Biomicroscopy of the tear film: aqueous and lipid tear substitutes in the normal and abnormal eye

Polarised light biomicroscopy was used to assess the behaviour and interactions of two polymer-containing preparations of artificial tears, and two lipid-containing ointments, with the normal and abnormal pre-corneal tear film. The preparations were used topically in six normal dogs, and three dogs with keratoconjunctivitis sicca. A transient morphological alteration of the surface lipid layer of the tear film was commonly observed after the use of polymer solutions. The ointments spread as a continuous or semi-continuous layer over the normal, or polymer-supplemented, aqueous tears. Further applications of artificial tears produced only a temporary disruption of this layer. In dogs with keratoconjunctivitis sicca, thickening of the lipid layer of the tear film was observed for over 20 hours after the administration of ointment. These observations are discussed in relation to the prospects for the improved medical treatment of keratoconjunctivitis sicca by the use of polymer-containing solutions and ointments in combination.     

Polarized light biomicroscopic observations on the pre-corneal tear film. 1. The normal tear film of the dog

Polarized light biomicroscopy was used to examine the normal pre-corneal tear film in 25 dogs. Blink rates were also determined. The normal range of surface morphology of the canine tear film was established. Three categories of surface patterning of the lipid layer of the tear film were identified. These were: (a) a wave-like pattern, (b) a pattern of irregular islets, and (c) a pattern resembling the polished surface of granite. All three patterns may occur in the same eye. The inter-blink thickness of the lipid layer of the pre-corneal tear film varied between 0·013 μm (the minimum thickness discernable by this method) and over 0·581 μm (the maximum thickness at which coloured interference fringes are clearly defined) in localized areas. The mean total blink rate was 14·2/min. Overall, incomplete blinks constituted approximately 66 per cent of the total blink rate, but the ratio of complete to incomplete blinks varied considerably between animals. By dividing the dogs into thick and thin lipid layer groupings, it was possible to demonstrate a statistically greater rate of complete blinks (P < 0·05, Mann-Whitney U test) in animals with a thinner lipid layer. The applicability of polarized light biomicroscopy to veterinary clinical ophthalmology is discussed.     

Peliosis hepatis-like lesion in a pekingese dog. A case report

Peliosis hepatis (PH) is a rare condition characterized by dilatation of hepatic sinusoids and the presence of blood-filled spaces within the liver. The disease occurs mainly in subjects exposed to toxic substances or estrogens, and is often asymptomatic. A 9 year-old pekingese female dog was sent to Division of Pathology for autopsy because of sudden death of unclear cause. Autopsy revealed severe hydroperitoneum (1500 ml of transsudate), dilatation of heart ventricles, passive hyperemia of lungs, spleen and kidneys. The liver was enlarged, with slightly wrinkled surface, with many red spots, of different size and shape. Microscopic examination of the liver revealed the presence of areas with severe dilatation of hepatic sinusoids, and multiple cystic spaces filled with blood. Some of these spaces were lined with endothelium and other were not. Spaces were different in shape and size and were localized in the areas between dilated sinusoids. Based on the macroscopic and microscopic examination, the liver lesions were identified as severe hepatic sinusoidal dilatation with formation of peliosis hepatis-like structures.     

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.     

Polarized light biomicroscopic observations on the pre-corneal tear film. 2. Keratoconjunctivitis sicca in the dog

Polarized light biomicroscopy was used to examine the pre-corneal tear film in 16 dogs which were suspected of, or confirmed to be, suffering from keratoconjunctivitis sicca. One animal displayed a normal tear film, while the other 15 showed variable degrees of abnormality. The abnormal features included: contamination of the ocular surface with particulate debris and plaques of mucus, discontinuity and excessive granularity of the surface lipid film, pronounced thinning of the aqueous phase, and breakup of the tear film. After parotid duct transposition, the tear film of some animals was almost indistinguishable from normal. However, in others the surface lipid layer was thinned, discontinuous, or virtual absent. Discontinuity of surface lipid was most obvious in animals with marked salivary epiphora, and was associated with a variable amount of crystalline concretion over the ocular surface. The clinical usefulness of polarized light biomicroscopy for the diagnosis, and subsequent monitoring, of keratoconjunctivitis sicca is discussed.     

