Anterior corneal dystrophy of American Dutch belted rabbits: biomicroscopic and histopathologic findings

Spontaneously occurring anterior corneal opacities were present in related, juvenile American Dutch belted rabbits. Slit lamp biomicroscopy revealed focal opacities of epithelium, basement membrane, and subepithelial corneal stroma. Lesions were characterized histologically by thin and disorganized surface epithelium, thickened and intensely staining epithelial basement membrane, fimbriated and irregular basement membrane-stromal juncture, and disorganized subepithelial stroma. Biomicroscopic and histopathologic features of anterior corneal dystrophy of American Dutch belted rabbits appear similar to those of human anterior corneal dystrophies.     

Histological and immunohistochemical evaluation of canine chronic superficial keratitis.

Chronic superficial keratitis (CSK) is an inflammatory disease of the canine cornea, characterised by infiltration of leucocytes into the anterior corneal stroma. The present study describes a quantitative histomorphometric analysis of the cell types infiltrating the corneal stroma in this disease. Infiltrating cells were quantified in samples taken at superficial keratectomy and processed for routine histology. Further characterisation of lymphocyte phenotypes was achieved by immunohistochemistry performed using a panel of monoclonal antibodies recognising canine lymphocyte CD antigens. Lymphocytes expressing the CD4 antigen were found to be the predominant infiltrating cell types in the CSK lesion. A significantly smaller number of lymphocytes expressed the CD8 antigen. The CD4/CD8 ratio was consistently above 2 and rose to above 4 at the advancing border of the lesion. A proportion of lymphocytes were shown by immunohistochemistry to contain gamma interferon. This study forms a basis for work further evaluating the cytological events central to the development of this spontaneous potentially auto-immune corneal disease.     

Rose bengal positive epithelial microerosions as a manifestation of equine keratomycosis

Purpose To describe the clinical appearance of corneal epithelial cell microerosions associated with keratomycosis in the horse. METHODS: Retrospective clinical study. RESULTS: Multifocal, punctate, superficial corneal opacities with positive rose bengal retention were noted in six horses with presumed 'viral keratitis'. Faint fluorescein staining was also present in three cases. Equine herpesvirus tissue culture inoculation was negative for a cytopathic effect in three cases. Aspergillus (n = 3), Curvularia (n = 1), and an unidentified fungus (n = 1) were cultured in five horses, and hyphae found on corneal cytology from the sixth. Mixed bacterial infections were present in three eyes. The eyes of two horses with Aspergillus progressed to deep melting corneal ulcers that required surgical therapy. The microerosions remained superficial, but persistent in the other four eyes. Natamycin was utilized topically in all six horses. Transmission electron microscopy from case 6 revealed mucin layer disruption, an intact corneal epithelial cell layer, and fungal attachment to degenerating epithelial cells. The visual outcome was positive in all six horses, although healing was prolonged (48.5 +/- 14.5 days on average in the horses with no surgery; 62 days on average in the two horses that required surgery). CONCLUSIONS: Complete removal or full-thickness penetration of the corneal epithelial cell barrier may not be necessary to allow fungal adherence and initiation of keratomycosis in the horse. Prior to colonization and invasion of the horse cornea, fungi may induce changes in the mucin layer of the tear film that result in or are associated with rose bengal positive microerosions of the superficial corneal epithelium. Horses with painful eyes, and eyes with superficial, multifocal corneal opacities should have their corneas stained with both fluorescein and rose bengal as fungal microerosions may stain weakly, or not at all, with fluorescein, and may thus be mistaken for presumed 'viral keratitis' of the horse.     

