Effects of anti-inflammatory drugs and preservatives on morphologic characteristics and migration of canine corneal epithelial cells in tissue culture

Objective To determine the effects of commonly used ophthalmic corticosteroids, suprofen, polysulfated glycosaminoglycan and preservatives on morphologic characteristics and migration of canine corneal epithelium grown in cell culture. Animals studied Corneal epithelial cells harvested from the corneas of euthanized dogs were propagated in cell culture. Procedures Canine corneal epithelium was grown in tissue culture. The cells were treated with different corticosteroids, polysulfated glycosaminoglycan, suprofen or preservatives at different concentrations after a defect was created in the monolayer. Cellular morphologic characteristics and closure of the defect were compared between test drugs and controls. Results Morphologically the cells treated with dexamethasone were essentially the same as controls. Prednisolone and hydrocortisone caused rounding and shrinkage of the cells. Both suprofen and polysulfated glycosaminoglycan caused no apparent changes in morphologic characteristics at the lowest concentrations tested, but at higher concentrations there was a concentration‐dependent degree of rounding and shrinkage. Benzylkonium chloride and thimerosal caused rounding and shrinkage of all the cells at all concentrations tested. Dexamethasone, hydrocortisone, and suprofen did not inhibit epithelial migration over the defects at the lowest concentrations tested. All other drugs and concentrations inhibited cellular migration. Conclusion Dexamethasone affected the morphologic characteristics and migration of corneal epithelial cells less than hydrocortisone and prednisolone; therefore, dexamethasone may be the drug of choice when a corticosteroid is indicated and an epithelial defect is present. Suprofen and polysulfated glycosaminoglycan caused a concentration‐dependent effect on morphologic characteristics and migration. The preservatives caused severe changes and inhibited migration of the canine corneal epithelial cells at all concentrations and may therefore contribute to poor epithelialization of ulcers treated with preservative‐containing drugs.     

Antibiotic susceptibility of bacteria isolated from cats with ulcerative keratitis in Taiwan

Objectives: To assess the antibiotic susceptibility of bacteria isolated from corneal ulcers in cats. Methods: A total of 92 cats with infected corneal ulcers were swabbed for bacterial culture and the antibiotic sensitivity of the isolates analysed. Results: Bacteria were isolated from 54 of 92 infected eyes with corneal ulcers and purulent discharge. A total of 59 bacterial isolates were obtained from the 54 ulcers. The ratio of Gram-positive to Gram-negative isolates was approximately 3:1. The most commonly isolated Gram-positive bacteria were Staphylococcus species (51 per cent of all isolates), while Pseudomonas aeruginosa (13.5 per cent of all isolates) was the most common Gram-negative bacteria isolated. The Gram-negative isolates demonstrated a greater incidence of antibiotic resistance than the Gram-positive ones. The most effective antibiotics against the isolates were ciprofloxacin, tobramycin and gentamicin, with erythromycin and lincomycin showing the greatest number of resistant isolates. Clinical Significance: Staphylococcus and Pseudomonas species were the most common Gram-positive and Gram-negative bacteria, respectively, isolated from feline eyes with ulcerative keratitis. The second-generation fluoroquinolone, ciprofloxacin and the aminoglycoside gentamicin were found to be highly effective against the majority of isolates.     

