Evaluation of tear film proteinases in horses with ulcerative keratitis

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.     

Profiles of matrix metalloproteinase activity in equine tear fluid during corneal healing in 10 horses with ulcerative keratitis

OBJECTIVE: Levels of tear film matrix metalloproteinases (MMPs) activity are significantly elevated in horses with ulcerative keratitis and contribute to the excessive breakdown of stromal collagen. Changes in the amount of proteolytic activity in horse tear film during corneal healing and stromal remodeling have not yet been reported, but we hypothesize they should decrease. In the present study we analyzed serial tear fluid from horses with ulcerative keratitis to identify any changes in MMP activity during corneal healing and stromal remodeling. PROCEDURES: Samples of tear fluid were obtained from both eyes of 10 horses with ulcerative keratitis on the day of admission (day 1) at the hospital and then at various time points until complete healing of the cornea. Tear film MMP2 and MMP9 activity was determined by quantitative gelatin zymography. In all cases medical treatment included topical applications of equine serum, antibiotics, atropine and systemic administration of anti-inflammatory drugs. Surgical procedures were performed in several cases on day 2 in addition to the medical treatment. RESULTS: The mean total MMP activity (+/- SD) measured in relative standard units (RSU) in the tear fluid of the ulcerated eye (2.44 +/- 1.44) of the 10 horses was significantly higher than the mean in the contralateral eye (0.81 +/- 0.68) (P = 0.006), on the day of admission at the VMTH. The mean MMP activity in these ulcerated eyes significantly decreased (-82.4%) between the first day of admission and the day when the ulcer had completely healed (P = 0.0002). The activity level in the healed eye (0.43 +/- 0.17) was not significantly different to the one in the contralateral eye (0.36 +/- 0.18) on the day of complete corneal healing (P = 0.374). The level of MMP activity in the contralateral eye also decreased from 0.81 +/- 0.68-0.36 +/- 0.18 but this decrease (56%) was not significant (P = 0.069). CONCLUSIONS: Ulcerative keratitis in horses is associated with initially high levels of tear film proteolytic activity that decrease as the ulcers heal. The success of medical and surgical treatment of the corneal ulcers is reflected by the enzyme activity in tears. In horses successful treatment does lead to a rapid reduction in tear film proteolytic activity that corresponded with the improvement in the clinical signs of corneal ulceration. Measurement of MMP activity in the tear film might represent a way to monitor the progression of corneal healing in horses with ulcerative keratitis.     

Evaluation of matrix metalloproteinase concentrations in precorneal tear film from dogs with Pseudomonas aeruginosa-associated keratitis

OBJECTIVE: To evaluate the changes in concentrations of matrix metalloproteinase (MMP)-2 and MMP-9 in the precorneal tear film of dogs with Pseudomonas aeruginosa-associated keratitis during corneal healing and stromal remodeling. ANIMALS: 10 dogs with unilateral P aeruginosa-associated keratitis and 10 clinically normal dogs. PROCEDURES: Precorneal tear film samples were collected from both eyes of 10 dogs with unilateral P aeruginosa-associated keratitis on the day of admission to the hospital and then at various time points until complete healing of the cornea was achieved. Precorneal tear film samples were also collected from both eyes of 10 clinically normal adult dogs (control group). Concentrations of MMP-2 and MMP-9 in precorneal tear film samples from each group were determined via gelatin zymography for comparison. RESULTS: The proteolytic processes in the ulcerated eyes decreased as corneal healing progressed. On the day of admission, concentrations of latent and active forms of MMP-2 and MMP-9 in ulcerated eyes were significantly higher than values in the contralateral unaffected eyes in dogs with P aeruginosa-associated keratitis; concentrations of latent MMP-2 and MMP-9 were also greater than control group values. Concentrations of latent and active forms of MMP-2 and MMP-9 in the healed eyes of dogs with P aeruginosa-associated keratitis were significantly lower than concentrations in the ulcerated eyes on the day of admission. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that reduction of precorneal tear film concentrations of MMPs by use of proteinase inhibitors may be effective in the treatment of dogs with P aeruginosa-associated keratitis.     