Iridociliary epithelial tumors in 100 dogs and 17 cats: a morphological study

The morphological features of iridociliary epithelial tumors in 100 dogs and 17 cats were reviewed. Twenty-seven cases were in either Golden Retrievers or Labrador Retrievers. Affected globes were stained for light microscopy with alcian blue, periodic acid Schiff (PAS) and hematoxylin and eosin stains. Selected tissues were examined by immunohistochemistry for vimentin, desmin, cytokeratin, S-100, neuron-specific enolase (NSE), and glial fibrillary acid protein (GFAP). The presence or absence of hyaluronic acid was recorded by staining with alcian blue before and after digestion of the tissue with hyaluronidase. Canine tumors were divided into papillary and solid tumors based on the pattern of growth. Twenty-eight of 57 papillary tumors exhibited invasive behavior including eight of the 57 which showed infiltration of the sclera. Twenty-nine of 43 solid tumors were invasive including 13 of 43 with scleral invasion. Tumors with scleral invasion were designated adenocarcinoma. Tumors of both types could be pigmented or nonpigmented and often contained smooth basement membranes reminiscent of the inner membrane of the nonpigmented ciliary body epithelial cell. All of the feline tumors were nonpigmented and 14 of 16 feline tumors were solid and two of the tumors were papillary. Eighteen of 20 canine tumors and three of four feline tumors stained positive for vimentin. Cytokeratin stain was positive only in a few of the highly aggressive tumors. The finding of pigmented epithelial cells, thick, smooth basement membrane structures, positive staining for vimentin, S-100, and NSE as well as hyaluronic acid deposition were considered to be features which define iridociliary epithelial tumors in dogs. The positive staining for vimentin and NSE are highly specific markers which help to characterize iridociliary epithelium and distinguish this tumor from metastatic epithelial tumors. The finding of solid nonpigmented tumors with small epithelial cells packeted by thin PAS-positive membranes staining positive for vimentin were considered significant features defining iridociliary epithelial tumors in cats. Follow-up information on survival and cause of death was obtained on 43 canine cases and only two feline cases. The average follow-up interval in dogs was 25 months and only two dogs died with lesions that could have been due to metastasis although neither was confirmed. We concluded that neither iridociliary adenomas nor adenocarcinomas is likely to metastasize.     

Major histocompatibility class II expression in the normal canine cornea and in canine chronic superficial keratitis

OBJECTIVE: To compare the expression of major histocompatibility complex (MHC) class II antigen in the corneas of normal dogs and dogs affected with chronic superficial keratitis (CSK). METHODS: MHC class II expression was determined in frozen sections of normal canine cornea and cornea from lesions of CSK by immunohistochemistry using a monoclonal antibody directed against the canine MHC class II molecule. Langerhans cell phenotype was determined morphologically and by histochemical determination of ATPase activity. To determine the influence of gamma interferon on expression of MHC class II molecules by corneal cells, corneal explants were cultured with the cytokine and MHC class II expression determined as above. RESULTS: Numerous MHC class II-expressing cells were demonstrated within the stroma and epithelium of the normal corneal limbus and conjunctival epithelium while very little MHC class II expression was detected in the central region of normal canine cornea. In limbal and conjunctival epithelium, cells expressing MHC class II antigen showed ATPase activity, suggesting that they were Langerhans cells. Corneas from dogs with CSK showed MHC class II expression associated with stromal cells, some of which exhibited a dendritic morphology while most were lymphocytic. Corneal epithelial cells within the lesion also aberrantly expressed MHC class II. Corneal explants expressed MHC class II to varying degrees after differing periods of incubation with the cytokine gamma interferon. CONCLUSIONS: While the normal central cornea has little MHC class II expression, aberrant expression occurs in CSK, associated with secretion of gamma interferon by infiltrating CD4-expressing lymphocytes. Although this change is likely to be a secondary feature of the CSK lesion, increased MHC class II expression may play a part in perpetuating the corneal inflammation seen in the disease.     

Diagnosis and management of chronic corneal epithelial defects (indolent corneal ulcerations)

Chronic corneal epithelial defects (CCEDs; indolent corneal ulcerations) are the most common refractory ulcerations in veterinary medicine and are diagnosed by their classic appearance. CCEDs are superficial ulcerations without stromal involvement and have a nonadherent epithelial border (lip). Fluorescein stain adheres to the exposed stroma and extends below the epithelial border, outlining the epithelial lip. CCEDs occur secondary to adnexal disease, keratoconjunctivitis sicca, exposure keratitis, neurotrophic keratitis, and primary corneal disease. In cats, herpes keratitis is associated with the development of CCEDs. Bacterial infections are not responsible for the refractory nature of CCEDs. Because of the refractory nature of CCEDs, treatment can be frustrating for both owner and veterinarian. Current treatment recommendations consist of identifying and treating the underlying cause and performing procedures that stimulate epithelialization and adhesion of the corneal epithelium. Initial treatment of CCEDs includes ulcer debridement and grid keratotomy. Superficial keratectomy is indicated in refractory cases.     

