Deep lamellar endothelial keratoplasty in 10 horses.

OBJECTIVE: To describe and evaluate a surgical technique utilized for the therapy of deep corneal stromal abscesses (DSA) in horses. The DSA is excised and replaced with a partial thickness corneal lamellar allograft. METHODS: A retrospective clinical study describing the indications for the surgical technique utilized and the outcomes of this procedure in 10 eyes of 10 horses. RESULTS: Each affected eye had a discrete DSA within the posterior stroma. An initial partial thickness semicircular corneal incision was made at the limbus, followed by anterior stromal lamellar dissection over the lesion. After excision of the DSA and replacement with a larger diameter split-thickness donor button, the anterior stroma was replaced into its original position and the initial corneal incision was repaired. All of the animals that underwent deep lamellar endothelial keratoplasty (DLEK) procedure healed appropriately and with subjectively less postoperative scarring and complications than previously described surgical approaches to DSA. CONCLUSIONS: This procedure is an effective technique for surgical removal of DSA in horses and, in most cases, results in a visual and cosmetically acceptable globe. The advantages of this technique compared to other surgical approaches to DSA are the peripheral location of the incision, shortened anesthesia times, the resultant minimal scarring and shorter healing times associated with DLEK.

Myocilin protein levels in the aqueous humor of the glaucomas in selected canine breeds.

OBJECTIVE: To compare aqueous humor myocilin protein levels in dogs with the primary glaucomas to those with the secondary glaucomas, primary cataracts, and diabetic cataracts. MATERIALS AND METHODS: Four groups were selected, based on diagnosis by the attending veterinary ophthalmologists and included: primary glaucoma (primary open-angle glaucoma (POAG) and primary closed angle glaucoma (PCAG); n = 155); secondary glaucoma (n = 94); primary (presumed inherited) cataract (n = 142), and diabetic cataract (n = 83). A total of 474 samples (187 males, 263 females, 24 unreported) with average ages of 117 months for the males and 101 months for the females were analyzed. Myocilin protein was measured using the Coomassie staining and Western blot methods relative to a myocilin control. RESULTS: Differences were seen between nonglaucomatous (cataractous) and glaucomatous dogs with myocilin levels in glaucomatous eyes being many times higher than those in the cataractous dogs. Primary glaucomatous dogs were found to have an aqueous humor myocilin protein level of 17.30 +/- 1.03 units. Secondary glaucomas had the highest level of myocilin in the aqueous humor with 19.27 +/- 1.41 units. Diabetic cataractous dogs had the lowest levels of myocilin reported with 6.60 +/- 0.88 (mean +/- SEM) units. Normal (cataractous) dogs had a myocilin level in the aqueous humor of 8.05 +/- 0.86 units. CONCLUSION: Aqueous humor protein levels were elevated, relative to the myocilin control, in both the primary and secondary glaucoma groups compared to the cataract and diabetic cataract groups. Like in the Beagle POAG, aqueous humor myocilin protein levels are increased. Further studies are indicated to investigate the exact role of the aqueous humor myocilin protein in the genesis in increased IOP in these primary glaucomatous breeds.

Corneal transplantation for inflammatory keratopathies in the horse: visual outcome in 206 cases (1993-2007).

OBJECTIVE: To evaluate the visual outcome of three techniques of corneal transplantation surgery in treating severe inflammatory keratopathies in the horse. DESIGN: Retrospective medical records study. ANIMALS STUDIED: Medical records of 206 horses that received corneal transplantation surgery at the University of Florida Veterinary Medical Center from 1993 to 2007 were reviewed. PROCEDURE: Data collected from the medical records included signalment, types of ocular lesions, type of transplant surgery performed, length of follow-up, complications, and visual outcomes. RESULTS: Full thickness penetrating keratoplasty (PK) was performed in 86 horses for melting ulcers, iris prolapse/descemetoceles, and medically nonresponsive full thickness stromal abscesses (SA). Posterior lamellar keratoplasty (PLK) and deep lamellar endothelial keratoplasty (DLEK) are split thickness penetrating keratoplasties that were utilized for medically nonresponsive deep stromal abscesses (DSA) in 54 and 66 eyes, respectively. The most common postoperative surgical complication was graft rejection and varying degrees of graft opacification. Wound dehiscence and aqueous humor leakage was also a common postoperative problem. A positive visual outcome was achieved for PK, PLK, and DLEK in 77.9%, 98.1%, and 89.4%, respectively. CONCLUSIONS: Corneal transplantation is a tectonically viable surgery in the horse with an overall success rate of 88.5% in maintaining vision when treating vascularized and infected corneal disease in the horse.

