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.     

Schirmer tear test values and tear film break-up times in cats with conjunctivitis

OBJECTIVES: (1) To document tear film break-up time (TFBUT) in a group of cats with conjunctivitis; (2) to determine if TFBUTs from cats with conjunctivitis vary significantly from previously established normal values for TFBUT in young cats without ocular disease; (3) to determine if a correlation exists between Schirmer tear test (STT) values and TFBUTs in cats with conjunctivitis; (4) to determine if the TFBUTs in cats with conjunctivitis are influenced by the detection of DNA from feline herpes virus-type 1 (FHV-1), Chlamydophila felis, Mycoplasma spp., and feline calicivirus. ANIMALS STUDIED: Fourteen cats between the ages of 0.8 years to 12 years with active, untreated conjunctivitis and without active keratitis or other ocular or systemic abnormalities were included in this study. Procedures Complete ophthalmic examinations, including TFBUT, were performed on all cats. Polymerase chain reaction (PCR) screening for FHV-1, Chlamydophila felis, Mycoplasma spp., and feline calicivirus was performed on conjunctival swabs from affected eyes and blood samples from all cats. RESULTS: Mean TFBUT for cats in this study was 8.9 (+/- 4.8) s in the right eye (OD) and 8.1 (+/- 4.6) s in the left eye (OS). No correlation existed between mean TFBUTs and mean STT values OD or OS. Conjunctival swabs from seven cats (n = 9 eyes) tested positive via PCR for one of the above infectious agents. Blood samples from nine cats tested positive for FHV-1. Mean TFBUTs for cats from which the DNA from FHV-1 was isolated from the blood were significantly lower than mean TFBUTs for cats from which no such DNA was isolated from the blood. CONCLUSIONS: In this study, the mean TFBUT in cats with conjunctivitis was significantly lower than previously established values for clinically healthy cats. This supports the theory that qualitative tear film deficiency, and thus tear film instability, may play a role in the pathogenesis of feline conjunctivitis. Qualitative tear film deficiency may predispose to the development of conjunctivitis or may occur secondarily to this condition.     

Tear-film osmolarity in normal cats and cats with conjunctivitis

Objective To compare the tear‐film osmolarity of normal cats and cats with conjunctivitis. Animal studied The population consisted of shelter, research, and privately owned cats. Procedures Cats were classified as normal or having conjunctivitis. An ophthalmic examination including Schirmer tear test (STT), fluorescein staining, tear‐film break‐up time (TFBUT), intraocular pressure (IOP), and slit‐lamp biomicroscopy of the anterior segment was performed. The severity of conjunctivitis was graded and assigned a numerical score. The Tear Lab^TM Osmolarity System was utilized to determine the tear‐film osmolarity. Unpaired t‐tests were used to compare tear‐film osmolarity, TFBUT, IOP, and STT of the two groups. Results A total of 93 cats (186 eyes) were examined. There were 37 normal cats (74 eyes) and 39 conjunctivitis cats (78 eyes). The mean age was 2.34 years. There was no statistical difference (P = 0.2065) between the median tear‐film osmolarity of normal cats (328.5 ± 17.94 mOsms/L) and conjunctivitis cats (325.0 ± 24.84 mOsms/L). Cats with conjunctivitis had an accelerated TFBUT (P < 0.0001) and lower IOPs (P < 0.0001) as compared to normal cats. No statistical difference was found between STT values (P = 0.1304). Conclusions The median tear‐film osmolarity of normal cats was 328.5 mOsms/L. Despite the accelerated TFBUT, conjunctivitis did not cause a statistically significant change in tear‐film osmolarity. The Tear Lab^TM Osmolarity System was easily used and well tolerated by the cats in the study.     

Qualitative tear film and conjunctival goblet cell assessment of cats with corneal sequestra