Spontaneous chronic corneal epithelial defects in dogs: a review

Spontaneous chronic corneal epithelial defects (SCCEDs) in dogs are typically found in middle-aged dogs of all breeds. These epithelial defects may be present for weeks to months, particularly if left untreated or if treated inappropriately. Typical histopathological findings include loss of the corneal epithelial basement membrane and formation of a superficial, acellular, hyalinized zone in the stroma. Together, these histological abnormalities lead to delayed wound healing and poor epithelial adhesion. Epithelial debridement, anterior stromal puncture, grid keratotomy, and superficial keratectomy are the most common treatment options applied to the defects. Procedures that address the stromal changes present generally have a higher success rate than epithelial debridement alone.     

Evaluation of commercially available antibodies to cytokeratin intermediate filaments and laminin in normal cat pinna.

The pattern of distribution of cytokeratin (CK) intermediate filaments can be used to characterize subsets of epithelial tissues. The purpose of the study was to examine the CK expression of feline pinna skin. Six normal feline pinnae were routinely processed in formalin. An immunohistochemical method was used to stain the pinnae with 8 commercially available anti-human CK antibodies (Abs) (PKK1, CAM 5.2, UCD 10/11, 35BH11, 34BE12, AE1/AE3, MAK 6, A575) and an anti-human laminin Ab. All the CK Abs selectively localized to epithelium except 35BH11, which did not react with any part of the pinna. Some epithelial subsets were identified by their unique staining pattern with CK Abs. Basal cells but not suprabasal cells of the epidermis stained with PKK1; basal but not lumenal cells of apocrine glands stained with 34BE12. Apocrine glands stained with all CK Abs except 35BH11. All epithelial structures were stained with A575. Basal lamina of epithelial and mesenchymal tissues was clearly identified by the anti-laminin Ab. The results indicate that in cat pinna some commercially available anti-human CK Abs selectively stain subsets of epithelium and adnexa. PKK1, 34BE12, and A575 were the CK Abs with the most consistent staining patterns, the other Abs stained more variably from pinna to pinna. The pattern of epithelial and adnexal staining was similar but not identical to that reported for humans.     

MHC class II risk haplotype associated with canine chronic superficial keratitis in German Shepherd dogs

Canine chronic superficial keratitis (CSK) is an inflammatory ocular disease of an autoimmune origin leading to blindness if untreated. The main symptoms of CSK are progressive, bilateral vascularisation, fibrous tissue formation and pigmentation of the anterior corneal stroma. Although CSK is found in many breeds it is most prevalent in German Shepherd dogs (GSDs). Since Major Histocompatibility Complex (MHC) class II is associated with several autoimmune diseases in dogs we investigated the possible role of DLA-DRB1, -DQA1 and -DQB1 in GSDs affected with CSK. Our study population included 25 healthy controls and 30 CSK dogs. Most of the affected dogs were females suggesting a female predisposition. We identified 11 unevenly distributed haplotypes of which DLA-DRB1*01501/DQA1*00601/DQB1*00301 was significantly associated with the CSK dogs (OR=2.67, CI=1.17-6.44, p=0.02). We also found that overall homozygosity of MHC class II increases risk for CSK (OR=4.37, CI=1.27-18.46, p=0.02) and homozygosity of the risk haplotype by over eight-fold (OR=8.5, 95% CI=1.4-224, p=0.017). This study identifies a MHC class II risk haplotype for CSK in GSD and further supports the autoimmune origin of the disease.     

Establishing a reproducible method for the culture of primary equine corneal cells

Objective  To establish a reproducible method for the culture of primary equine corneal epithelial cells, keratocytes, and endothelial cells and to describe each cell's morphologic characteristics, immunocytochemical staining properties and conditions required for cryopreservation.
Procedures  Corneas from eight horses recently euthanized for reasons unrelated to this study were collected aseptically and enzymatically separated into three individual layers for cell isolation. The cells were plated, grown in culture, and continued for several passages. Each cell type was characterized by morphology and immunocytochemical staining.
Results  All three equine corneal cell types were successfully grown in culture. Cultured corneal endothelial cells were large, hexagonal cells with a moderate growth rate. Keratocytes were small, spindloid cells that grew rapidly. Epithelial cells had heterogenous morphology and grew slowly. The endothelial cells and keratocytes stained positive for vimentin and were morphologically distinguishable from one another. The epithelial cells stained positive for cytokeratin. Keratocytes and endothelial cells were able to be cryopreserved and recovered. The cryopreserved cells maintained their morphological and immunocytochemical features after cryopreservation and recovery.
Discussion  This work establishes reproducible methods for isolation and culture of equine corneal keratocytes and endothelial cells. Cell morphology and cytoskeletal element expression for equine corneal epithelial cells, keratocytes, and endothelial cells are also described. This has not previously been reported for equine corneal cells. This report also demonstrates the ability to preserve equine keratocytes and endothelial cells for extended periods of time and utilize them long after the primary-cell collection, a feature that has not been reported for veterinary corneal cell culture.     