First Report of the Presence of Tomato apical stunt viroid on Tomato in Sénégal.

Tomato apical stunt viroid (TASVd) was initially discovered in the Ivory Coast (2). It was later reported in Indonesia and more recently was found to be responsible for severe outbreaks in protected tomatoes in Israel (1) and Tunisia (3). Although not of quarantine status, TASVd is included in the EPPO alert list. In 2005, severe arrest of apical growth and leaf chlorosis were observed in tomato samples from northern Sénégal. Tomato yellow leaf curl virus was initially identified in some samples, but since the symptoms observed were reminiscent of those associated with viroid infection, samples were analyzed by return-polyacrylamide gel electrophoresis and molecular hybridization with a Potato spindle tuber viroid (PSTVd) probe. Positive results prompted a reanalysis by reverse transcription-PCR assays specific for PSTVd or TASVd. Positive amplification was only obtained with the TASVd-specific primers (Vir+ GGGGAAACCTGGAGGAA and Vir- GGGGATCCCTGAAGGAC), and the identity of the viroid confirmed by sequencing of the amplified fragment. The complete genome sequence obtained (GenBank Accession No. EF051631) shows 94 to 96% identity with other TASVd sequences in the databases, the highest homology being with the original Ivory Coast isolate (96%, 11 mutations, and 4 indels for the 362-nt genome). These results provide new information on the diversity of TASVd and of its detrimental potential for tomato crops and represent, to our knowledge, the first report of the presence of TASVd in Sénégal. References: (1) Y. Antignus et al. Phytoparasitica 30:502, 2002. (2) C. R. Walter. Acad. Sci. 292:537, 1981. (3) J. Th. J. Verhoeven et al. Plant Disease 90:528, 2006.

[Study conducted at the Yaoundé University Hospital on anti-retroviral treatment compliance (Cameroon)]. / Etude sur l'observance des traitements antirétroviraux au Centre hospitalier universitaire de Yaoundé (Cameroun).

To build the knowledge base on anti-retroviral (ARV) treatment compliance in Africa, a study has been carried out at Yaoundé University Hospital. It documented and studied the follow up of 231 patients under tri-therapy at the time of the study (December 2003). Respecting the strict time schedules is a crucial issue, especially for taking the morning and evening doses of medication. This is true for all patients, regardless of their social or economic status. The majority of the patients request more thorough information about their treatment. Some patients face economic problems which are more often related to the indirect costs rather than to the cost of treatment. Finally, the difficulty in confronting the treatment's side effects or the galenic nature of ARV treatment itself is noted. Nevertheless, it seems that the patients could better manage these issues and deal with the ensuing challenges if better communication with more complete information was provided to them at the beginning of their treatment, accompanied by written fact sheets specific to each type of treatment.

Comment on "Acoustical observation of bubble oscillations induced by bubble popping".

We have reproduced the experiment of acoustic monitoring of spontaneous popping of single soap bubbles standing in air reported by Ding et al. [2aaPhys. Rev. E 75, 041601 (2007)]. By using a single microphone and two different signal acquisition systems recording in parallel the signal at the microphone output, among them the system used by Ding et al., we have experimentally evidenced that the acoustic precursors of bubble popping events detected by Ding et al. actually result from an acausal artifact of the signal processing performed by their acquisition system which lies outside of its prescribed working frequency range. No acoustic precursor of popping could be evidenced with the microphone used in these experiments, whose sensitivity is 1VPa-1 and frequency range is 500 Hz-100 kHz.

A national network of infectious diseases experts.

