Glaucoma is fundamentally an optic neuropathy in which retinal ganglion cells and their axons perish. Retinal ganglion cells are neurons that receive visual information from photoreceptors via two intermediate neuron types (bipolar cells and amacrine cells).The axons arise from the ganglion cells and converge at the optic disc where they exit the globe through the cribiform plate as the optic nerve. Retinal ganglion cells vary significantly in terms of their size, connections, and responses to visual stimulation but they all share the similarity of having a long axon that extends into the brain, transmitting visual information from the retina. Large myelinated axons predominate in the optic nerve of the horse (8). The intraretinal segment of a ganglion cell axon tends to be unmyelinated, acquiring a myelin sheath at the level of the optic papilla. Intra-retinal myelination does occur as a normal variation at the temporal and nasal aspect of the optic nerve in some equine fundi.

The scleral lamina cribrosa (LC) is a specialized extracellular matrix that spans the scleral canal as a complex mutilayered set of collagenous plates (9) which form pores through which optic nerve fibre bundles pass. Two distinct pressure domains exist, one to either side of LC. In separating the intra-ocular from the orbital pressure domains, pore size of the lamina cribrosa is crucial. Pores must be large enough to permit unimpeded egress of groups of nerve fibre bundles, yet be small enough to contain the pressure of the eye. The macromolecular structure of the equine lamina cribrosa suggests that it is a very resilient structure that may provide some protection to the optic nerve axons during episodes of elevated intraocular pressure (9). However, the intralaminar optic nerve of horses with glaucoma have a significantly larger number of laminar pores which are significantly smaller and rounder than healthy horses as well as having a higher percentage of connective tissue. These changes may present a design feature that predisposes some horses to developing glaucoma or that these changes occur within the LC due to the effects of raised IOP. Pathological IOP results in compression and distortion of the scleral lamina cribrosa leading to rotation, misalignment and collapse of the laminar pores and laminar channels which causes interruption to retrograde axonal transport within the optic nerve, eventually causing RGC death. Other mechanisms that lead to retinal cell death in mammals are under study including  the effects of vascular compromise, glutamate neurotoxicity, the effect of nitric oxide and immune dysfunction within the hypertensive eye. It is likely that damage to RGC’s is mutifactorial in cases of glaucoma.

Retinal ganglion axons are clinically recognisable where they aggregate to form the optic nerve head (ONH). The ONH is described in terms of the neuroretinal rim and the optic cup. The characteristic anatomical change in the optic nerve in glaucoma is a "cupping" of the optic. Where ganglion cell axons have been lost, the area of the neuroretinal rim decreases with a corresponding increase in the area of the optic cup. The ratio of cup-to-disc area is used to monitor the progression of glaucoma in humans using scanning laser tomography although this is not clinical tool available to veterinary practitioners. The optic nerve axon density in horses with glaucoma has been shown to be reduced by up to 65% (10) which contributes to the clinical appearance of optic nerve atrophy, cupping and exposure of the laminar cribrosa.

Glaucomas are divided into three broad categories: congenital, primary, and secondary. Congenital glaucoma caused by developmental abnormalities of the ICA (goniodysgensis) have been reported in Thoroughbred, Arabian and Standardbred foals foals (11,12) presenting as buphthalmos at birth. Primary glaucomas  are bilateral in nature and have heritable characteristics with no overt ocular abnormality to account for the increase in IOP. They have not been conclusively reported in the horse. The secondary glaucomas have an identifiable cause such as intra-ocular neoplasia, uveitis, post traumatic lens luxation/rupture, neoplasia and septic endophthalmitis. Recurrent Uveitis is the primary cause of secondary glaucomas causing aqueous outflow blockage due to the formation of posterior synechiae, iris bombe, blockage of the aqueous outflow channels through preiridial fibrovascular membranes (PIFM) formation or inflammatory exudate accumulation. Uveal atrophy secondary to uveitis may predispose the ICA to collapse.

The diagnosis of equine glaucoma is made by demonstrating raised IOP in conjunction with specific clinical signs. Large diurnal variations in IOP have been demonstrated in horses with glaucoma which can necessitate multiple tomometry readings throughout the day to demonstrated spikes in IOP (13) . This diagnostic challenge can also complicate how response to therapy is interpreted.

Clinical features of equine glaucoma arranged in anatomical locations include :

Cornea - oedema, ranging from mild oedema to bullous keratophy (which may be associated with unresponsive corneal ulcers), linear band opacities (which represent thinning of Decemets membrane appearing as white lines that typically span the deep cornea from limbus-to-limbus), vascularisation, ulcerative exposure keratitis

Anterior chamber - aqueous flare

Iris - synechia, iridocyclitis

Pupil size - from miosis (in cases of ERU) to degrees of mydriasis (which can range form marginal dilatation to fixed and dilated pupils)

Lens - lenses may be subluxated (demonstrating aphakic crescents) or luxated.

Retina - optic nerve cupping, areas of retinal degeneration, tapetal hyper-reflectivity

Globe size - buphthalmia is consistent with end stage glaucoma.

Functional tests - such as dazzle response and menace response may still be present in glaucomatous eyes. Loss of vision is the natural progression in uncontrolled glaucoma.

Pain- mild to severe

The ultimate aim of glaucoma treatment is to retain useful vision and control pain. Medical treatment is aimed at reducing IOP by decreasing aqueous production and increasing aqueous outflow. Iridocyclitis should be addressed when present. The long-term prognosis for maintaining vision with medical therapy alone is guarded, although the initial response to early cases is often good. An IOP of ................
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