Morphology of the pig cornea in normal conditions and after incubation in a perfusion apparatus

The fine structure of the pig cornea in normal conditions and after being used in a perfusion apparatus, for 4 h, is described. Earlier reports on the normal morphology of the pig cornea were partly not confirmed. Thus the number of cell layers in the epithelium was found to be 19-23 (a basal cell layer, 4–5 polyhedral cell layers and 14–17 squamous cell layers) compared to earlier reported 6–9 layers.The mean thickness of normal and perfused corneas were 722 μm and 752 μm respectively. Normal corneas had a hydration level of 77.2 % and after perfusion 78.5 %. The normal morphology and morphological changes due to exposure to perfusion were studied by light and electron microscopy. The differences observed between normal and perfused corneas have to be considered limited, and restricted mainly to the anterior squamous epithelium and the endothelium.Taken together our results indicate that the corneas used in the apparatus still had functional integrity.     

Corneal sensitivity in healthy, immature, and adult alpacas

Objective To determine the corneal sensitivity of healthy, immature, and adult alpacas to establish normal reference values for this species. Animals studied Six normal crias and 18 normal adult alpacas. Procedures Corneal sensitivity was determined by evaluating the corneal touch threshold (CTT) in five regions of the cornea using a Cochet–Bonnet aesthesiometer. The nylon filament length in cm was then converted to applied pressure values in mg/0.0113 mm² and g/mm² using a conversion chart. Results The central region of the cornea was the most sensitive, and the dorsal and temporal regions were the least sensitive in alpacas. There were no significant differences between the right and left eyes or between values in males and females at any site. Crias had significantly greater corneal sensitivity compared with adult alpacas in all five regions of the cornea that were evaluated. Conclusions Normal reference values for CTT in alpacas were established using a Cochet–Bonnet aesthesiometer. The central region of the cornea is the most sensitive in alpacas, and crias have higher corneal sensitivity than adult alpacas.     

Corneal innervation in mesocephalic and brachycephalic dogs and cats: assessment using in vivo confocal microscopy

Objective To determine the density of the canine and feline corneal neural network in healthy dogs and cats using in vivo confocal microscopy (IVCM). Animals examined A total of 16 adult dogs (9 Mesocephalic breeds, 7 Brachycephalic breeds) and 15 cats (9 Domestic Short-haired cats (DSH), 6 Persian cats) underwent IVCM. Procedure Animals were examined with a confocal corneal microscope (HRTII/RCM; Heidelberg Retina Tomograph II/Rostock Cornea Module^®, Heidelberg Engineering, Dossenheim, Germany). The investigations focused on the distribution of the corneal nerves and quantification of central subepithelial and subbasal nerve plexus. Results The corneal stromal nerve trunks, subepithelial and subbasal nerve plexus were observed. The nerve fiber density (NFD) quantified in nerve fiber length in mesocephalic dogs were 12.39 ± 5.25 mm/mm² in the subepithelial nerve plexus and 14.87 ± 3.08 mm/mm² in the subbasal nerve plexus. The NFD of the subepithelial nerve plexus in DSH cats was 15.49 ± 2.7 and 18.4 ± 3.84 mm/mm² in the subbasal nerve plexus. The subbasal NFD of DSH cats was significantly higher than in mesocephalic dogs (P = 0.037). The subepithelial NFD in brachycephalic dogs, and Persian cats were 10.34 ± 4.71 and 9.50 ± 2.3 mm/mm², respectively. The subbasal NFD measured 11.80 ± 3.73 mm/mm² in brachycephalic dogs, and 12.28 ± 4.3 mm/mm² NFD in Persian cats, respectively. The subepithelial and subbasal NFD in Persian cats were significantly lower than in DSH cats (P = 0.028, respectively, P = 0.031), in contrast to brachycephalic vs. mesocephalic dogs. Conclusion The noninvasive IVCM accurately detects corneal innervation and provides a reliable quantification of central corneal nerves.     

Pyogenic granuloma of the cornea in a pony: Uncommon complication of corneal wound healing

A 6-year-old miniature Shetland pony mare was referred for evaluation of a left corneal mass, which developed from the healing tissue of a corneal traumatic ulceration that had occurred 4 weeks previously. On gross examination, a spherical, smooth-surfaced, and pink-colored lesion of about 1 cm in diameter was protruding from the left palpebral fissure. Ophthalmic examination revealed that it was attached to the scar tissue of the cornea, and that one corpora nigra was adherent to the posterior face of corneal wounded area, without sign of uveitis. The remainder of the ophthalmic examination was unremarkable. The mass was excised, and cryotherapy was used as an adjunctive therapy. Histopathology of the resected mass was consistent with a pyogenic granuloma on the basis of radially oriented proliferating capillaries, embedded in immature granulation tissue containing an infiltrate of neutrophils, plasma cells and eosinophils. There were no histological features of malignancy. 2 months after surgery, the ventral part of the fibrotic corneal scar was slightly raised by a pink tissue, suggesting possible recurrence of the initial lesion. A second cryotherapy was performed over the leukoma area. No recurrence has been noted for a follow-up period of more than 25 months. Pyogenic granuloma is a benign proliferative fibrovascular response that typically develops after trauma or surgery. Corneal involvement is rare in humans, and to the authors' knowledge has never been documented in veterinary ophthalmology.     