Isolation and characterization of primary canine lens epithelial cells

OBJECTIVE: The purpose of this study was to characterize the proliferation and differentiation of primary canine lens epithelial cells (LEC) under standard culture conditions. PROCEDURE: Canine LEC were isolated by mechanical dissection of the canine globe and enzymatic digestion of the lens capsule from fresh lenses. Isolated capsules and cell suspensions were seeded in laminin-coated culture flasks. Canine LEC proliferated and formed monolayers, which could be passaged and maintained for approximately 2 weeks. Cells were characterized morphologically and cell lysates examined for expression of protein markers of epithelial origin and differentiation. RESULTS: Canine LEC exhibit morphologic characteristics of epithelial cells when cultured on laminin/lysine coated flasks. Expression of epithelial cell marker, cytokeratin 5, was highest at passage 1 and diminished with increasing passage number. Expression of gamma-crystallin, a protein found only in differentiated lens fiber cells, increased at passage 6. A laminin/lysine-coated surface supported optimal proliferation of canine LEC. Both an initial seeding density of 1 x 10(5) cells/cm(2) and culture in Dulbecco's modified essential media (DMEM) supplemented with 10% FBS supported a doubling time of less than 48 h in canine LEC. CONCLUSION: This study demonstrates that primary canine LEC retain the characteristics of lens epithelial cells prior to passage 6 under the described culture conditions and represent a suitable in vitro model for investigating lens physiology and cataractogenesis.     

Detection of cell detachment activity induced by Moraxella bovis

OBJECTIVE: To characterize the effect that filtrate obtained from cultures of Moraxella bovis has on cultured corneal epithelial cells and other types of cultured mammalian cells. SAMPLE POPULATION: Cultured hamster corneal epithelial cells, bovine epithelial cells, and several transformed cell lines exposed to culture filtrate from a pathogenic isolate of M bovis. PROCEDURE: Moraxella bovis was cultured, and bacteria were removed by filtration. The resulting bacterial culture filtrate was incubated with various types of cultured cells, and effects of the filtrate on detachment of various mammalian cell types was quantified by the use of neutral red dye. Additionally, bacterial culture filtrate was treated with protease inhibitors as well as trypsin and heat prior to incubation with cultured mammalian cells. RESULTS: Bacterial culture filtrate of M bovis caused all types of cultured cells to detach from each other and from the substrate, with the maximal effect evident 2 hours after initiating incubation. Detached cells were alive, and detachment was reversible. Serine protease inhibitors (phenylmethylsulfonylfluoride and alpha2-macroglobulin) inhibited cell detachment attributable to bacterial culture filtrate. Heating and treatment with trypsin also inhibited cell detachment. CONCLUSIONS AND CLINICAL RELEVANCE: Moraxella bovis produces a soluble factor that causes reversible detachment of cultured cells. This activity may play a role in the pathogenesis of infectious bovine keratoconjunctivitis.     

Cytopathic effects of Moraxella bovis on cultured bovine neutrophils and corneal epithelial cells

The effects of Moraxella bovis on the morphologic features of purified bovine neutrophils and bovine corneal epithelial cells were examined, using transmission and scanning electron microscopy and light microscopy. Within 2 minutes after incubation of bovine neutrophils with living M bovis, electron microscopic cellular changes included vacuolation, swelling, and loss of microplicae. Most of the neutrophils were lysed by 10 minutes of incubation. Human neutrophils phagocytosed the M bovis and remained intact, even after 30 minutes of incubation with the bacteria. Living M bovis killed bovine corneal epithelial cells in vitro. Sterile filtrates prepared from 6-hour shaker cultures of M bovis also killed bovine corneal epithelial cells, but the cytotoxic activity was less than that produced by the living bacteria. Cellular changes were first observed in specimens collected 1 hour after corneal cell monolayers were inoculated with sterile culture filtrates. The changes in these cells included pit-like lesions on the cellular surface, cellular separation, and vacuolation.     

