Canine corneal epithelial cells possess a sustained proliferative capacity and generate a spontaneously derived cell line.

We have previously reported characteristics of canine corneal epithelial cells in vitro and found that canine corneal epithelial cells could maintain their proliferative capacity even after continuous culture without the use of feeder cells and growth promoting additives. The objective of this study was to elucidate proliferative characteristics of canine corneal epithelial cells independent of feeder cells and growth promoting additives, with the aim of developing a spontaneously derived corneal epithelial cell line. Canine and rabbit corneal epithelial cells were harvested from the limbus and cultured with, or without, feeder cells and growth promoting additives, and both were passaged continuously until growth arrest. Canine corneal epithelial cells could proliferate independently, and could be passaged more times than rabbit cells. A canine corneal epithelial cell line, cCEpi, which could be passaged more than 100 times without using feeder cells and growth promoting additives, was established. cCEpi cells maintained a cell morphology close to the primary culture and expressed p63, cytokeratin 15 (K15), and K3. Although changes in colony morphology, shortening of the population doubling time and a heteroploid karyotype were observed, cCEpi was not tumorigenic. Stratified cell sheets cultured from cCEpi were morphologically and immunohistologically similar to sheets cultivated from early passage cells. In conclusion, canine corneal epithelial cells can proliferate independent of feeder cells and growth promoting additives. cCEpi maintains properties similar to normal corneal epithelial cells and could be a useful source for studies in cellular biology and for developing novel therapies.

Evaluation of ABCG2 and p63 expression in canine cornea and cultivated corneal epithelial cells.

OBJECTIVE: To examine the expressions of ABCG2 and p63 in canine corneal epithelia and to evaluate their significance in corneal regeneration. PROCEDURES: Canine corneal and limbal epithelial cells were obtained from five healthy beagle dogs. We analyzed the morphological properties of cultivated limbal and corneal epithelial cells. We compared the expressions of ABCG2 and p63 in the limbus and central cornea by immunohistochemistry and real-time quantitative PCR. We analyzed the expression of these markers in cultivated cells by immunocytochemistry and real-time quantitative PCR. RESULTS: The limbal epithelial cells were smaller and proliferated more rapidly than the corneal epithelial cells in primary cultures. The corneal cells failed to be subcultured, whereas the limbal cells could be subcultured with increasing cell size. ABCG2 was localized in the basal layer of the limbal epithelium, and p63 was widely detected in the entire corneal epithelia. ABCG2 expression was significantly higher, and p63 was slightly higher in the limbus compared with the central cornea. ABCG2 was detected only in limbal cells in primary culture, not in corneal cells or passaged limbal cells. p63 was detected in both limbal and corneal cells and decreased gradually in the limbal cells with the cell passages. CONCLUSIONS: ABCG2 was localized in canine limbal epithelial cells, and p63 was widely expressed in canine corneal epithelia. ABCG2 and p63 could prove to be useful markers in dogs for putative corneal epithelial stem cells and for corneal epithelial cell proliferation, respectively.

Comparison of the canine corneal epithelial cell sheets cultivated from limbal stem cells on canine amniotic membrane, atelocollagen gel, and temperature-responsive culture dish.

OBJECTIVE: The current study compared canine corneal epithelial cell sheets cultivated from limbal stem cells on amniotic membrane, atelocollagen gel, and temperature-responsive culture dish. PROCEDURES: We collected limbal epithelial cells from the intact eyes of beagles and cultivated the cells on denuded canine amniotic membranes, temperature-responsive cell culture labware, and collagen gel with 3T3 feeder cells. Immunofluorescence staining for Ki-67 was used to analyze the capacity of cell proliferation in the sheets. Immunofluorescence staining was also performed for the corneal epithelium-specific marker cytokeratin 3 and putative stem cell markers ABCG2 and p63. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to detect ABCG2 and p63. RESULTS: The growth rates of the cultivated cells, or the times it took them to reach confluency, were different for the three scaffolds. The cultivated sheet on the temperature-responsive dish consisted of 2-3 layers, while those on the collagen gel and on the amniotic membrane consisted of 5-8 layers. The basal layer cells grown on all three scaffolds expressed putative stem cell markers. In real-time RT-PCR analysis, the highest level of p63 was observed in the sheets grown on collagen gel. CONCLUSIONS: In this study, the cells cultured on the collagen gel demonstrated a capacity for cell proliferation, and the expressions of stem cells in the sheets suggested that collagen gel is the most suitable carrier for clinical use.

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.

Investigation of fellow eye of unilateral retinal detachment in Shih-Tzu.