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

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% alpha1-proteinase inhibitor (PI), 0.5% alpha1-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 activity (sum of all bands detected) observed on the gelatin zymogram gels was reduced by 99.4% by EDTA, 96.3% by doxycycline, 98.8% by NAC, 98.9% by ilomostat, 52.4% by 0.1% alpha1-PI, 93.6% by 0.5% alpha1-PI, and 90.0% by ES. CONCLUSIONS AND CLINICAL RELEVANCE: We documented that EDTA, doxycycline, NAC, ilomostat, alpha1PI, and ES inhibited MMP activity in vitro. Because these compounds use different mechanisms to inhibit various families of proteases in the tear film of horses, a combination of these protease inhibitors may be beneficial for treatment of corneal ulcers in horses.     

Matrix metalloproteinase inhibition in corneal ulceration.

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.     

The precorneal tear film in horses: its importance and disorders.

The precorneal tear film (PTF) is of critical importance in the physiologic and pathologic findings of the cornea. Clinicians should recall that quantitative and qualitative disorders of the PTF can be a cause of corneal diseases as well as a clinical sign of ocular surface diseases. It is also important to consider that some systemic and topical treatments may affect the PTF volume and composition. Not all interactions are known at this time. There is a continued need for basic research into PTF components in healthy and diseased equine eyes, because much remains unknown. Until additional basic information about the biochemical composition and pH of the PTF as well as the interactions between equine corneal pathogens and specific PTF deficits becomes available, it will not be possible to define the cause and effect relations precisely between the various ocular surface diseases and deficiencies,excesses, and imbalances of PTF components. It is quite possible that a number of equine corneal diseases may be manifestations of qualitative PTF disorders.     

Inflammatory stromal keratopathies: medical management of stromal keratomalacia, stromal abscesses, eosinophilic keratitis, and band keratopathy in the horse.

This article discusses the diagnosis and medical treatment of stromal keratomalacia or "melting ulcers," stromal abscesses, eosinophilic keratitis (EK), and calcific band keratopathy. These are common and important inflammatory keratopathies of the equine corneal stroma. Keratomalacia and stromal abscesses are associated with infection, leukocytic invasion of the stroma, and loss of tissue and tear film proteinase homeostasis. Eosinophils infiltrate the stroma in response to unknown stimuli in EK. Calcium is deposited in the stroma and epithelium secondary to chronic equine recurrent uveitis in calcific band keratopathy. They are all associated with varying degrees of iridocyclitis.     

Feline corneal disease

The cornea is naturally transparent. Anything that interferes with the cornea's stromal architecture, contributes to blood vessel migration, increases corneal pigmentation, or predisposes to corneal edema, disrupts the corneas transparency and indicates corneal disease. The color, location, and shape and pattern of a corneal lesion can help in determining the underlying cause for the disease. Corneal disease is typically divided into congenital or acquired disorders. Congenital disorders, such as corneal dermoids are rare in cats, whereas acquired corneal disease associated with nonulcerative or ulcerative keratitis is common. Primary ocular disease, such as tear film instability, adenexal disease (medial canthal entropion, lagophthalmus, eyelid agenesis), and herpes keratitis are associated with the majority of acquired corneal disease in cats. Proliferative/eosinophilic keratitis, acute bullous keratopathy, and Florida keratopathy are common feline nonulcerative disorders. Nonprogressive ulcerative disease in cats, such as chronic corneal epithelial defects and corneal sequestration are more common than progressive corneal ulcerations.     

Qualitative tear film disease

Abnormalities of the lipid and mucin components of the preocular tear film may result from diseases of the eyelid margins and conjunctiva. Chronic keratoconjunctivitis with epithelial edema and superficial corneal neovascularization, with or without ulceration, characterizes qualitative tear diseases. Tear components other than lipid and mucin that carry probable clinical significance include tear proteins, all-trans retinal, cholesterol, glucose, and electrolytes. Although less common than quantitative or aqueous deficiencies, qualitative abnormalities are recognized as primary or secondary causes of ocular surface disease in companion animals.     