OBJECTIVES: To evaluate the tear film qualitatively and conjunctival goblet cell numbers in cats with and without corneal sequestra. ANIMALS STUDIED AND PROCEDURES: This was a prospective evaluation of 11 cats with corneal sequestra and 14 control eyes that were either the contralateral normal eye when the sequestrum was unilateral or from control cats of similar age with no ocular disease. All cats in this study were examined by a veterinary ophthalmologist. The ophthalmic examinations included a neuro-ophthalmic evaluation, Schirmer tear tests, fluorescein staining, tear film break-up times, applanation tonometry, biomicroscopy, and indirect ophthalmoscopy. The palpebral conjunctiva at the dorsal nasal, ventral nasal, dorsal temporal and ventral temporal fornices were biopsied after topical anesthetic was applied to the cornea and conjunctiva. The conjunctival biopsies were fixed in formalin and sectioned routinely and stained with hematoxylin and eosin, and periodic acid-Schiff. These slides were examined by light microscopy by a blinded examiner. Goblet cell numbers were compared to conjunctival basal epithelial cell numbers by region. The goblet cell numbers by region from the eyes with sequestra was statistically compared to those from eyes without sequestra, with a student's paired t-test. Conjunctival swabs were collected from the cats with corneal sequestra and submitted for polymerase chain reaction for Herpes felis, Chlamydia psiitticia, and Mycoplasma felis. The corneal sequestra were removed by surgical keratectomy and fixed and stained routinely, and examined by light microscopy. RESULTS: No neurologic abnormalities were detected in any of the cats. The Schirmer tear tests (eyes with sequestra 14+/-5.1 mm/min; normal eyes 15+/-6.8 mm/min) and intraocular pressures (eyes with sequestra 21+/-6.6; normal eyes 22+/-5.8) were within normal reference ranges for cats. Biomicroscopic examinations revealed varied sizes and depths of brown- and amber-colored corneal sequestra. No abnormalities were noted on indirect ophthalmoscopic examinations. The tear film break-up time was 21 s (+/-12) for the normal eyes (n=14) and 14 s (+/-13) in eyes with corneal sequestra (n=11). The average goblet/epithelial cell ratios by region for the normal eyes and the eyes with sequestra respectively were 0.66, 0.56 for the dorsal nasal fornix, 0.68, 0.57 for the ventral nasal fornix, 0.63, 0.48 for the temporal dorsal fornix, and 0.55, 0.49 for the temporal ventral fornix. There were no significant differences in tear film break-up times and goblet cell numbers in eyes with corneal sequestra and those without sequestra. Three conjunctival swabs from two of 11 cats with sequestra were positive with PCR for Herpes felis virus. These included one cat with bilateral sequestra and one cat with unilateral corneal sequestrum. CONCLUSIONS: The pathogenesis of feline corneal sequestra does not appear to be linked primarily to abnormal goblet cell numbers, qualitative tear film abnormalities, and accelerated tear film break-up time.     

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.     

Determination of tear break-up time reference values and ocular tolerance of tetracaine hydrochloride eyedops in healthy horses

Reasons for performing study: Tetracaine hydrochloride (THCl) has been reported to cause irritation in dogs. In man, some topical anaesthetics have been shown to disrupt the tear film. Tear break‐up time (TBUT) is a useful test allowing an assessment of the quality of the precorneal tear film. Only one TBUT value has been reported in horses with no information on the technique used. Objectives: To provide a method for performing the TBUT in horses and to report any side effects of a single application of THCl in clinically normal horses, particularly on the stability of the tear film. Methods: In Study 1, one drop of 0.5 or 1% THCl was applied to one eye of 20 horses divided in 2 groups. Treated eyes were assessed for the development of side effects 2.5 and 5 min after treatment. In Study 2, the TBUT was measured in both eyes of 2 groups of 10 horses, before and 2.5 and 5 min after, instillation of one drop of either 0.5 or 1% THCl. Results: No animals developed any ocular side effect after instillation. Basal TBUT was 8.3 ± 1.3 s. TBUT decreased from baseline 5 and 2.5 min after application of one drop of 0.5% THCl and one drop of 1% THCl, respectively. Conclusions: A technique to measure the TBUT in healthy horses is described and normal range values that could be used as a reference were obtained. Potential relevance: THCl is well tolerated in horses but lowers the TBUT.     

Effects of trimethoprim-sulfadiazine on tear production and the fluctuations of Schirmer tear test values in horses

The objectives of this study were to observe the effects of trimethoprim-sulfadiazine on equine tear production and to determine normal fluctuations in Schirmer tear test (STT) values in horses. A randomized, placebo-controlled, blinded clinical trial measuring STT values in 15 horses over an 8-week period was performed. The treatment group (eight horses) received 30 mg/kg trimethoprim-sulfadiazine orally once a day and the control group (seven horses) received placebo (flour) at the same time. All horses were housed outdoors throughout the study. Schirmer tear test values were measured at 0, 2, 4, 6 and 8 weeks, and 4 weeks after discontinuation of treatment. There were no significant differences in tear production between the treated and control groups. Fluctuations in STT were observed and may result from individual and environmental variations. Trimethoprim-sulfadiazine did not decrease tear production in the horses in this study. Horses normally experience periodic fluctuations in STT values.     