Keratoprosthesis with retrocorneal fixation: preliminary results in dogs with corneal blindness

Objective To evaluate the use and complications of a penetrating keratoprosthesis implantation in the management of corneal opacification in dogs. Methods A retrospective clinical study describes the indications for the surgical technique utilized and the outcomes of this procedure in 20 eyes of 19 dogs with blindness of corneal origin. A successful surgical outcome was defined as a clear keratoprosthesis optic and improvement or restoration of functional vision over a follow‐up period ranging from at least 8 months to a maximum of 7 years. Results Eyes with total corneal opacification resulting from chronic superficial keratitis (n = 11), keratoconjunctivitis sicca (n = 5), endothelial dystrophy (n = 3) and chemical burn (n = 1) were treated by unilateral (n = 18) or bilateral (n = 1) full‐thickness implantation of a keratoprosthesis. Keratoprostheses were retained in 15 eyes (75%) which regained vision to the date of reporting. Among these eyes, six had uncomplicated postoperative course, five developed retroprosthetic membranes and four developed granulation tissue over the optic of the keratoprostheses. These complications were successfully removed surgically in the nine eyes. The five remaining eyes (25%) developed serious early postoperative complications, for which enucleation had to be performed. Conclusion In keratopathies in which the corneal opacification could not be treated by standard medical or surgical procedures, this keratoprosthesis appears to be promising to restore vision in chronic superficial keratitis and deep corneal dystrophy. It appears to have a poor prognosis in keratoconjunctivitis sicca in brachycephalic dogs. The post operative complications retro‐prosthetic membranes and granulomatous overgrowth could be treated well.     

Heterologous penetrating keratoplasty for treatment of a corneal sequestrum in a cat

A corneal sequestrum was diagnosed in an 8-year-old, neutered male Burmese cat. A heterologous penetrating keratoplasty (PK) (fresh canine corneal tissue) was performed to restore a clear visual axis. A heterograft was selected in order to decrease the risk of viral transmission as a screened donor was not available. One month postoperatively the graft was vascularized and opaque. The owner failed to return for recheck examinations until 16 months postoperatively at which time only a faint central nebula remained.     

Early postnatal development of central corneal thickness in dogs

Objective To investigate the changes in corneal thickness that occur during maturation of the canine eye over the first months of life. Animals studied Dogs of two different breeds with ages ranging from 14 days to 42 weeks of age. Procedures The central corneal thickness was measured by ultrasonic pachymetry every week for the first month after eyelid opening (around 14 days) and then every month until 42 weeks of age. Segmented regression was applied to capture the two phases observed in the central corneal thickness plotted against age. Breed, eye and gender were also included in the model. Results Mean central corneal thickness (CCT) values initially decreased following eyelid opening, with the lowest point being reached at around 6 weeks of age. Then CCT gradually increased as the dogs matured. Differences between left and right eye were not significant. Breed and gender effects were significant factors in the statistical model. Conclusions Following eyelid opening there is an initial decrease in corneal thickness until approximately 6 weeks of age, which presumably mirrors maturation of corneal endothelial cell function. After 6 weeks of age the CCT increases with age until approximately 30 weeks of age after which there was only a gradual increase over the remainder of the study period. A similar pattern of changes in corneal thickness in humans has been previously recorded.     

Profiles of matrix metalloproteinase activity in equine tear fluid during corneal healing in 10 horses with ulcerative keratitis

OBJECTIVE: Levels of tear film matrix metalloproteinases (MMPs) activity are significantly elevated in horses with ulcerative keratitis and contribute to the excessive breakdown of stromal collagen. Changes in the amount of proteolytic activity in horse tear film during corneal healing and stromal remodeling have not yet been reported, but we hypothesize they should decrease. In the present study we analyzed serial tear fluid from horses with ulcerative keratitis to identify any changes in MMP activity during corneal healing and stromal remodeling. PROCEDURES: Samples of tear fluid were obtained from both eyes of 10 horses with ulcerative keratitis on the day of admission (day 1) at the hospital and then at various time points until complete healing of the cornea. Tear film MMP2 and MMP9 activity was determined by quantitative gelatin zymography. In all cases medical treatment included topical applications of equine serum, antibiotics, atropine and systemic administration of anti-inflammatory drugs. Surgical procedures were performed in several cases on day 2 in addition to the medical treatment. RESULTS: The mean total MMP activity (+/- SD) measured in relative standard units (RSU) in the tear fluid of the ulcerated eye (2.44 +/- 1.44) of the 10 horses was significantly higher than the mean in the contralateral eye (0.81 +/- 0.68) (P = 0.006), on the day of admission at the VMTH. The mean MMP activity in these ulcerated eyes significantly decreased (-82.4%) between the first day of admission and the day when the ulcer had completely healed (P = 0.0002). The activity level in the healed eye (0.43 +/- 0.17) was not significantly different to the one in the contralateral eye (0.36 +/- 0.18) on the day of complete corneal healing (P = 0.374). The level of MMP activity in the contralateral eye also decreased from 0.81 +/- 0.68-0.36 +/- 0.18 but this decrease (56%) was not significant (P = 0.069). CONCLUSIONS: Ulcerative keratitis in horses is associated with initially high levels of tear film proteolytic activity that decrease as the ulcers heal. The success of medical and surgical treatment of the corneal ulcers is reflected by the enzyme activity in tears. In horses successful treatment does lead to a rapid reduction in tear film proteolytic activity that corresponded with the improvement in the clinical signs of corneal ulceration. Measurement of MMP activity in the tear film might represent a way to monitor the progression of corneal healing in horses with ulcerative keratitis.     