OBJECTIVES: The aim of this study was to consider the implementation of a network of infectious diseases (ID) experts to optimize the antibiotic prescriptions of community and hospital practitioners. METHODS: An observational prospective study was conducted among ID experts in the Pays-de-la-Loire Region to evaluate the number of calls and to determine the practitioner's reasons for soliciting ID expertise. For each phone consultation, four criteria were recorded during 5 days: origin of the call (internal/external), kind of question (diagnostic/therapeutic) time spent for the advice provided, type of advice. RESULTS: A total of 386 phone consultations for 20 infectious disease specialists were recorded during the study period (5 days); 81% were internal to the hospital, 7.7% from another hospital, and 11.3% from private practice, 56.3% of the questions concerned a therapeutic strategy, 21% a diagnostic advice, and 22.6% concerned both diagnosis and therapy. Two third of the questions were answered within 10minutes. In 68.7% of cases, the ID specialist answered immediately, 19.8% of calls required following-up the patient, 6% led to refer the patient to an ID consultation, and 5.5% to hospitalization. CONCLUSION: The survey results stress the important need for such ID expertise, both in hospitals and in ambulatory medicine. Collaboration of ID specialists in a regional network would allow an easy and permanent access to antibiotic therapy advice for prescribers. This network would improve the quality and safety of care.

Proteinases of the cornea and preocular tear film.

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.

Combined keratectomy, strontium-90 irradiation and permanent bulbar conjunctival grafts for corneolimbal squamous cell carcinomas in horses (1990-2002): 38 horses.

OBJECTIVE: The purpose of this study was to evaluate the effectiveness of postoperative beta-irradiation with strontium-90 as an adjunctive treatment to superficial keratectomy and permanent bulbar conjunctival graft for removal of equine corneolimbal squamous cell carcinoma (SCC), in decreasing recurrence rate. STUDY: The retrospective case study included 38 horses diagnosed and treated for SCC of the eye that involved the limbus and/or cornea. The patients were treated between 1990 and 2002, with strontium-90 irradiation immediately after corneal and conjunctival graft surgery. Recurrence was defined as the postoperative and postirradiation regrowth of SCC in the same site and globe that was previously treated. RESULTS: The Appaloosa was the most commonly represented breed and horses that had more than one base coat color represented the majority of the cases (53%). The coat colors of white, chestnut/sorrel and gray were the most commonly represented colors of the horses treated. Eight horses (21%) could not be assessed for tumor recurrence due to lack of two or more post-treatment examinations, and another horse was enucleated 6 days postoperatively due to progressive corneal ulceration. Twenty-four horses (63% of the entire study population; 83% of the followed cases) had a mean +/- SD of 1754 +/- 1319 days without tumor recurrence, ranging from 14 days to 5110 days. Five horses (13% of the entire study population; 17% of the assessed horses) had tumor recurrence at a mean +/- SD of 449 +/- 339 days with a range of 29 days to 900 days. For the five recurrences, treatment included local excision (n = 1), enucleation (n = 2), and additional strontium-90 therapy (n = 3). CONCLUSIONS: The combination of superficial keratectomy, beta-irradiation and permanent bulbar conjunctival grafts for limbal, corneal or corneolimbal SCC in horses is effective in at least 83% of the horses. Recurrence occurred in about 17% of the horses. Multiple biannual re-examinations are recommended to observe for tumor recurrence.

Amniotic membrane transplantation for corneal surface reconstruction after excision of corneolimbal squamous cell carcinomas in nine horses.

OBJECTIVE: The purpose of this study was to evaluate the usefulness and effectiveness of permanent amniotic membrane transplantation as an adjunctive treatment to superficial keratectomy alone or combined with strontium-90 irradiation for treatment of equine corneolimbal squamous cell carcinoma (SCC) to decrease corneal scarring and recurrence rate. STUDY: The retrospective case study included 11 horses (n = 12 eyes) diagnosed and treated for ocular SCC that involved the limbus and cornea. Nine of those horses (n = 9 eyes) were treated between 2002 and 2006, with superficial lamellar keratectomy alone or combined with strontium-90 irradiation and followed by placement of a permanent amniotic membrane graft in the surgical defect. The level of scarring (i.e. the clarity of the cornea) resulting with the use of amniotic membrane was subjectively compared to cases where a permanent bulbar conjunctival graft was performed following keratectomy combined with strontium-90 irradiation or cryotherapy (n = 3 eyes). Recurrence was defined as the postoperative and postirradiation regrowth of SCC in the same site and globe. RESULTS: The nine horses that received an amniotic membrane graft after keratectomy alone or combined with irradiation showed a minimal level of scarring in a cornea that regained a greater transparency in comparison to the horses that were treated with a bulbar conjunctival graft. All of the horses that received an amniotic membrane graft had 226 +/- 218 days of follow-up without tumor recurrence (mean +/- SD), ranging from 21 days to 778 days. CONCLUSIONS: The combination of superficial keratectomy alone or associated with beta-irradiation and permanent amniotic membrane transplantation is an effective treatment of corneal or corneolimbal SCC in horses. The placement of an amniotic membrane material represents an alternative surgical procedure to bulbar conjunctival grafts, especially if there is a lack of bulbar conjunctiva tissue available after tumor resection or if a particularly large corneal resection is necessary. The amniotic membrane is incorporated into the corneal defect and seems to create noticeably much less scarring than a corneal defect covered by bulbar conjunctiva.