Establishing and functional testing of a canine corneal construct

Purpose  To provide a model to be used for in vitro studies on drug effects in dogs, this study was conducted to establish a protocol for the construction of a three-dimensional corneal construct. Primary canine corneal cells and a rabbit corneal epithelial (RCE) cell line were used in comparison.
Methods  The corneal construct was assembled step by step in membrane inserts of a six-well plate over a total of 5 weeks, including culture at the air–liquid interface to allow a differentiation of the epithelial cells. The constructs were studied histologically.
Single cell cultures of canine corneal cells as well as RCE cells were stimulated with lipopolysaccharides (LPS) and sodium dodecyl sulfate (SDS). Treatment with different concentrations of dexamethasone was used to test its effects on the cellular prostaglandin E₂ (PGE₂) production. The same experiments were repeated with the corneal constructs and the reactions compared. Expression of the glucocorticoid receptor (GR) in the constructs was studied using immunohistochemistry.
Results  A protocol for the construction of a vital corneal construct was established and the morphological similarity to the canine cornea in vivo shown. The GR protein was detected in all three cell types of the constructs. Stimulation with LPS and SDS led only in the corneal constructs to a significantly increased PGE₂ production, which could be reduced by dexamethasone.
Conclusions  The corneal construct is an interesting system to test drug effects on corneal cells. It allows studies on a cornea-like system including all three major cell types.     

Basic fibroblast growth factor stimulates epithelial cell growth and epithelial wound healing in canine corneas

Objective  To evaluate the effect of basic fibroblast growth factor (bFGF) on the proliferation of canine corneal epithelial cells and epithelial wound healing.
Animal studied  Canine corneal epithelial cells from the corneas of euthanized dogs and corneal epithelial wounds on one eye from each of 24 dogs.
Procedures  The proliferation of corneal epithelial cells in vitro was measured using the methylthiazolyl-tetrazolium (MTT) assay. A corneal wound on one eye of each dog was made with a corneal trephine (6 mm diameter). Four concentrations of bFGF, 0, 100, 500, and 1000 ng/mL, were applied to the affected eyes of dogs, t.i.d. Fluorescein staining was used to assess closure of the corneal epithelial wound.
Results  The addition of bFGF resulted in a significant increase in epithelial proliferation at 24 h after culture, except 1 ng/mL bFGF. Cells with all bFGF treatments proliferated significantly at 48 and 96 h compared to those in the non-bFGF group. bFGF at a concentration of 10 ng/mL promoted cell proliferation maximally. The wound healing rate in the bFGF-treated groups was greater than that in the control. All corneal wounds in bFGF-treated corneas closed by day 7, whereas two of six corneal wounds in the control showed poor healing. None of the eyes developed corneal clouding or neovascularization during the experiment.
Conclusions  Basic fibroblast growth factor accelerated the proliferation of canine epithelial cells and effectively promoted corneal epithelial wound healing.     

Culture of feline corneal epithelial cells and infection with feline herpesvirus-1 as an investigative tool

OBJECTIVE: To isolate and characterize pure cultures of feline corneal epithelial cells and to assess the extent and nature of feline herpesvirus (FHV)-1 infection in these cells. SAMPLE POPULATION: Healthy eyes from 23 recently euthanatized cats. PROCEDURE: Stroma and epithelium of the rostral portion of the cornea were surgically isolated, and epithelial cells were detached from the stroma by enzymatic incubation. Epithelial cells were cultured in hormone-supplemented media. Cells were passaged, and cytokeratin expression was assessed. Cells were then infected with FHV-1, and cytopathic effects were determined. RESULTS: Cell cultures were readily established from samples obtained from each eye and could be maintained through 6 passages. Cultured cells expressed cytokeratins 3 and 12 but not other cytokeratins. Infection with FHV-1 was rapid and caused widespread cytopathic effects. CONCLUSIONS AND CLINICAL RELEVANCE: Feline corneal cells cultured in vitro during multiple passages maintain consistent morphologic characteristics and intermediate filament expression. They are susceptible to infection with FHV-1 and may provide a useful in vitro model for investigation of ocular drugs.     