Topical effect of various agents on gelatinase activity in the tear film of normal dogs

OBJECTIVE: To evaluate the topical effect of various agents, currently used in the treatment of melting ulcers, on gelatinase activity present in the tear film of normal dogs. ANIMAL STUDIED: Eight normal adult beagles. PROCEDURES: Each animal received the following agents: cyclosporine A 1%, N-acetylcysteine 10%, ciprofloxacin 0.3%, ethylenediaminetetraacetic acid (EDTA) 1%, doxycycline 0.001%, polysulfated glycosaminoglycans (PSGAG) 5%, autoserum, and artificial tears during a 48-h period following a Latin square design. Tear samples were collected with micro-capillary pipettes following a corneal surface irrigation of each eye with sterile saline on four different occasions. Basal and total gelatinase activities were evaluated by optical density after processing in a commercial gelatinase activity assay. From the optical density ratio, a semi-quantitative measure of gelatinase activity was obtained. Basal and total activities were measured in all samples. RESULTS: The lowest total gelatinase activity, representing a percent decline in the enzyme activity although not significant, was observed 1 h after the last treatment in seven out of the eight ophthalmic agents; EDTA (68%), ciprofloxacin (76%), cyclosporine A (68%), doxycycline (47%), artificial tears (26%), PSGAG (25%), and N-acetylcysteine (20%). However, only the reduction observed with EDTA 6 h after the last treatment was significantly lower compared to the reduction observed with the artificial tears. CONCLUSION: This study indicated that only EDTA was able to significantly reduce the gelatinase activity in a persistent manner in the tear film of normal canine eyes. Further studies will be required to evaluate the effect of EDTA under ulcerative conditions and to more accurately ascertain the potential in vivo effect of the other agents.     

Characterization of cytokines associated with Th17 cells in the eyes of horses with recurrent uveitis

Objective Equine recurrent uveitis (ERU) is a spontaneous disease that is the most common cause of blindness in horses, affecting up to 15% of the horse population. Th17 cells are a major cell population driving the pathogenesis in several mouse models of autoimmune inflammation, including experimental autoimmune uveitis. The purpose of this study is to investigate the role a Th17 cell‐mediated response plays in the pathogenesis of ERU. Procedure Banked, Davidson’s‐fixed equine globes histopathologically diagnosed with ERU (n = 7) were compared immunohistochemically with healthy control globes (n = 7). Immunohistochemical staining was performed using a pan‐Leptospira antibody and antibodies against IL‐6, IL‐17, and IL‐23. Additionally, immunostaining was performed for T‐cell (CD3) and B‐cell (CD79α) markers. Specificity of immunoreactivity was confirmed by western blot analysis. Results Immunohistochemical staining was positive for IL‐6, IL‐17, and IL‐23 within the cytoplasm of nonpigmented ciliary epithelial cells and mononuclear inflammatory cells infiltrating the iris, and ciliary body of ERU horses (n = 7) but negative in controls (n = 7). ERU‐affected eyes were CD3 positive (n = 7) and CD79α negative (n = 7). Staining for Leptospira was negative in all ERU and control globes. Conclusions Strong immunoreactivity for IL‐6, IL‐17, and IL‐23, in conjunction with the fact that T lymphocytes are the predominating inflammatory cells present in ERU, suggests that IL‐17‐secreting helper T‐cells play a role in the pathogenesis of ERU. These findings suggest that horses with ERU may serve as a naturally occurring animal model for autoimmune uveitis.     