Pars plana vitrectomy for the treatment of ophthalmomyiasis interna posterior in a dog.

Unilateral chorioretinitis caused by ophthalmomyiasis interna posterior in a 5-month-old, female spayed Dachshund is reported. Larva removal by pars plana vitrectomy was performed after an unsuccessful photocoagulation of the organism. The larva was intact after surgical removal and was identified as a first stage instar Cuterebra spp. larva. The eye remains visual and comfortable with no signs of residual or recurrent inflammation postoperatively. This report describes, for the first time, the successful surgical retrieval of an intravitreal fly larva in a dog using vitrectomy techniques without sacrificing either the globe or vision, and taxonomic identification of the parasite.

Optic nerve head neuroretinal rim blood flow differences in monkeys with laser-induced glaucoma.

INTRODUCTION: The blood flow of the neuroretinal rim (NRR) of the optic nerve head (ONH) of the rhesus monkey with laser-induced glaucoma was examined. METHODS: Argon laser photocoagulation of the trabecular meshwork to induce elevated intraocular pressure (IOP) was performed in one eye of nine normal male rhesus monkeys. The nasal and temporal NRR of the monkey ONH were examined by the Heidelberg retina tomograph/flowmeter (HRT/HRF) under neuromuscular blockade. A mixed effect analysis of variance was used to determine significant differences between eyes and between locations in the eyes. RESULTS: The average IOP in the hypertensive glaucoma and normal eyes was 34.8 +/- 7.2 and 16.0 +/- 1.9 mmHg, respectively. The HRT determined average overall cup to disc (C/D) area ratio in the glaucoma and normal eyes, which was 0.49 +/- 0.28 and 0.22 +/- 0.16, respectively. The mean temporal NRR HRF flow in the hypertensive eyes was significantly greater than in the normotensive eyes (P < 0.0001), than in the nasal NRR of the hypertensive eyes (P < 0.0001) and than in the nasal NRR of the normotensive eyes (P < 0.01). The mean nasal NRR HRF flow in the hypertensive eyes was significantly less than in the nasal NRR of the normotensive eyes (P < 0.01). There was no statistical difference between the mean HRF flow of the temporal and nasal NRR of the normotensive eyes. The elevated IOP positively influenced the flow values in the hypertensive eye (r = 0.724). CONCLUSIONS: The capillary microcirculation of the temporal NRR of the rhesus monkey ONH with laser-induced glaucoma has significantly increased blood flow, and the nasal NRR significantly reduced blood flow compared to blood flow in the NRR of normal normotensive monkey eyes.

Tolerance of the rabbit cornea to an n-butyl-ester cyanoacrylate adhesive (Vetbond).

OBJECTIVE: An n-butyl-ester cyanoacrylate adhesive available for veterinary surgery (Vetbond, 3M) was tested in rabbits for corneal irritation. PROCEDURES: Two experimental procedures were used on 24 rabbits: injection of the adhesive into an intralamellar corneal pocket (n = 10) and application of the glue to a mid-stromal corneal defect (n = 14). In both experiments the eyes were examined for 20 days for evidence of corneal irritation and tolerance. At the end of each experiment, histopathologic studies were performed on all corneas. RESULTS: The corneal reaction to the intrastromally injected cyanoacrylate was characterized clinically by slight edema and vascularization localized to the vicinity of the adhesive. A moderate foreign body-type reaction was found histologically. Following application of the adhesive to a central stromal defect, the treated corneas remained totally clear and histopathologic examination showed that the healing process was not altered compared to the controls. The mean retention time of the glue patch was 14 days. CONCLUSIONS: Intrastromal injection and surface application to a corneal defect of n-butyl-ester cyanoacrylate to a corneal defect induced only a mild inflammatory response and did not interfere with the reparative process. These findings suggest that this surgical adhesive would be acceptable for treating corneal ulcerations in animals.