Polarized light biomicroscopic observations on the pre-corneal tear film. 3. The normal tear film of the cat

Polarized light biomicroscopy was used to examine the pre-corneal tear film in 14 normal cats. Blink rates were also determined. The normal range of surface morphology of the feline tear film was established. Two principal categories of surface patterning of the lipid layer of the tear film were identified. These occurred in all eyes examined and were: (a) a wave-like pattern, and (b) a pattern of variable character resembling the polished surface of granite. Over most of the area of the tear film, the thickness of its lipid layer between blinks was under 0·089 μm in all cats examined. However, narrow streaks of thicker lipid (up to 0·511 μm) were occasionally observed. The mean total blink rate was 2·4/min. Overall, incomplete blinks constituted approximately 50 per cent of the total blink rate. The tear film of the cat is compared with that of man and the dog.     

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.     

The tear film and ocular mucins

Abstract The trilaminar tear film, composed of the lipid, aqueous and mucin layers, has many functions including defending the ocular surface. The aqueous layer has several soluble antimicrobial factors that protect the ocular surface. Ocular mucins have recently been studied with regard to their role in the defense of the eye as well as in dry eye syndromes. To date, 15 mucin genes have been identified, and six of these mucin genes are localized to or secreted by ocular glands or epithelia. Understanding the production, secretion and function of ocular mucins will aid in the treatment of dry eye syndromes and ocular surface microbial infections.     

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.     

Decreased tear production associated with general anesthesia in the dog.

Schirmer I tear tests were done on 20 dogs before and during general anesthesia. Atropine sulfate reduced tear production from a mean base-line value of 20.35 +/- 3.33 mm/minute to 14.10 +/- 6.13 mm/minute, 10 minutes after its subcutaneous administration. The combination of subcutaneous atropine, intravenous thiamylal sodium, and inhalation anesthesia reduced tear production to a mean value of 0.53 +/- 1.12 mm/minute at 60 minutes after induction.     

Daytime profile of the intraocular pressure and tear production in normal dog

Objective  To evaluate the circadian rhythms of intraocular pressure (IOP) and tear production in dog exposed to a natural photoperiod.
Animals studied  We used 12 clinically healthy Beagles dog housed under natural photoperiod at indoor temperature and humidity.
Procedure  Intraocular pressure and Schirmer tear test (STT) I were measured every 4 h over a 48-h period in both eyes in each animal. Statistical analysis of the data was performed by one-way repeated-measures anova , Student's t -test, and single cosinor method.
Results  On each day, there was a highly significant effect of time on both parameters. A statistically significant difference of STT I values was observed comparing left and right eyes ( P  < 0.0001). Robust daily rhythms were observed for both parameters, IOP values showed diurnal acrophase (left eye: 09:33 ± 00:50 h; right eye: 09:25 ± 00:22 h), while STT I values showed nocturnal acrophase (left eye: 20:27 ± 00:46 h; right eye: 20:00 ± 00:05 h).
Conclusion  This study has demonstrated circadian rhythms in both IOP and STT I.     

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.     

Jejunal incarceration by an omental tear in a dog.

A 2-year-old male mixed-breed dog was examined after being hit by a car, and exploratory celiotomy revealed jejunal incarceration through an omental tear. The affected intestinal segment was distended and black, and had small serosal tears. Clinical signs of disease and hematologic, serum biochemical findings during surgery were consistent with concurrent diagnosis of sepsis. The dog responded to supportive treatment and resection of the affected intestinal segment by jejunal end-to-end anastomosis.     

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.     

Ulcerative keratitis associated with qualitative tear film abnormalities in cats

Three cats with indolent corneal ulcers and one cat with bilateral corneal sequestration and normal aqueous tear production were found to have rapid tear break-up times (BUTs). Tear BUTs in clinically affected cats averaged 2.5 ± 1.29 s and 2.33 ± 0.58 s for the right and left eyes, respectively. Palpebral conjunctival biopsies were harvested from consistent sites from each eye of affected cats ( n  = 7 affected eyes), and age-and breed-matched controls ( n  = 2 unaffected eyes). Light microscopy revealed a marked decrease to complete absence of conjunctival goblet cells (average goblet cell (GC):epithelial cell (EC) density = 18:50), conjunctival epithelial dysplasia, squamous metaplasia, and neutrophilic and mononuclear cell submucosal infiltration in affected cats. Specimens from the control cats had an average GC:EC density of 34:50, and minimal submucosal inflammatory infiltrate. The corneas ( n  = 7 eyes) healed following surgical keratectomy with ( n  = 2 eyes) or without ( n  = 1 eye) conjunctival pedicle flaps, superficial keratectomy and striate keratotomy with ( n  = 2 eyes) or without ( n  = 2 eyes) third eyelid flaps, and mucinomimetic tear supplementation ( n  = 5 eyes). Goblet cell regeneration was confirmed after 5 months of mucinomimetic supplementation ( n  = 2 eyes). The etiology for these mucin deficiencies remains unknown.     

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.     

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.     

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.