An Overview of Proteinase Inhibitors

Proteinase inhibitors are proteins in the body that regulate the catalytic activity of proteinases. They are important in a large variety of physiologic processes including coagulation, digestion, tumor metastasis and immunity. Proteinase inhibitors are categorized as either nonspecific proteinase inhibitors or class-specific proteinase inhibitors. Nonspecific proteinase inhibitors are comprised soley of the alpha macroglobulins, most notably alpha2-macroglobulin. Class-specific proteinase inhibitors are subcategorized as serine proteinase inhibitors, aspartic proteinase inhibitors, metalloproteinase inhibitors, and cysteine proteinase inhibitors. Each subcategory is made up of numerous inhibitors. As the roles of individual proteinase inhibitors are determined, the therapeutic use of natural and synthetic proteinase inhibitors is also being investigated. The purpose of this article is to review the history and classification of proteinase inhibitors and their relevance to veterinary medicine.     

Corneal diagnostic procedures

The cornea is the anterior, transparent portion of the fibrous tunic of the eye. It is continuous with the sclera at a transition called the limbus. In healthy conditions, the transparency of the cornea is maintained by the smooth, nonkeratinized, squamous epithelium, which is further enhanced by the precorneal tear film, the lack of corneal vascularization or pigmentation, the size and regular arrangement of the collagen fibrils that make up the corneal stroma, and the relative dehydration of the cornea (which is maintained by the endothelium and epithelium). The cornea can respond to adverse stimuli through vascularization, pigmentation, fibrosis, accumulation of cellular or noncellular infiltrate, and/or edema. Because of these limited responses, routine diagnostic procedures are critical in the diagnosis and treatment of corneal disorders. This article discusses tests of the precorneal tear film, corneal staining procedures, culture and sensitivity, cytology, and a few other procedures that are performed less commonly or require specialized instrumentation.     

Equine corneal surgery and transplantation.

Corneal disease is common in equine ophthalmology and requires vigilant monitoring and appropriate therapy to optimize the outcome.Many equine corneal diseases, particularly those that progress rapidly, may benefit from surgical intervention. These include descemetoceles, deep corneal lacerations and ulcers, corneal perforation/iris prolapse, ulcerative keratitis, corneal stromal abscesses, and corneoscleral neoplasia. Indications for corneal transplantation include optical, tectonic, therapeutic, and cosmetic purposes. Corneal transplantation is most often implemented in equine patients for tectonic and therapeutic reasons when a cornea is compromised by corneal stromal abscess, iris prolapse, or neoplasia.This article provides an outline of when to consider surgical intervention for corneal disease, the procedures available and expected outcomes, and how appropriate early surgical intervention can dramatically improve the end result.     

Proteinase, lipase and amylase activities in the small intestine of pigs suffering from colienterotoxaemia.

The activities of proteinases, lipases, amylases and the activities of proteinase inhibitors, as well as the numbers of Escherichia coli in the contents from the small intestine were examined for pigs suffering from colienterotoxaemia and for healthy pigs. Enzyme activities were determined using an agar diffusion test.Compared with healthy animals the activities of proteinases and amylases in diseased animals were reduced while lipases showed increased activity. In pathologically changed contents showing large numbers of E. coli, proteinases could not be demonstrated; however, proteinase inhibitors were found in these contents. In healthy animals, proteinase inhibitors were not demonstrated in ingesta-con-taining contents.In diseased animals, E. coli were found in large numbers in all parts of the small intestine. In healthy animals, E. coli was demonstrated especially in the posterior part of the small intestine and regularly in small numbers.The possible influence of digestive enzymes, especially proteinases and their inhibitors, on enterotoxins from E. coli is discussed.     

Doxycycline levels in preocular tear film of horses following oral administration

Objective To determine the concentration of doxycycline in preocular tear film following oral administration in horses as a possible therapeutic modality for infectious and keratomalacic equine keratitis. Procedure Eight broodmares without ocular disease from a Thoroughbred breeding facility were included in this study. Each mare received 20 mg/kg of doxycycline by mouth once daily in the morning for five consecutive days. Tears were collected 1 h after doxycycline administration starting on day one of administration and continuing for 10 consecutive days. Doxycycline levels in the tears were measured using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS). Results Doxycycline was present in the tears of each mare at low µg/mL levels with the highest concentration appearing on the third to fifth days (8.21–9.83 µg/mL). Doxycycline levels had fallen below quantifiable ranges by day 10. No systemic side-effects were noted in any of the horses included in this study. Conclusions Oral doxycycline is present in preocular tear film of normal horses with noninflamed eyes and may be useful as treatment in equine ulcerative keratomalacia. The oral dose listed was tolerated well by the horses in this study. The drug levels attained at 20 mg/kg once daily orally of doxycycline may aid in the treatment of corneal ulceration in horses, but further study is warranted.     