Comparative analysis of canine dermatophytosis and superficial pemphigus for the prevalence of dermatophytes and acantholytic keratinocytes: a histopathological and clinical retrospective study

Acantholytic dermatophytosis is a rarely reported condition of dogs that clinically and histopathologically mimics superficial pemphigus (erythematosus, foliaceus). Histologically, periodic acid-Schiff (PAS) and Grocott's methenamine-silver (GMS) are often necessary to show the fungus. A retrospective histopathological study was conducted on 190 canine skin biopsy specimens: 95 each with the diagnosis of canine dermatophytosis or of superficial pemphigus. All specimens were stained with haematoxylin and eosin, PAS, and GMS. Dermatophytes were not seen in any superficial pemphigus cases. Acantholytic keratinocytes were noted in 14% of the dermatophytosis cases, none of which had clinical signs consistent with superficial pemphigus. Among cases with acantholytic keratinocytes, superficial pemphigus had significantly more acantholytic cells than dermatophytosis (P = 0.02). When comparing face and nonface cases, there was no difference in prevalence of acantholytic keratinocytes in dermatophytosis or number of acantholytic keratinocytes in superficial pemphigus. All dermatophyte cases were both GMS and PAS positive with neither stain being visually superior. No dermatophyte cases where acantholytic keratinocytes were noted had a history, clinical signs and histopathological features compatible with acantholytic dermatophytosis.     

Corneal anatomical differences in the aetiology of chronic superficial keratitis

The apparently normal corneas of 14 diseased eyes of German shepherd dogs (GSDs) were shown to possess very thin epithelial layers on microscopy. Normal corneas of 38 GSDs and 113 dogs of various breeds and crossbred animals were compared for epithelial thickness, structure of corneal stroma and corneal strength. Due to lower values for these criteria found in the GSDs they are considered to play an important role in the aetiology of chronic superficial keratitis.     

Histopathologic evidence of capecitabine corneal toxicity in dogs

In an experimental model of transplant rejection, renal transplants were performed on 6 mixed-breed dogs. Capecitabine (CPC) was administered as an oral immunosuppressive agent. All recipients received systemic CPC, cyclosporine (CSA), prednisolone, and famotidine throughout the study. Two dogs developed superficial keratitis, which was characterized by multifocal geographic erosions, superficial corneal epithelial pigmentation, and corneal neovascularization. These clinical signs correlated with the dose of CPC given, whereas other drug doses remained unchanged. After euthanasia, routine histologic sections were stained with hematoxylin and eosin and with alcian blue periodic acid-Schiff for light microscopic evaluation. Ocular histopathologic abnormalities were limited to neovascularization and inflammatory infiltrate of the anterior corneal stroma and abnormal basal cell morphology, disorganization, thinning, and pigmentation of the corneal epithelium. The purpose of this communication is to describe the clinical and histopathologic evidence of CPC corneal toxicity in dogs.     

Fine structure of the reindeer cornea in normal conditions and in keratitis

The fine structure of the reindeer cornea in normal conditions and in spontaneous keratitis was described. There were more layers of squamous and fewer layers of polyhedral cells than in pigs, cattle and horses.Mild cases of corneal opacity just had some swollen superficial epithelial cells.When fully developed the keratitis had all the characteristics of an acute inflammation. Swelling or shrinking of the epithelial cells with or without degenerative nuclear changes were common. A striking alteration of the epithelial cells was the irregular appearance of the filaments which was frequently observed. In some cases there was pronounced intercellular oedema with desquamation of the superficial squamous cells and occasional neutrophils in the intercellular space. The stroma was the site of oedema, infiltration of leukocytes and vascularization.The aetiology and pathogenesis of the disease are not yet established but experimental research on these problems is in progress.     

Spongiotic vesicular dermatitis as a cutaneous reaction pattern in seven horses

Over a 6-year period seven adult horses of different breeds and genders developed multifocal, exudative, oozing dermatitis characterized histologically by epidermal spongiotic vesicles and perivascular eosinophilic, neutrophilic and mixed mononuclear inflammation. Three horses were pruritic. Systemic disease was not noted. Two horses had a history of recurrent urticaria (hives) and one horse had nodules or welt-type lesions that progressed to exudative, oozing lesions. Interepithelial immunoglobulin (Ig)G was detected by avidin-biotin complex-peroxidase staining, but the pattern of staining was more consistent with epithelial oedema than specific IgG deposition associated with pemphigus. The exudative oozing lesions developed under circumstances suggesting that dermal oedema progressed to intracellular and intercellular epidermal oedema, which in turn progressed to the spongiotic vesicular epidermal lesions.     