Detection of Resistance to Aminoglycoside Antibiotics and Four Resistance Genes of Canine Escherichia coli

The objective of this paper was to investigate the drug resistance of canine Escherichia coli (E.coli) strains and the carrying rates of four resistance genes,and explore the relationship between resistance phenotypes and resistance genes.This article chose the aminoglycoside antibiotics including gentamicin,amikacin,spectinomycin and tobramycin to carry out the antibiotics sensitivity test.According to the established PCR assays,we detected the molecular characteristics of the 156 strains of isolates.The positive fragments of four kinds of resistance genes were cloned and sequenced,and the relationships between antibiotics sensitivity test and the resistance genes were analyzed.The results showed that the resistance rates of canine E.coli strains to gentamycin,tobramycin,spectinomycin and amikacin were 55.8%,32.7%,25.0% and 20.5%,respectively.The detection rates of resistance genes aacC2,aphA3, aadA and aacC4 were 55.8%,26.3%,23.1% and 9.0%,respectively.Two strains carried all four kinds of resistance genes,eight strains carried two kinds of resistance genes,and the strains carried two or more resistance genes accounted for 40.4% (63/156).The sequence analysis showed that the amplified gene fragments had higher homology compared with the sequences from GenBank.The main resistance gene in canine E.coli was aacC2 and there was a positive correlation between resistance rates and resistance genes compliance rate.     

两例犬化脓性子宫内膜炎的病原分离鉴定及药敏试验

本研究对两例确诊为化脓性子宫内膜炎的犬进行了病原的研究。用阴道采样法采集子宫分泌物进行细菌培养和生化鉴定,结果第1例因连续使用抗生素,而分离不到细菌;第2例分离到葡萄球菌和链球菌;药敏试验结果表明,犬子宫内膜炎的主要病原菌对临床使用较多的青霉素和链霉素有很大的耐药性,而对头孢唑啉、环丙沙星、恩诺沙星等药物较为敏感,为临床首选药物。     

In vivo confocal microscopy in the normal corneas of cats, dogs and birds

OBJECTIVE: To evaluate the applicability of in vivo confocal microscopy (IVCM) in veterinary ophthalmology and analyze the morphology of living, healthy cornea. ANIMALS EXAMINED: Thirty-seven dogs, 34 cats and five birds. PROCEDURE: Various corneal sublayers were visualized in the central region using an in vivo confocal corneal microscope (HRTII/RCM). RESULTS: An investigation method was developed and adapted for use on animals with varying skull forms and eye positions. Real-time images of the epithelial cells, the corneal stroma and the endothelial layer were obtained. The corneal stromal nerve trunks and the subepithelial and basal epithelial nerve plexus were visualized. In dogs, full corneal thickness (FCT) was 585 +/- 79 microm (mean +/- SD) and endothelial cell density (ECD) 3175 +/- 776 cells/mm(2) (mean +/- SD). In cats, FCT was 592 +/- 80 microm and ECD 2846 +/- 403 cells/mm(2). There were no significant differences between canine and feline FCT and ECD and no morphologic differences could be seen between dogs and cats. The bird images revealed a number of structural differences. CONCLUSION: Noninvasive IVCM allows accurate detection of corneal sublayers, corneal pachymetry, endothelial cell density and corneal innervation in various animal species. For clinical usage, patients must be under general anesthesia. The confocal images provided anatomic reference images of various healthy corneal structures in dogs, cats and birds.     

Identification and antimicrobial susceptibility of coagulase positive staphylococci isolated from healthy dogs and dogs suffering from otitis externa

A total of 67 strains of coagulase positive staphylococci isolated from healthy dogs and dogs suffering from otitis externa were studied. Twenty-two isolates were from healthy dogs (five from hound dogs and 17 from companion dogs) and 45 from dogs suffering otitis externa (14 from hound dogs and 31 from companion dogs). Presumptive identification was attempted using the following tests: production of acetoin, anaerobic utilization of mannitol, acid production from mannitol, presence of beta-galactosidase, and growth on P agar supplemented with different concentrations of acriflavine. Susceptibility of staphylococci to 16 antibiotics was determined. Most effective antibiotics were imipenem, amoxycillin/clavulanic acid, ciprofloxacin, tobramycin, gentamicin and marbofloxacin. Penicillin, ampicillin and polymyxin B showed the lowest activity. There were no significant differences in antimicrobial susceptibility among isolates from healthy dogs and dogs suffering from otitis externa.     

