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.     

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.     

Successful treatment of an unusually large corneal epithelial inclusion cyst using equine amniotic membrane in a dog

A 10‐year‐old intact male Yorkshire Terrier was referred for investigation of a large raised and nonpainful corneal lesion oculus dexter. Clinical examination revealed a pale, translucent corneal mass, which occupied half of the corneal surface and measured 11 mm × 11 mm × 13 mm. The mass was removed by superficial keratectomy and equine amniotic membrane (AM) was transplanted into the large corneal defect to cover the wound and provide tectonic support for the remaining cornea. The mass was histologically confirmed as a corneal epithelial inclusion cyst. There was no evidence of recurrence or complication at the surgical site 100 days postoperatively. Corneal epithelial inclusion cysts are uncommon in dogs. Although superficial keratectomy is the recommended treatment for corneal inclusion cyst, the combination of superficial keratectomy and AM transplantation had to be considered as an alternative for repair of large corneal defects. This is the first case report of the combined application of AM and superficial keratectomy to successfully treat a corneal inclusion cyst in a dog.     

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.     

Histologic evaluation of the immediate effects of diamond burr debridement in experimental superficial corneal wounds in dogs

Purpose To evaluate the corneal changes immediately after diamond burr debridement of superficial corneal wounds in dogs. Spontaneous chronic corneal epithelial defects (SCCEDs) are the most common form of canine recurrent corneal ulcers. The diamond burr has been used in the management of corneal lesions in humans since 1983. Recently, it has been successfully used in the treatment of SCCEDs in dogs; however, little has been documented as to its mechanism of action. Methods Five adult female research dogs euthanized for reasons unrelated to the study were included, providing 10 normal eyes. An excimer laser spatula was used for epithelial removal after delineation with an 8 mm punch biopsy trephine. Diamond burr debridement was performed for 30 and 45 s in five eyes each (groups 1 and 2 respectively). The procedure was performed on the ventral half of the experimental defect as well as ventral normal cornea, immediately after euthanasia, and prior to enucleation. Samples were processed routinely for histologic evaluation and stained with periodic acid–Schiff. Results No stromal defects could be identified under light microscopy. In experimental corneal wounds, multi‐focal areas remained covered by the epithelial basement membrane (BM) after diamond burr treatment in both groups (group 1 = 48%±16SD, group 2 = 26%±12SD). Removal of BM on group 2 was significantly higher than group 1 (P < 0.05). Conclusions The diamond burr allows a safe method of debridement and does not create defects beyond the epithelial BM in corneal wounds in normal dogs. Evaluation of the diamond burr debridement in cases of SCCEDs is warranted.     

Immune-mediated canine and feline keratitis.

Although the normal cornea is devoid of vasculature and lymphatics, there are still several immune-mediated corneal conditions that can occur in dogs and cats. An overview of corneal immunology is presented. Diseases of dogs, including chronic superficial keratitis, superficial punctate keratitis, and canine adenovirus endotheliitis, as well as feline diseases, including eosinophilic keratitis and herpesvirus-related conditions, are discussed.     

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.     

Corneal squamous cell carcinoma in a dog: a case report

Purpose:  To report a case of primary corneal squamous cell carcinoma (SCC) in an English Bulldog. In addition, immunohistochemistry of the corneal tissue mass was performed using a panel of antibodies. A prominent feature of the present case was the clinical history of chronic keratitis due to eyelid abnormalities.
Results:  No papillomavirus antigen was detected in section of normal or neoplastic corneal tissue. The corneal epithelial cells were positive for pancytokeratins AE1/AE3 and MNF116, and E-cadherin. The neoplastic cells in close proximity to the normal epithelial lining were positive for both pancytokeratins and E-cadherin with gradual loss of staining toward the center of the neoplastic mass. Rare neoplastic cells demonstrated positive staining for caspase 3 and a large number was strongly positive for GADD45 and p53.
Conclusion and discussion:  The observed loss of the various cytokeratins, the strong p53 expression, and low numbers of caspase 3 positive cells were suggestive that a p53 mutation may have caused this primary corneal SCC. Over-expression of the tumor-suppressor gene p53 is likely to be a consequence of ultraviolet radiation exposure. Two factors, however, may have played a role in the formation of this primary corneal SCC: chronic irritation of the corneal surface (microtrauma) and exposure to UV radiation.     