OBJECTIVE: To investigate disease in the fellow eye, and consider the relation to rhegmatogenous retinal detachment (RRD) in Shih-Tzus. ANIMALS STUDIED: The fellow eyes of 49 Shih-Tzus (27 male, 22 female; median age: 6.8 years) with unilateral RRD diagnosed by funduscopy or ultrasonography at Rakuno Gakuen University Teaching Animal Hospital were assessed in this study. PROCEDURES: Ophthalmic examinations (including menace response, pupillary light reflex, slit-lamp biomicroscopy, and funduscopy) were performed in the subjects. Electroretinography was performed in 12 eyes that developed retinal degeneration. Maximum follow-up period was 42 months. RESULTS: Cataracts and vitreous opacity were observed in 26 (53%) and 32 eyes (65%), respectively, by slit-lamp biomicroscopy. Retinal degeneration with various degrees of hyper-reflectivity of the tapetal fundus and/or attenuation of retinal vessels was observed in 35 eyes (71%) on funduscopy. A reduction of amplitude in rod, standard combined and 30 Hz flicker electroretinogram was detected in 5 (42%), 10 (83%), and 6 eyes (50%), respectively. During the follow-up period, RRD was detected in six eyes. CONCLUSION: Retinal degeneration was frequently detected by funduscopy and electroretinograms in the fellow eye in Shih-Tzus with RRD. In our subjects, vitreous degeneration was also observed frequently. It has been reported that peripheral retinal degeneration is one of the causes of RRD associated with vitreous degeneration in humans. We assume that primary retinal degeneration with secondary vitreous degeneration is one of the causes of RRD in Shih-Tzus.

Morphological analysis of corneal opacity in Shiba dog with GM1 gangliosidosis.

GM1 gangliosidosis is one of the inherited metabolic lysosomal storage disorders characterized by neurological symptoms caused by beta-galactosidase deficiency and consequent accumulation of GM1 ganglioside in neuronal cells. Shiba dogs affected with GM1 gangliosidosis have been found to suffer from corneal opacity. In our morphological analysis, keratocyte enlargement was induced by abnormal intracellular accumulation of neutral carbohydrates, resulting in the loss of normal arrangement of collagen fibrils in the opaque cornea was found to be associated with the disorder. We therefore conclude that corneal opacity in this Shiba dog with GM1 gangliosidosis may be caused by neutral carbohydrate accumulation in lysosomes, swelling and dysfunction of keratocytes, and subsequent irregular arrangement of collagen fibrils in the corneal proper substance.

A preliminary study of direct application of atelocollagen into a wound lesion in the dog cornea.

PURPOSE: To evaluate the effectiveness of atelocollagen for canine corneal wound healing. MATERIALS AND METHODS: Atelocollagen was used to fill a transplant bed in the central cornea, which was then covered with a contact lens. The wound healing process was analyzed clinically, morphologically, and biochemically. RESULTS: At the early healing stage, both the pupillary zone and details of the iris were observed. The stromal collagen fibrils normalized in a time-dependent manner. Type III collagen in the wound area was detected faintly throughout the experimental period. CONCLUSIONS: This novel method is advantageous for accelerating wound healing without causing inflammation.

Microstructure and glycosaminoglycan ratio of canine cornea after reconstructive transplantation with glycerin-preserved porcine amniotic membranes.

OBJECTIVE: Although amniotic membranes of canine, feline, and equine species have some advantages as corneal transplantation material in many canine ocular diseases, their softness, thinness, and low availability can pose problems. As an alternative, the more abundant porcine amniotic membranes may be used. This paper describes the use of glycerin-preserved porcine amniotic membranes in corneal transplantation in eight normal dogs. METHOD: A 0.4-mm deep recipient bed in the axial cornea of the OS of all dogs was created using an 8-mm Barron radial vacuum trephine. The recipient bed was then filled with amnion, and the entire cornea was covered with another piece of the glycerin-preserved membrane. The ocular signs evaluated were corneal opacity and corneal vascularization. The dogs were euthanized on days 5, 10, 20, or 40 after surgery, and samples were collected to evaluate corneal thickness, parenchymal cell number, mean collagen fibril diameter, collagen fibril content and the glycosaminoglycan (GAG) ratio. RESULTS: Corneal opacity was observed immediately after surgery. Restoration of corneal transparency, regression of corneal vascularization, and visualization of the pupil and iris were noted on day 40. CONCLUSIONS: The clinical observations were supported histologically by regained corneal thickness, parenchymal cell number, mean collagen fibril diameter, collagen fibril content, and GAG ratio, suggesting that this technique may be a novel method for the treatment of ocular surface disorders.