In vivo confocal microscopy of the normal equine cornea and limbus

Objective  To describe morphologic features, pachymetry and endothelial cell density of the normal equine cornea and limbus by in vivo confocal microscopy.
Animals studied  Ten horses without ocular disease.
Procedure  The central and peripheral corneas were examined with a modified Heidelberg Retina Tomograph II and Rostock Cornea Module using a combination of automated and manual image acquisition modes. Thickness measurements of various corneal layers were performed and endothelial cell density determined.
Results  Images of the constituent cellular and noncellular elements of the corneal epithelium, stroma, endothelium, and limbus were acquired in all horses. Corneal stromal nerves, the subepithelial nerve plexus, and the sub-basal nerve plexus were visualized. Cells with an appearance characteristic of Langerhans cells and corneal stromal dendritic cells were consistently detected in the corneal basal epithelium and anterior stroma, respectively. Median central total corneal thickness was 835 μm (range 725–920 μm) and median central corneal epithelial thickness was 131 μm (range 115–141 μm). Median central endothelial cell density was 3002 cells per mm² (range 2473–3581 cells per mm²).
Conclusions  In vivo corneal confocal microscopy provides a noninvasive method of assessing normal equine corneal structure at the cellular level and is a precise technique for corneal sublayer pachymetry and cell density measurements. A resident population of presumed Langerhans cells and corneal stromal dendritic cells was detected in the normal equine cornea. The described techniques can be applied to diagnostic evaluation of corneal alternations associated with disease and have broad clinical and research applications in the horse.     

Bacterial isolates and antimicrobial susceptibilities in equine bacterial ulcerative keratitis (1993--2004)

REASONS FOR PERFORMING STUDY: Bacterial ulcerative keratitis is a common and often vision-threatening problem in horses. Emerging bacterial resistance to commonly used topical antibiotics has been demonstrated. Previous antibiotic use may alter the antimicrobial susceptibility of bacterial isolates. OBJECTIVES: To document aerobic bacterial isolates and associated bacterial susceptibilities from horses with ulcerative keratitis treated at the University of Tennessee between January 1993 and May 2004 and determine whether prior antibiotic therapy affected antimicrobial susceptibility of the isolates. METHODS: Medical records from horses with ulcerative keratitis and positive aerobic bacterial cultures and antimicrobial susceptibility were evaluated. Clinical history regarding antibiotic therapy prior to culture was documented. RESULTS: Fifty-one aerobic bacterial isolates from 43 horses were identified. Streptococcus equi subspecies zooepidemicus was the most commonly isolated organism, accounting for 33.3% of all isolates, followed by Pseudomonas aeruginosa (11.8%), Staphylococcus spp. (11.8%) and Gram-negative nonfermenting rods (7.8 %). No resistance was noted amongst S. equi ssp. zooepidemicus to cephalothin, chloramphenicol or ciprofloxacin. Only 64 % of S. equi ssp. zooepidemicus isolates were sensitive to bacitracin. No resistance was noted among P. aeruginosa to gentamicin, tobramycin or ciprofloxacin. Antibiotic therapy with neomycin-polymixin B-bacitracin prior to presentation and culture was documented in 11/17 horses in which S. equi ssp. zooepidemicus was isolated and in 4/6 horses in which P. aeruginosa was isolated. Three horses received topical corticosteroids prior to culture, of which 2 had polymicrobial infections. CONCLUSIONS: S. equi ssp. zooepidemicus and P. aeruginosa were the most frequently isolated bacterial organisms in equine ulcerative keratitis. No significant trends in aminoglycoside or fluoroquinolone resistance were noted among these organisms. However, the resistance of S. equi ssp. zooepidemicus to bacitracin with common use of this antibiotic suggests that previous antibiotic therapy probably affects antimicrobial resistance. POTENTIAL RELEVANCE: Therapy prior to culture may play an important role in antimicrobial susceptibility of corneal bacterial isolates. Corticosteroid use may increase the risk of polymicrobial infections of corneal ulcers, leading to a worse prognosis. Although significant fluoroquinolone resistance has not been documented in the veterinary literature, these antimicrobials should be reserved for known infected corneal ulcers and not used for prophylaxis. Empirical antibiotic therapy should not only be guided by clinical signs, but also take into consideration previous antimicrobial and corticosteroid therapy.     