Feline corneal disease

The cornea is naturally transparent. Anything that interferes with the cornea's stromal architecture, contributes to blood vessel migration, increases corneal pigmentation, or predisposes to corneal edema, disrupts the corneas transparency and indicates corneal disease. The color, location, and shape and pattern of a corneal lesion can help in determining the underlying cause for the disease. Corneal disease is typically divided into congenital or acquired disorders. Congenital disorders, such as corneal dermoids are rare in cats, whereas acquired corneal disease associated with nonulcerative or ulcerative keratitis is common. Primary ocular disease, such as tear film instability, adenexal disease (medial canthal entropion, lagophthalmus, eyelid agenesis), and herpes keratitis are associated with the majority of acquired corneal disease in cats. Proliferative/eosinophilic keratitis, acute bullous keratopathy, and Florida keratopathy are common feline nonulcerative disorders. Nonprogressive ulcerative disease in cats, such as chronic corneal epithelial defects and corneal sequestration are more common than progressive corneal ulcerations.     

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.     

熊胆滴眼液对兔角膜烧伤和角膜翳的治疗实验研究

为研究熊胆滴眼液对角膜烧伤和角膜翳的治疗效果 ,采用“电热角膜瘢痕复制器”制作家兔角膜烧伤与斑模型 ,以生理盐水、去熊胆滴眼液、氯霉素眼药水和狄奥宁眼药水作对照 ,用熊胆滴眼液进行治疗实验。实验结果表明 :熊胆滴眼液对家兔角膜烧伤及并发结膜炎的治疗效果显著 ,优于去熊胆滴眼液和氯霉素眼药水 (P<0 .0 5 ) ;对角膜翳的治疗效果非常显著 ,优于去熊胆滴眼液和狄奥宁眼药水 (P<0 .0 1)。扫描电镜观察了熊胆滴眼液对角膜翳的治疗作用 ,结果显示 :治疗 2月后 ,角膜上皮细胞呈现更新速率较快的结构像 ,治疗 5月后 ,上皮细胞表面的微皱襞及微绒毛排列规则、细密 ,与正常角膜上皮细胞的超微结构相一致。表明复方熊胆滴眼液具有使角膜翳处的角膜上皮恢复正常的作用。     

Multiple striate keratotomy: a treatment for corneal erosions caused by epithelial basement membrane disease

Objective To study the efficacy of multiple striate keratotomy for the treatment of persistent corneal erosions suspected to be caused by primary corneal epithelial basement membrane disease.
Design A retrospective study.
Animals 16 dogs, three cats and one Australian dingo.
Procedure A technique called multiple striate keratotomy was used to treat twenty animals suffering from persistent corneal erosions.
Results All persistent corneal erosions healed with only one treatment. Most cases healed within 2 weeks. One case developed a second erosion in the same eye but in a different position to the original erosion.
Conclusions Multiple striate keratotomy is a safe, effective and well tolerated technique for the treatment of persistent corneal erosions thought to be caused by corneal epithelial basement membrane disease.     

In vitro measurement of rabbit corneal epithelial thickness using ultrahigh resolution optical coherence tomography

The objective of this study was to reproducibly measure corneal epithelial thickness centrally and at the limbus in the rabbit cornea using ultrahigh resolution optical coherence tomography (OCT). Twelve freshly enucleated New Zealand white rabbit eyes were kept in a moist chamber at 4 degrees C. An ultrahigh resolution OCT system with a spatial resolution of 1.3 microm was used to image the cornea and its component layers. The central and peripheral (limbal) regions of all the samples were scanned within 6 h of harvest in order to minimize the post-mortem degradation of the corneal epithelium. The thickness of the corneal epithelium was determined by measuring the pixel equivalents of the obtained image. Unpaired Student's t-test was used to evaluate differences. The epithelial thickness centrally was found to be 45.8 +/- 2.2 microm, and 37.6 +/- 1.4 microm at the limbus (P < 0.001). Rabbit corneal epithelium is thicker centrally than at the limbus when measured by ultrahigh resolution OCT. This technique will aid in delineating the pathophysiology of diseases of the anterior cornea.     

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.     

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.     

Bacterial corneal diseases in dogs and cats

Corneal diseases are very common in small animals. Corneal disease associated with bacterial agents is frequent in the dog and maybe less frequent in the cat. The medical history, important steps of the ophthalmic examination, and the ophthalmic diagnostic tests that are relevant in such corneal conditions are outlined. Bacterial corneal diseases in dogs and cats are most commonly considered in one of two categories--bacterial ulcerative keratitis and corneal abscesses. The clinical aspects of these two entities as well as the therapeutic strategies available for general practitioners and ophthalmologists are discussed. Ulcerative keratitis is frequent; it represents the most common ocular diseases in dogs and cats. Because some of these corneal ulcers can be very severe, progress rapidly, and therefore are sight threatening, the crucial steps of their diagnosis and management are stressed. The use of a magnification system, fluorescein dye, and corneal cytology and culture, if indicated, is necessary for diagnosis at an early stage of the disease. The treatment of bacterial ulcerative keratitis should eradicate the infection, reduce or stop the corneal destruction and support the corneal structures, control the uveal reaction and the pain associated with it, and minimize the scarring. The prognosis depends on the stage and the severity of the corneal ulceration, the etiology of the condition, and the therapeutic choice. A close follow-up of animals with corneal ulceration is highly recommended because corneal ulcers can progress rapidly.     

