Maintenance and repair of corneal stromal extracellular matrix (ECM) requires a tightly coordinated balance of ECM synthesis, degradation and remodeling in which proteolytic enzymes (proteinases) perform important functions. There are natural proteinase inhibitors present in preocular tear film (PTF) and cornea simultaneously with proteinases that prevent excessive degradation of normal healthy tissue. Disorders occur when there is an imbalance between proteinases and proteinase inhibitors in favor of the proteinases, causing pathologic degradation of stromal collagen and proteoglycans in the cornea. Two matrix metalloproteinases (MMPs), MMP-2 and MMP-9, are of major importance in terms of remodeling and degradation of the corneal stromal collagen. Immunohistochemical studies have shown different origins of MMP-2 and -9. MMP-2 is synthesized by corneal keratocytes and performs a surveillance function in the normal cornea, becoming locally activated to degrade collagen molecules that occasionally become damaged. Alternatively, MMP-9 may be produced by epithelial cells and polymorphonuclear neutrophils following corneal wounding. Because the cornea is in close contact with the preocular tear film (PTF), proteinases have been evaluated in the PTF. In damaged corneas, total proteolytic activity in the tear fluid was found to be significantly increased compared to normal eyes and contralateral eyes. Studies analyzing the proteolytic activity in serial PTF samples during corneal healing led to the following conclusions: ulcerative keratitis in animals is associated with initially high levels of tear film proteolytic activity, which decrease as ulcers heal; proteinase levels in melting ulcers remain elevated leading to rapid progression of the ulcers. The success of medical and surgical treatment of the corneal ulcers is reflected by the proteolytic activity in tears. In animals, successful treatment leads to a rapid reduction in tear film proteolytic activity that corresponds with the improvement in the clinical signs of corneal ulceration. The in vitro effects of various compounds on proteolytic activity in the tear fluid of animals with ulcerative keratitis have been evaluated and their important inhibitory effects have been confirmed. Because these various compounds utilize different mechanisms to inhibit various families of proteinases, a combination of these proteinase inhibitors may be beneficial.

Proteinases of the cornea and preocular tear film

In this paper, the basics, screening technique and major indications of ocular ultrasound are described. This diagnostic technique proved to be an essential tool to examine intraocular structures when opacified ocular media inhibit direct examination and to evaluate retrobulbar structures when exophthalmos is evident.

Review of ocular ultrasonography.

Resistive index (RI) and pulsatility index (PI) are indirect measurements of blood flow resistance that may be used to evaluate vascular changes in renal and ophthalmologic diseases. To our knowledge, no reports are available describing values for renal and ocular PI index in the unsedated dog and ocular RI and PI indices in the unsedated cat. The purpose of this study was to measure normal values for both intrarenal and ocular RI and PI within the same subject in unsedated clinically normal dogs and cats. Twenty-seven dogs and 10 cats were considered healthy by means of physical examination, CBC, biochemical profile, urinalysis, and ultrasonography. Systolic blood pressure was measured by Doppler ultrasonography. Intrarenal and ocular arteries were scanned by pulsed Doppler ultrasonography to calculate RI and PI. No significant differences were noted between the values obtained for the right vs. the left kidney and eye. The upper values of these indices were calculated as mean + 2 standard deviations resulting in 0.72 and 1.52 for dog renal RI and PI; 0.7 and 1.29 for cat renal RI and PI; 0.76 and 1.68 for dog ocular RI and PI; and 0.72 and 1.02 for cat ocular RI and PI.

Doppler ultrasonographic estimation of renal and ocular resistive and pulsatility indices in normal dogs and cats

Objective—To examine in vitro effects of various antiproteolytic compounds on activity of matrix metalloproteinase (MMP)-2 and -9 in the tear film of horses with active corneal ulcers. Sample Population—Samples of tear film obtained from the eyes of 34 horses with active ulcerative keratitis. Procedure—Horses were sedated, and tear samples were collected from the lower fornix of 34 ulcerated eyes by use of capillary tubes. The protease inhibitors 0.2% EDTA, 0.1% doxycycline, 10% N-acetylcysteine (NAC), 0.1% solution of a modified dipeptide that contains hydroxamic acid (ie, ilomostat), 0.1% α1-proteinase inhibitor (PI), 0.5% α1-PI, and 100% fresh equine serum (ES) were used to treat pooled samples. Amount of latent and active MMP-2 and -9 was measured by optical density scanning of gelatin zymograms of treated and untreated tear samples. Results—Pooled tear samples obtained from ulcerated eyes contained the latent and active forms of MMP-2 and -9. Compared with MMP activity in untreated samples, total MMP...