Topical treatment of non-healing corneal epithelial ulcers in dogs with aminocaproic acid

The potential efficacy of topical epsilon-aminocaproic acid, an antiplasmin agent, in the treatment of persistent corneal epithelial defects was evaluated in a study of the medical records of 44 dogs, in which 51 eyes had been diagnosed with a corneal epithelial defect lasting more than 10 days, with no apparent underlying cause. At an initial examination all the affected eyes had had non-adherent epithelium removed. Thirty-four of the eyes in 28 dogs examined between January 2000 and March 2003 were also treated by the topical application of a solution of 35.7 mg/ml ophthalmic aminocaproic acid three times a day; the other 17 eyes in 16 dogs treated between October 1997 and March 1999 had received only topical treatment with gentamicin in addition to the debridement. Both groups were assessed clinically at weekly intervals for a maximum of three weeks. The two groups had approximately the same breed distribution, and there were no statistically significant differences between them in terms of their age, sex, affected side or duration of the corneal erosions. After three weeks, 32 of the 34 eyes treated with aminocaproic acid (94.1 per cent) had been cured, compared with seven of the 17 eyes treated with gentamicin (41.2 per cent) (P=0.0001). No adverse drug reactions were reported.     

Ultrastructure of cultured canine oral keratinocytes

Keratinocytes from explants of the oral mucosa of dogs were grown in culture for five passages. The ultrastructure of primary cultures and fully developed subcultures passaged 1, 3, and 5 times was examined. At every stage, the cells had the morphologic characteristics of epithelial cells and formed a multilayered squamous epithelium. The basal cells had the characteristics of metabolically active cells, whereas the suprabasal cells and the cells at the media interface expressed many, but not all, of the organelles and cell surface characteristics associated with keratinocyte differentiation. Keratohyalin granules were located in the suprabasal and superficial cells. Cell size and shape and the relationship between cells in the layers also reflected the morphologic characteristics of the parent tissue. Cells maintained this typical structure through all passages and the cultures changed minimally for up to a week after development.     

Antibacterial susceptibility patterns of Pseudomonas strains isolated from chronic canine otitis externa

Development of antibiotic resistance in bacteria is a problem of great concern. It is important to establish the convenience of antimicrobial susceptibility tests in animal infections. The aim of this study was to test the susceptibility to antibiotics of Pseudomonas strains isolated from chronic canine otitis externa. We tested 23 strains of Pseudomonas: 19 Ps. aeruginosa, three Ps. fluorescens and one Pseudomonas spp. The most effective antibiotics were tobramycin (100% susceptible), marbofloxacin (91.3%) and ceftazidime (91.3%). Ticarcillin and gentamicin, commonly used for the treatment of otitis externa also showed good results (susceptibility of strains was 86 and 65.2% respectively). Lower susceptibility was found using enrofloxacin (52.1%) probably due to its indiscriminate use. We emphasize the need for a rational policy of antibiotic prescribing in order to prevent the selection of resistant strains.     