Effects of conditioned media from human amniotic epithelial cells on corneal alkali injuries in rabbits

This study was performed to evaluate the effects of conditioned media (CM) from human amniotic epithelial cells (HAECs) on the corneal wound healing process. Eighteen rabbits (36 eyes) were used and randomly assigned to three groups according treatment: CM from HAECs (group 1), vehicle alone (group 2), and saline (group 3). Corneal alkali injuries were induced with 1 N sodium hydroxide. Each reagent used for treatment evaluation was injected into the dorsal bulbar subconjunctiva and the area of the corneal epithelial defect was measured every other day. Two animals from each group were euthanized at a time on days 3, 7, and 15, and the cornea was removed for histological examination. The sum of the epithelial defect areas measured on day 0 to day 6 as well as day 0 to day 14 in group 1 was significantly smaller than those of other groups. Histological examination revealed that the group 1 corneas had less inflammatory cell infiltration and showed more intact epithelial features compared to the other groups. These results suggest that CM from HAECs promote corneal wound healing in rabbits.     

Effects of radiofrequency hyperthermia on the healthy canine cornea

Radiofrequency hyperthermia was used to induce axial corneal lesions in the eyes of 10 dogs. Clinical observations were continued for up to 6 months, using biomicroscopy and indirect ophthalmoscopy. Eyes were harvested at intervals for light and electron microscopic evaluation. Clinical alterations included immediate corneal opacification and epithelial disruption at the site of electrode contact. Ulcerative keratitis persisted for 4 to 6 days, accompanied by anterior uveitis. Additional corneal changes included stromal thinning, edema, and vascularization. Final evaluation revealed negligible alterations in corneal contour or clarity 6 months after treatment. Microscopically, epithelial and superficial stromal necrosis preceded epithelial loss. Stromal alterations included edema (associated with focal endothelial detachments), vascularization, and inflammatory cell infiltration. Recovery was characterized by keratocytic hyperplasia and hypertrophy, epithelial proliferation, and stromal condensation.     

A novel herpesvirus associated with chronic superficial keratitis and proliferative conjunctivitis in a great horned owl (Bubo virginianus)

An adult great‐horned owl (Bubo virginianus; GHOW) presented with a history of recurrent corneal ulceration of the right eye (OD). Findings included ulcerative superficial keratitis, proliferative conjunctivitis, and iris pigmentary changes. The ulcer was initially nonresponsive to medical therapy, but showed rapid and appropriate healing following diamond burr debridement. Proliferative conjunctivitis markedly improved following topical antiviral therapy with cidofovir 1%, interferon alpha 2B ophthalmic solutions, and oral l ‐lysine. Histopathologic evaluation of a conjunctival biopsy revealed epithelial features suspicious for viral cytopathic changes and intranuclear structures suspicious for viral inclusions, suggestive of a possible viral‐induced papillomatous conjunctivitis. A novel alphaherpesvirus, referred to as Strigid Herpesvirus 1 (StrHV1), was identified using PCR and gene sequencing. This case represents a new clinical manifestation of a previously unreported herpesvirus in the GHOW. Identification of the herpes virus was critical to administration of appropriate therapy and resolution of the conjunctivitis, and corneal epithelial debridement promoted resolution of the chronic corneal epithelial defect.     

Corneal epithelial inclusion cyst in a Llama

A 13-year-old, female Llama presented for evaluation of a limbal based corneal mass involving the OD of 4 months duration. The mass was excised en bloc by a nonpenetrating keratectomy, followed by placement of a conjunctival advancement flap covering the keratectomy site. The mass was submitted for histological evaluation. Histopathology identified the mass to be a corneal epithelial inclusion cyst filled with necrotic squamous and neutrophilic debris. Surgical excision was complete and considered curative with no signs of recurrence 3 months postoperatively. There was no known prior ocular trauma; however, a previously performed corneal biopsy for evaluation of recurrent epithelial erosions may have been an initiating cause.