Morphology,cytochemical staining and ultrastructural characteristics of reindeer (Rangifer tarandus) leukocytes

Peripheral blood smears from four adult reindeer (Rangifer tarandus) were examined after staining with Romanowsky's stain and cytochemical stains, including alpha-napthyl butyrate esterase (alpha-NBE), Sudan black B (SBB), chloroacetate esterase (CAE) and alkaline phosphatase (ALP). Romanowsky-stained eosinophils, neutrophils, lymphocytes and monocytes resembled those of cattle, sheep and goats. Basophils had two different staining patterns with Romanowsky's stain. Basophils that we termed "grey basophils" were similar in appearance to grey eosinophils in Greyhound dogs, with medium blue-grey to lavender-grey cytoplasm containing varying numbers of clear vacuoles or granules and variable numbers of small, intensely basophilic, perinuclear granules. The second basophil staining pattern was more typical of ruminant basophils, with uniform, pale to dark basophilic cytoplasmic granules. Basophils stained positive for alpha-NBE, SBB, CAE, and ALP. Eosinophils stained positive for SBB, and were negative for alpha-NBE, CAE, and ALP. Neutrophils were negative for SBB, CAE, and ALP. Monocytes stained positive for alpha-NBE, were rarely positive for CAE and SBB, and were negative for ALP. Transmission electron microscopy revealed matrix within all granulocytes granules, including those of basophils.     

Chronic superficial keratitis in dogs: detection of cellular hypersensitivity.

Dogs affected with chronic superficial keratitis (CSK) and clinically normal dogs were tested for cellular hypersensitivity, using the leukocyte migration-inhibition (LMI) technique to 3 ocular antigens (Staphylococcus aureus and corneal and iridal proteins). Affected dogs had statistically significant increases in hypersensitivity cellular responses against corneal and iridal antigens. Affected dogs did not differ from clinically normal dogs in their cell response to S aureus.     

Amyloid-producing odontogenic tumor in a Shih-Tzu dog

A 9-month-old male Shih-Tzu dog had a right mandibular tumor composed of strands, or nest-like proliferation of epithelial cells with abundant fibrous stroma characterized by spheroid to large nodular deposition of amyloid with Congo-red stain. Globule calcification was also seen throughout the tumor tissue and the spheroid depositions often had a concentrically laminated structure (Liesegang rings). The case was diagnosed as amyloid-producing odontogenic tumor in a dog.     

The effect of topical pimecrolimus on keratoconjunctivitis sicca and chronic superficial keratitis in dogs: results from an exploratory study

OBJECTIVE: Pimecrolimus is an ascomycin derivative that interferes selectively with the activation of T cells and mast cells and inhibits the production of inflammatory cytokines. This study evaluated the efficacy of an experimental ophthalmic formulation of pimecrolimus in treating keratoconjunctivitis sicca (KCS) and chronic superficial keratitis (CSK) in dogs. ANIMALS AND PROCEDURES: Eight dogs with KCS and six with CSK were included. The dogs were of various breeds, suffered from chronic conditions, and had been pretreated unsuccessfully. The affected eyes were treated with 1 drop of an experimental, corn oil-based pimecrolimus 1% formulation three times a day. Parameters evaluated included Schirmer tear test (STT), ocular discharge, conjunctival inflammation, corneal inflammatory cell infiltrate and scarring, and comfort level. RESULTS: The effect of pimecrolimus 1% was pronounced (increase in STT values to higher than 4 mm/min, no signs of inflammation) or moderate (increase in STT values of 3-4 mm/min, mild signs of corneal/conjunctival inflammation) in a total of 6/8 animals with KCS. In 4/6 animals with CSK, the effect was either pronounced (total regression of fibrovascular infiltration into the cornea, no corneal scarring) or moderate (distinct regression of pannus, mild corneal scarring). The response to treatment was unsatisfactory in four of 14 animals. CONCLUSION: Results of this exploratory study suggest that topical 1% pimecrolimus may be a new effective treatment for keratoconjunctivitis sicca and chronic superficial keratitis in dogs.