扎布耶盐山羊金黄色葡萄球菌分离鉴定及药敏试验

为调查西藏仲巴县帕江乡羊场金黄色葡萄球菌感染情况,采集仲巴县帕江乡羊场的扎布耶盐山羊脓汁、乳汁、血样及部分病变组织进行金黄色葡萄球菌分离鉴定及药敏试验。结果表明,该羊场金黄色葡萄球菌检出率16．24％（19/117）,对诺氟沙星、氯霉素、庆大霉素、苯唑西林、头孢西丁、万古霉素、氨苄西林、头孢唑林、头孢拉定、复方新诺明、恩诺沙星、氧氟沙星、环丙沙星13种抗生素均高度敏感,说明在藏西北养羊生产过程使用抗生素较少,更没有滥用药物治疗。该调查研究为仲巴县帕江乡羊场金黄色葡萄球菌病诊治提供了诊断方法和治疗用药方案。     

Efficacy of two chondroitin sulfate ophthalmic solutions in the therapy of spontaneous chronic corneal epithelial defects and ulcerative keratitis associated with bullous keratopathy in dogs

OBJECTIVE: To determine the efficacy of two antimicrobial-chondroitin sulfate ophthalmic solutions in the therapy of spontaneous chronic corneal epithelial defects (SCCED) and ulcerative keratitis associated with bullous keratopathy in dogs. ANIMALS STUDIED: Eighty dogs with SCCED and 14 dogs with ulcerative keratitis associated with bullous keratopathy. PROCEDURE: Following manual debridement of nonadherent epithelium, dogs were treated topically with a chondroitin sulfate ophthalmic solution containing either tobramycin or ciprofloxacin. Patients were re-evaluated at 2-week intervals for 4 weeks. RESULTS: After 2 weeks of treatment, 53.6% of eyes with SCCED and 17.6% of eyes with ulcerative keratitis associated with bullous keratopathy had healed. After 4 weeks of treatment, 81.0% of eyes with SCCED and 23.5% of eyes with ulcerative keratitis associated with bullous keratopathy had healed. There were no statistically significant differences in healing percentages between the tobramycin-chondroitin sulfate solution treatment groups and the ciprofloxacin-chondroitin sulfate solution treatment groups. Two dogs with SCCED, one treated with the tobramycin-chondroitin sulfate solution and the other treated with the ciprofloxacin-chondroitin sulfate solution, developed sterile corneal stromal abscesses during the study. CONCLUSIONS: Topical therapy with an antimicrobial-chondroitin sulfate ophthalmic solution combined with manual debridement of nonadherent epithelium compares favorably with other published medical and surgical therapies for SCCED; however, these compounds are only equivocally more effective than therapy with manual debridement alone. These solutions appear to be ineffective in the treatment of ulcerative keratitis associated with bullous keratopathy. The significance of the two cases of corneal stromal abscessation is unknown at this time and warrants further investigation.     

Cultivation of corneal epithelial cell sheets on canine amniotic membrane

Objective To develop and assess canine corneal epithelial cell sheets cultivated from limbal stem cells on amniotic membrane. Procedures Canine corneal limbal segments were obtained from six beagle dogs. Cryopreserved denuded amniotic membranes (obtained from Miniature Dachshund and Cavalier King Charles Spaniel breeds) from which the epithelial cells were removed were used as scaffolds. The limbal segments were cultured on these amniotic membranes with 3T3 feeder cells for 2 weeks. The harvested corneal epithelial cell sheets were stained with H&E for histologic analysis. The harvested sheets were analyzed immunohistochemically using a corneal epithelium‐specific marker keratin 3(K3) and putative stem cell markers ABCG2, p63, and vimentin. Results Cultivated cells from the corneal limbal tissues reached confluency in 7–8 days. The cultivated cells adhered to the denuded amniotic membrane and formed a sheet. The cultivated cell sheet was transparent and consisted of five to eight layers. K3 was observed in all layers and ABCG2, p63, and vimentin were notably present in the basal layer of the cultivated canine epithelium by immunofluorescence. Conclusions Canine corneal epithelial cells were successfully cultivated on the canine amniotic membrane. The cultivated epithelial sheets contained putative stem cells in the basal layer and had a stratified epithelium.