Comparative morphology of the tapetum lucidum (among selected species).

OBJECTIVES: The phenomenon of 'eye-shine' is seen in a variety of animal species, and is generally thought to be related to the presence of an intraocular reflecting structure, the tapetum lucidum. The tapetum lucidum is a biologic reflector system that is a common feature in the eyes of vertebrates. It normally functions to provide the light-sensitive retinal cells with a second opportunity for photon-photoreceptor stimulation, thereby enhancing visual sensitivity at low light levels. The tapetum lucidum is presented here according to a classification based on the location, as well as the composition, of this reflective layer. Finally, the physical and chemical properties, as well as the origins of the different tapeta lucida, are discussed and compared. METHODS: The anatomic and biochemical aspects of the tapetum lucidum in various vertebrates are examined. Morphologic observations were made from paraffin and plastic embedded specimens. Specimens were treated with traditional stains and observed by light and transmission electron microscopy. RESULTS: Some species (primates, squirrels, birds, red kangaroo and pig) do not have this structure and they usually are diurnal animals. In vertebrates, the tapetum lucidum exhibits diverse structure, organization and composition. Therefore, the retinal tapetum (teleosts, crocodilians, marsupials, fruit bat), the choroidal guanine tapetum (elasmobranchs), the choroidal tapetum cellulosum (carnivores, rodents, cetacea), and the choroidal tapetum fibrosum (cow, sheep, goat, horse) are described. CONCLUSIONS: The tapetum lucidum represents a remarkable example of neural cell and tissue specialization as an adaptation to a dim light environment and, despite these differences, all tapetal variants act to increase retinal sensitivity by reflecting light back through the photoreceptor layer. These variations regarding both its location and structure, as well as the choice of reflective material, may represent selective visual adaptations associated with their feeding behavior, in response to the use of specific wavelengths and amount of reflectance required.

Corneal thickness and endothelial cell density measured by non-contact specular microscopy and pachymetry in Rhesus macaques (Macaca mulatta) with laser-induced ocular hypertension.

PURPOSE: Sustained increase in intraocular pressure (IOP) in humans results in a loss of corneal endothelial cells and an increase of corneal thickness. The effects of chronically elevated IOP on the corneal endothelium of monkeys with laser-induced ocular hypertension, a commonly used animal model of human glaucoma have not been documented. This study examined the central corneal thickness (CCT), the corneal endothelial cell density (ECD), and the corneal endothelial cell size (ACS) in Rhesus monkeys with experimental ocular hypertension. Materials and methods. Ten male monkeys with argon laser-induced ocular hypertension in one eye for an average duration of 2.4+/-0.7 years, were sedated with ketamine hydrochloride, and the CCT, ECD, and ACS measured at the center of the cornea of both eyes with a Topcon SP-2000P non-contact specular microscope (Topcon America Corporation((R)), Paramus, NJ, USA). CCT was also measured using a DHG-500 Pachette ultrasonic pachymeter (DHG Technology Inc., Exton, PA, USA). Mean and standard deviation (S.D.) of CCT, ECD and ACS for each eye was calculated and statistically compared.Results. Mean CCT in the hypertensive and normal eyes measured by specular microscopy was 0.477+/-0.023mm and 0.468+/-0.020 mm, respectively. Mean ECD in the hypertensive and normal eyes was 2601.7+/-631.8 and 3990.2+/-402.9 cells mm(-2), respectively. The mean size of the endothelial cells was 252.4+/-23.9 micro m(2) in the normal eye and 408.7+/-115.0 microm m(2) in the hypertensive eye. No significant difference in the measurement of CCT was observed between the specular microscope and the pachymeter (p=0.46). No significant difference in the mean CCT was observed between the two eyes (p=0.4820), whereas the mean ECD was significantly lower in the hypertensive eye than in the normal eye (p<0.001). The ECD was inversely related to the length of IOP elevation (p<0.001). CONCLUSIONS: No difference in the corneal thickness measurement was observed between the specular microscopy and the pachymetry techniques. Chronic ocular hypertension did not significantly affect the CCT, but caused a significant loss of endothelial cells in the center of the cornea of the laser treated eyes compared to the normotensive eyes. The duration of elevated IOP was the most important factor affecting the ECD.