Evaluation of various compounds to inhibit activity of matrix metalloproteinases in the tear film of horses with ulcerative keratitis

The primary objective of current treatment strategies for infectious keratitis is to sterilize the ulcer as rapidly as possible with topically administered antibiotics. Ulcerative processes can proceed in some cases, despite the absence of microbes, as a result of remaining corneal and tear film MMPs. Combining antibiotic therapy with MMP inhibitors can speed corneal healing, because MMPs play an important role in corneal ulceration and stromal liquefaction. MMPs from the rabbit, horse, and human being are inhibited by metal-binding agents EDTA, NAC, and doxycycline as well as by the serum antiprotease alpha2-macroglobulin. It is not yet certain which proteinase inhibitor has the most favorable therapeutic index for clinical use, although we prefer serum because of its effects on multiple types of proteinases. The MMP inhibitors do have significant therapeutic promise in the treatment of corneal ulceration.

Matrix metalloproteinase inhibition in corneal ulceration.

The objective of this study was to compare orbital and ocular vasculature velocity, measured by Doppler imaging, in normal Beagles and Beagles with inherited primary open-angle glaucoma. Eight normal Beagles and 13 Beagles with different stages of primary open-angle glaucoma were evaluated twice with a 2-4-week period between measurements. Doppler imaging was performed with the dogs anesthetized, and the Doppler transducer applied directly on the corneal surface. The majority of the orbital vasculature (external ethmoidal artery; internal ophthalmic artery and vein; and external ophthalmic artery and vein) and ocular blood vessels (anterior ciliary artery and veins; long posterior ciliary arteries; short posterior ciliary arteries; primary retinal arteries; and the vortex veins) were identified and Doppler blood velocity parameters were determined. The glaucomatous dogs demonstrated significant differences in the Doppler velocity parameters of several orbital vessels (external ethmoidal, external ophthalmic, and internal ophthalmic arteries), and several ocular vessels (anterior ciliary, short posterior ciliary, and long posterior ciliary arteries). These differences included decreased blood velocities, and increased pulsatility and resistive indexes. The Doppler blood flow velocities of the primary retinal arteries were unchanged between the normal and glaucomatous dogs. In the glaucomatous dogs, the Doppler imaging suggests increased vascular resistance downstream in both the orbital and ocular vasculature. These blood velocity parameter changes may be primary or secondary, and may offer therapeutic opportunities to increase perfusion, prolong the retina and optic nerve head function, and maintain vision in the canine glaucomas.

Comparative Doppler imaging of the ophthalmic vasculature in normal Beagles and Beagles with inherited primary open-angle glaucoma.

Ulcerative keratitis is a common and potentially blinding ocular disease of horses, capable of progressing to corneal perforation in as little as 24 h. This rapid stromal degeneration is mediated in part by exogenous and endogenous proteinases. We measured and compared the concentrations of two matrix metalloproteinases (MMP-2 and MMP-9) and a serine proteinase (neutrophil elastase) present in the precorneal tear film of normal horses and horses with rapidly progressing ulcerative keratitis. Precorneal tear film samples were collected from 23 ulcerated and 21 unaffected eyes of 23 horses with unilateral ulcerative keratitis, and from 33 normal eyes of 17 control horses. MMP-2, MMP-9, and neutrophil elastase were identified by casein and gelatin zymography and quantified by computerized image analysis. Median MMP-9 levels were significantly higher in the precorneal tear film of young control horses vs. older control horses (P= 0.005). Median MMP-2, MMP-9, and neutrophil elastase levels were significantly higher in the precorneal tear film of ulcerated eyes when compared to age-matched normal controls (P= 0.004, P= 0.001, and P= 0.012, respectively). Median MMP-2 levels were also significantly higher in the precorneal tear film of contralateral eyes of affected horses when compared to age-matched normal controls (P= 0.004). No significant differences in median proteinase levels were detected between ‘sterile’ ulcers and those from which bacteria or mixed infections (bacteria and fungi) were isolated. However, median MMP-2 and neutrophil elastase levels were significantly higher in the precorneal tear film of eyes with ‘sterile’ ulcers when compared with ulcerated eyes from which fungi were isolated (P < 0.05). The results of this study support the use of topical antiproteinase therapy which targets both MMPs and serine proteinases in progressive equine ulcerative keratitis.

Evaluation of tear film proteinases in horses with ulcerative keratitis.

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Comparative Doppler imaging of the ophthalmic vasculature in normal Beagles and Beagles with inherited primary open-angle glaucoma.

Evaluation of tear film proteinases in horses with ulcerative keratitis.