The effects of cataract stage, lens-induced uveitis and cataract removal on ERG in dogs with cataract.

OBJECTIVE: The purpose of this study was to determine the effects of cataract stage, lens-induced uveitis and cataract removal on the electroretinogram (ERG) of dogs with cataract. ANIMALS STUDIED: Fifty-seven dogs diagnosed with unilateral or bilateral cataract whose ERG was recorded at Rakuno Gakuen University Teaching Animal Hospital from 2001 to 2004. PROCEDURES: Four responses were recorded during the ERG: rod ERG, standard combined ERG, single-flash cone ERG and 30-Hz flicker ERG. Cataracts were divided into four stages: incipient, immature, mature and hypermature, and with or without lens induced uveitis (LIU). Noncataractous eyes of dogs with unilateral cataract were used as the control. We compared ERG amplitude, implicit time, and the b- to a-wave amplitude ratio of cataractous vs. noncataractous eyes, preoperative vs. postoperative cataractous eyes, and cataractous eyes with and without LIU. RESULTS: No significant difference was found in ERG amplitude between incipient, immature and hypermature cataractous eyes, while in mature cataractous eyes decreased amplitude was confirmed in all responses compared with control eyes. However, no significant difference in b/a ratio was found at any stage of cataract. In postoperative eyes, increased amplitude was recorded in all responses compared to preoperative values. In eyes with LIU a decreased amplitude in the rod ERG and b-wave of standard combined ERG was recorded and, furthermore, a significant decline was confirmed in b/a ratio. CONCLUSION: ERG values were influenced by cataract stage and LIU. LIU was associated with a reduction in the b/a ratio.

Indocyanine green angiography for examining the normal ocular fundus in dogs.

In dogs, a variety of diseases of the retina and choroid have been reported, either separately or concomitantly; however, the canine choroid is difficult to evaluate by veterinary techniques currently available. Indocyanine green (ICG) angiography is widely used in human ophthalmology, but has not been investigated for use in canine ophthalmology. The aim of this study was to apply a new approach to ICG angiography and compare the resulting angiograms with fluorescein (FLUO) angiograms of the ocular fundus in dogs. With a fundus camera equipped with an infrared-sensitive charged coupled device (CCD), we performed angiography on eight healthy beagles under inhalation anesthesia. ICG angiography enabled clear visualization of the choroidal vasculature, whereas FLUO angiography showed only the retinal vessels. At 8.4 +/- 3.6 sec after administration of ICG dye into the cephalic vein, the choroidal arteries could be seen extending radially from the optic disc, then the choroidal veins became apparent at 10.2 +/- 4.1 sec, coursing alongside the choroidal arteries. Gradual fading of the choroidal vessels began 13.2 +/- 2.2 min after the dye was administered, and overall diffuse fluorescence of the fundus appeared. Diffuse fluorescence of the fundus continued after the choroidal vessels and optic disc faded at about 58.3 +/- 5.3 min from administration of the dye. In conclusion, ICG angiography provides clear resolution and is reliable and simple, thus offering promise as a diagnostic aid for clinical evaluation of the choroid in dogs.

Detection of cone dysfunction induced by digoxin in dogs by multicolor electroretinography.

It is difficult to detect discrete cone function with the present conventional electroretinography (ERG) examination. In this study, we developed contact electrodes with a built-in color (red (644 nm), green (525 nm), or blue (470 nm)) light source (color LED-electrode), and evaluated an experimental model of digoxin in the dog. First, 17 normal Beagle dogs were used to determine which electrode works well for color ERG measurement on dogs. Then, color ERG was performed on seven normal Beagle dogs at various points during a 14-day period of digoxin administration. A single daily dose of 0.0125 mg/kg/day, which is within the recommended oral maintenance dosage range for dogs, was administered orally for 2 weeks. Ophthalmic examination, measurement of plasma concentration of digoxin, and color ERG examination were performed. On first examination, amplitudes of all responses were significantly (P < 0.01) lower with the red, than with the blue and green electrodes during ERG recording. In ERG using the red electrode, the standard deviation was large. According to these preliminary results, the red electrode was not used in the experimental dog model with digoxin. In the digoxin administrated animals, no significant change was observed in the ophthalmic examination findings. The digoxin level increased steadily throughout the dosing period but was always within the therapeutic range for dogs. In rod ERG, no abnormalities were detected with any electrode. In standard combined ERG, decreased amplitude of the a-wave was detected with every electrode. In single flash cone ERG, prolongation of implicit time was detected by color ERG with the blue and green electrodes. In 30-Hz flicker ERG, decreased amplitude was detected only by color ERG with the blue electrode. The decreased amplitude and prolonged implicit time recovered after termination of digoxin administration. Cone dysfunction induced by digoxin in the dog was revealed by multicolor ERG using blue and green LED-electrodes. Multi-color ERG was useful for detecting cone type-specific dysfunction in the dog.