Modulation of proteolytic activity associated with persistent corneal ulcers in dogs

Canines affected with corneal lesions demonstrate increased proteolytic activity at the affected site. Canines that develop persistent corneal ulcers and maintain elevated levels of proteolytic activity respond to protease inhibitor therapy with polysulpated glycosaminoglycan, PSGAG. In this study, the proteolytic activity expressed in lacrimal fluid of canines was evaluated as normal (1.15 U mL⁻¹) or healed (1.19 U mL⁻¹). Six of the 26 dogs affected with persistent corneal ulcers, however, had a protease level consistent with the unaffected control animals. These dogs did not appear to respond to PSGAG therapy. Two pathophysiologies for persistent corneal ulcers are indicated. Identifying which mode is active could help determine the therapeutic treatment needed.     

Pseudallescheria boydii keratomycosis in a horse

The fungal organism Pseudallescheria boydii was isolated from the cornea of a Quarter Horse with ulcerative keratitis. Despite aggressive hourly medication through a subpalpebral lavage system, with drugs including miconazole and natamycin, the cornea developed a stromal abscess. Orbital exenteration was performed after 3 weeks. The fungal isolate was later determined to be resistant to all 8 antifungal drugs tested. Microscopic examination of the cornea revealed fungal hyphae throughout the corneal stroma and penetrating the Descemet membrane. Pseudallescheria boydii has not been implicated previously as a cause of keratomycosis in horses or in other domestic animals, although cases in human beings have been described.     

Cochet-Bonnet aesthesiometer-determined corneal sensitivity in neonatal foals and adult horses

Corneal touch threshold (CTT) was measured in sick neonatal foals, healthy foals, and healthy adult horses with a Cochet-Bonnet aesthesiometer. The mean overall CTT for the adult horses, sick foals, and healthy foals was 4.82 +/- 0.87 cm, 3.21 +/- 0.24 cm, and 5.01 +/- 0.61 cm, respectively. The central cornea of adult horses was more sensitive than the limbal cornea. Corneal sensitivity was significantly reduced in sick neonatal foals compared to adults. The mean Schirmer I tear test values were significantly lower in foals than adults, and were 14.2 +/- 1.0 mm, 12.8 +/- 2.4 mm, and 18.3 +/- 2.1 mm wetting in sick neonatal foals, normal neonatal foals, and adult horses, respectively. Reduced corneal sensation and lower tear production may be associated with ulcerative keratitis and slow corneal healing in some foals.     

Toxocara canis: proteinases in perivitelline fluid from hatching eggs

Developing larvae of Toxocara canis may secrete several kinds of enzymes within the egg perivitelline fluid (EPF) prior to and during hatching. In particular, proteinases in EPF could play a role in larval emergence within the host gastrointestinal lumen but its presence and nature is unknown. In this work, proteolytic activities in hatching fluid of T. canis were identified and analysed by substrate gel electrophoresis at different pH values and by using type specific protease inhibitors. Three bands of 91, 68 and 38 kDa showed gelatinolytic activity and all proteinase activity from EPF was of the aspartic-type since it was inhibited by pepstatin A. Interestingly, a significantly higher proteolytic activity was observed at acidic pH (< or =5.5). These data suggest that T. canis developmentally secretes and accumulates in EPF aspartic proteinases with a pH-dependent activity that might help the parasite to take advantage of conditions in the host gastrointestinal microenvironment where egg hatching is induced and executed.     

Aspergillus flavus keratomycosis in a cat treated with topical 1% voriconazole solution

An 8-year-old male castrated Domestic Short-haired cat was examined for a 1-week history of blepharospasm and mucoid ocular discharge OS. Examination revealed ulcerative keratitis with stromal loss, stromal infiltrate, corneal edema, perilimbal vascularization and miosis. Cytology of the cornea revealed multiple dichotomously branching, septate fungal hyphae and severe, predominantly neutrophilic inflammation. PCR of the cytology samples confirmed the presence of Aspergillus flavus while fungal and bacterial cultures were negative. Treatment with topical 1% voriconazole solution was successful in resolving the keratomycosis.