The effects of lactated Ringer's solution (LRS) or LRS and 6% hetastarch on the colloid osmotic pressure, total protein and osmolality in healthy horses under general anesthesia

ObjectiveTo investigate changes in colloid osmotic pressure (COP), total protein (TP) and osmolality (OSM) during anesthesia in horses given intravenous lactated Ringer’s solution (LRS) or LRS and hetastarch (HES).Study designProspective, clinical trial.AnimalsFourteen horses presented for surgery. Mean age 8.3 ± 1.9 years; mean weight 452 ± 25 kg.MethodsHorses were premedicated with xylazine intravenously (IV); anesthesia was induced with ketamine and diazepam IV, and maintained with sevoflurane. Butorphanol was administered IV with pre-medications or immediately after induction. Xylazine was administered IV for recovery if necessary. LRS was administered IV to all horses with a target rate of 5–10 mL kg^?1 hour^?1. Half of the horses also received 6% HES, 2.5 mL kg^?1 over 1 hour in addition to LRS. Horses that received LRS only were considered the LRS group. Horses that received both LRS and HES were considered the LRS/HES group. Blood was drawn pre- and post-anesthesia, immediately following induction, and every 30 minutes throughout anesthesia. COP, TP and OSM were measured.ResultsCOP and TP significantly decreased at similar rates for both treatment groups from pre-anesthetic values. Pre-anesthetic COP was significantly greater in the LRS group when compared to the LRS/HES group pre-, post- and throughout anesthesia. In the LRS group post-anesthetic OSM was significantly different than the pre-anesthesia value and that for the LRS/HES group.Conclusions and clinical relevanceAdministration of IV HES (2.5 mL kg^?1, over 1 hour) in combination with LRS does not attenuate the decrease in COP typically seen during anesthesia with crystalloid administration alone. Based on these results, administration of HES at this rate and total volume would not be expected to prevent fluid shifts into the interstitium through its effects on COP.     

Normal reference intervals and the effects of sample handling on dynamic viscoelastic coagulometry (Sonoclot) in healthy adult horses

Objective

To determine reference intervals and the effect of sample agitation and rest time on Sonoclot analysis in healthy adult horses.

Design

Original prospective study.

Setting

University veterinary medical teaching hospital.

Animals

Sixty healthy adult horses.

Interventions

Blood was collected for assessment of complete blood count, serum biochemical analysis, and Sonoclot analysis.

Measurements and Main Results

Horses were determined to be healthy based upon physical examination, CBC, and serum biochemistry analysis. Blood was analyzed in a glass bead‐containing cuvette using the Sienco Sonoclot analyzer following 2 rest periods (30 mins and 240 min) and with 2 sample handling interventions (agitated and nonagitated), to obtain values for clot rate, time‐to‐peak, activated clotting time, and platelet function. This study failed to detect a significant difference when a rest time of 30 minutes was compared with 240 minutes, but based on wide limits of agreement the 2 rest times were not considered interchangeable. Agitation at both rest times significantly affected all Sonoclot analyses leading to changes indicative of hypercoagulability.

Conclusions

Sample agitation and rest time should be taken into consideration when developing preanalytical guidelines for Sonoclot analysis in horses. Calculated reference intervals were relatively wide. Further research is needed to evaluate the clinical utility of Sonoclot analysis in horses.     

Serum concentration of surfactant protein D in horses with lower airway inflammation

Reasons for performing study: Surfactant protein D (SP‐D), mainly synthesised by alveolar type II cells and nonciliated bronchiolar cells, is one important component of innate pulmonary immunity. In man, circulating concentrations of SP‐D are routinely used as biomarkers for pulmonary injury. To date, serum SP‐D levels have only been investigated in horses in an experimental model of bacterial airway infection. Objectives: To compare serum SP‐D concentrations at rest and after exercise in horses with and without inflammatory airway disease (IAD). Methods: Venous blood samples were collected from 42 Standardbred racehorses at rest and 60 min after performing a standardised treadmill exercise test. Tracheal wash and bronchoalveolar lavage fluid (BALF) samples were collected after exercise. Based on BALF cytology, 22 horses were defined as IAD‐affected and 20 classified as controls. Serum SP‐D concentrations were assessed using a commercially available ELISA kit and statistically compared between groups of horses and sampling times. Results: Serum concentrations of SP‐D in IAD‐affected horses were significantly higher than those of control horses, both at rest and after exercise. Within the IAD‐affected group, no significant correlation was found between serum SP‐D concentrations and BALF cytology. Within each group of horses (IAD and control), no significant influence of exercise was found on serum SP‐D levels. Conclusions: This is the first study determining serum SP‐D concentrations in a noninfectious, naturally occurring form of lower airway inflammation in horses. The results highlight that IAD is associated with a detectable, though moderate, increase of circulating SP‐D levels. Potential relevance: Serum concentration of surfactant protein D could represent a potentially valuable and readily accessible blood biomarker of equine lower airway inflammation.