Electroretinography using contact lens electrode with built-in light source in dogs.

Electroretinography (ERG) is an effective method for the diagnosis of retinal disease. In the dog, dependable ERG recording is difficult without the use of an expensive device like a Ganzfeld full-field stimulator. The International Society for Clinical Electrophysiology of Vision has defined the standard flash stimulus condition (SF) and evaluation of the retina using the b/a ratio in humans. In dogs, evaluation using the b/a ratio has not been reported, whereas the intensity of SF has been defined. In this study, we performed a convenient ERG recording method using a contact lens electrode with a built-in light source (LED-electrode), and confirmed SF as reported previously. ERG recordings were performed on 15 healthy beagle dogs under sedation. We performed bilateral ERG at 12 different intensities after 30 min dark adaptation. After 10 min light adaptation, we recorded single flash cone and flicker cone response using the SF determined in this study. In this study, SF of 3.0 cd/m(2)/sec (6,000 cd/m(2), 0.5 msec) resulted in b/a=2. The intensity for rod response that recorded only the b-wave was 0.0096 cd/m(2)/sec (80 cd/m(2), 0.12 msec). We could achieve ERG for each response easily and smoothly under sedation, and without general anesthesia. Using an LED-electrode, we could perform more quantitative and reproducible ERG examinations than with traditional methods. We propose that the b/a ratio is the most useful parameter in ERG reporting for evaluating retinal function.

Anesthetic and cardiovascular effects of balanced anesthesia using constant rate infusion of midazolam-ketamine-medetomidine with inhalation of oxygen-sevoflurane (MKM-OS anesthesia) in horses.

The anesthetic sparring and cardiovascular effects produced by midazolam 0.8 mg/ml-ketamine 40 mg/ml-medetomidine 0.05 mg/ml (0.025 ml/kg/hr) drug infusion during sevoflurane in oxygen (MKM-OS) anesthesia was determined in healthy horses. The anesthetic sparring effects of MKM-OS were assessed in 6 healthy thoroughbred horses in which the right carotid artery was surgically relocated to a subcutaneous position. All horses were intubated and ventilated with oxygen using intermittent positive pressure ventilation (IPPV). The end-tidal concentration of sevoflurane (ET(SEV)) required to maintain surgical anesthesia was approximately 1.7%. Heart rate and mean arterial blood pressure averaged 23-41 beats/min and 70-112 mmHg, respectively. All horses stood between 23-44 min after the cessation of all anesthetic drugs. The cardiovascular effects of MKM-OS anesthesia were evaluated in 5 healthy thoroughbred horses ventilated using IPPV. Anesthesia was maintained for 4 hr at an ET(SEV) of 1.7%. Each horse was studied during left lateral (LR) and dorsal recumbency (DR) with a minimum interval between evaluations of 1 month. Cardiac output and cardiac index were maintained between 70-80% of baseline values during LR and 65-70% of baseline values during DR. Stroke volume was maintained between 75-85% of baseline values during LR and 60-70% of baseline values during DR. Systemic vascular resistance was not different from baseline values regardless of position. MKM-OS anesthesia may be useful for prolonged equine surgery because of its minimal cardiovascular depression in both of lateral and dorsal recumbency.

Axial correction of pes varus by transverse-opening wedge osteotomy and T-plate fixation with beta-tricalcium phosphate (beta-TCP) transplantation in dachshunds.

Axial correction was performed surgically in two miniature dachshunds presenting with lateral patellar dislocation and limping caused by pes varus. Pes varus had resulted from asymmetric closure of the physis of the distal tibia. Prior to surgery, osteotomy was simulated by measuring X-ray films to determine the distance required for the wedge opening. Transverse-opening wedge osteotomy was performed on the medial side of the distal tibia, and beta-tricalcium phosphate (beta-TCP) was inserted in a wedge shape into the area created by the cuneiform osteotomy. Finally, the tibia was fixed by a veterinary 1.5/2.0-mm T-plate. Both dogs were able to walk a few days after surgery, and the lateral dislocation of the patella normalized almost completely in about one month. At two months, X-ray films showed that the implant had remained in position without any dislocation, and the beta-TCP had fused with the surrounding bone.