Time-specific intraocular pressure curves in Rhesus macaques (Macaca mulatta) with laser-induced ocular hypertension.

OBJECTIVE: To detect and categorize time-specific variations in daytime intraocular pressure (IOP) found in Rhesus monkeys with laser-induced ocular hypertension. PROCEDURES: Ten male monkeys with argon laser-induced ocular hypertension in one eye were anesthetized with ketamine hydrochloride, and the IOP measured in both eyes at 7 a.m., 7.30 a.m., and then hourly until 1 p.m. with a Tonopen trade mark XL applanation tonometer. Intraocular pressure time profiles for both eyes in each animal were developed. The means +/- SD of the IOPs for both eyes were calculated for the whole 6-h study period, and the values compared statistically. The difference between the lasered eye mean IOP standard deviation and the normal eye mean IOP standard deviation for each animal during the 6-h follow-up was also calculated and compared. RESULTS: Mean IOP (+/- SD) in the glaucoma and normal eyes for the 10 animals during the 6-h study was 32.6 +/- 2.5 and 14.9 +/- 2.5 mmHg, respectively. The IOP was significantly higher in the experimental eye than in the normal eye (P = 0.0008). The mean IOP in the lasered eye did not significantly change during the study period, whereas a slight but significant increase in IOP of the normal eye over the study period was recorded (P = 0.003). The variance in IOP in the hypertensive eyes was considerably greater than that in the untreated control eyes. From 7 a.m. to 1 p.m. the IOP declined in five eyes and increased in the other five eyes with laser-induced ocular hypertension. CONCLUSIONS: The time-specific IOP variation pattern in the daytime in the laser treated eyes is significantly greater than the variation in the normotensive eyes. This shows that in order to detect statistical differences between IOP variations induced by an IOP-reducing drug, and the exaggerated spontaneous IOP variations present in the laser-induced hypertensive eye, sufficient animals should be included in any study. Understanding the time-specific IOP variation present in a group of monkeys with laser-induced ocular hypertension is essential prior to using the model for the evaluation of IOP-reducing drugs.

Connective tissue growth factor in tear film of the horse: detection, identification and origin.

BACKGROUND: Healing of corneal ulcers in horses is often associated with profound corneal stromal fibrosis and scar formation resulting in visual impairment. Connective tissue growth factor (CTGF) is a fibrogenic cytokine involved in wound healing and scarring. The purpose of this study was to determine whether CTGF was present in the tear fluid of normal horse eyes and the eyes of horses with corneal ulcers in order to evaluate the role of CTGF in corneal wound healing and corneal scar formation. METHODS: Tear fluid samples were collected from 65 eyes of 44 horses; 32 samples from normal eyes, 21 samples from eyes with corneal ulceration, and 12 samples from the unaffected contralateral eyes of horses with ulcers. CTGF levels in the tears were determined by enzyme immunoassay using goat IgG against human CTGF. Antigenetic similarity of human and horse CTGF was established in a bio-equivalence assay. The identity of horse CTGF was confirmed by western blot. Lacrimal and nictitating membrane glands were investigated by immunohistochemistry in the attempt to clarify the origin of tear fluid CTGF. RESULTS: CTGF was detected in tear film of 23 normal unaffected eyes (72%) and 8 normal contralateral eyes (67%), with the mean CTGF levels (+/- SEM) being 51.5+/-19.2 and 13.4+/-3.9 ng/ml respectively. CTGF was found in 8 eyes with corneal ulcers (38%) with the mean CTGF concentration of 26.3+/-14.8 ng/ml. Western blot identified the protein detected as CTGF. The identification of CTGF in lacrimal glands suggests a major role of these glands in the presence of CTGF in tears. CONCLUSIONS: CTGF is present in horse tear fluid and derives, at least partly, from the lacrimal gland. Equine CTGF has strong antigenic similarity with human CTGF. Corneal disease leads to a decrease of CTGF concentrations in tears. The possible role of CTGF in the healing process of ocular surface requires further investigation.

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.