Keratometry in normal cats: a cross-sectional study in Japan using an automated handheld keratometer

Keratometry was performed in 73 domestic cats of varied signalment in Japan using an automated handheld keratometer. The mean corneal curvature radius was significantly lower for cats younger than 1 year than for those older than 2 years (8.04 mm vs. 8.80−8.99 mm, P<0.01). The radius was significantly greater in males than in females among the cats older than 11 years (9.22 mm vs. 8.84 mm, P=0.01), while the age distributions of the males and females were similar. Corneal astigmatism did not significantly differ across the gender and age groups. The predictability of the corneal curvature and astigmatism was approximately 41−43% and less than 3%, respectively, as a function of age and bodyweight. The results highlighted some age- and sex-related keratometric variations in domestic cats in Japan.     

Ultrastructural findings in feline corneal sequestra

OBJECTIVES: (1) To describe the ultrastructural features of corneal sequestra in cats; and (2) to enhance our understanding regarding the pathogenesis of feline corneal sequestration. METHODS: Nine corneal sequestra were harvested via keratectomy from globes of nine cats. The sequestra were routinely fixed then postfixed for high resolution light and transmission electron microscopy (HR-LM and TEM, respectively). The tissues were embedded in Epon/Araldite. Sections of 0.5-microm thickness were cut and stained with 1% toluidine blue in 1% sodium tetraborate solution for HR-LM. Ultrathin sections were collected on copper grids and stained with uranyl acetate and Sato's lead stain for TEM. Ultrathin sections were examined and the images were captured on an Advantage HR CCD camera using a Hitachi 7500 electron microscope operated at 80 kV. Two healthy corneas from two cats were harvested immediately following euthanasia. These corneal tissues (control samples) were processed in the same manner as the corneal sequestra for HR-LM and TEM. A portion of each sequestrum was also submitted for polymerase chain reaction (PCR) testing for infectious agents including feline herpesvirus-1 (FHV-1), Toxoplasma gondii, Chlamydophila felis and Mycoplasma spp. RESULTS: Ultrastructure of healthy corneal tissues revealed basal corneal epithelial cells aligned adjacent to a thin acellular layer similar to Bowman's layer with underlying tightly packed, regularly arranged, collagen fibrils oriented in different planes. Keratocytes were elongated and had long and irregularly shaped nuclei, and cytoplasm contained rough endoplasmic reticulum and abundant membrane-bound vesicles. In contrast, corneal sequestra contained varying amounts of an amorphous, electron-dense substance, continuous with intact basal epithelial basement membranes peripherally, and overlying corneal ulceration and loosely packed collagen fibrils. Remnants of necrotic keratocytes were seen in spaces between disarranged collagen layers. In all samples, occasional keratocytes exhibited morphology indicative of apoptosis including clumping and margination of chromatin, and shrunken cytoplasm. Varying degrees of inflammation were noted on HR-LM and TEM of affected corneas including peri- and intralesional neutrophils, lymphocytes, plasma cells, and macrophages. Corneal sequestra were FHV-1-positive (n = 3), FHV-1- and T. gondii-positive (n = 1), T. gondii-positive (n = 3), or negative for DNA of these infectious agents (n = 2) using PCR. All corneal sequestra were negative for DNA of Chlamydophila felis and Mycoplasma spp. using PCR. CONCLUSIONS: Apoptosis may play a role in the pathogenesis of feline corneal sequestration independent of the presence of DNA of these infectious organisms. Prospective clinical studies are warranted to further understand the significance of T. gondii in relation to feline corneal sequestration.     

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.     

Postnatal development of corneal curvature and thickness in the cat

Objective To evaluate the postnatal development of central corneal curvature and thickness in the domestic cat. Animals studied Six Domestic Short‐haired (DSH) kittens starting at 9 weeks of age and 6 adult cats. Procedures Kittens were evaluated biweekly to monthly for a 12‐month period, starting at age 9 weeks. Corneal development was monitored by hand‐held keratometry and ultrasound biomicroscopy. Standard regression analysis using a nonlinear least squares method was used to generate a formula that would predict corneal curvature as a function of age. Results Mean keratometry (K) values for the 9‐week‐old cats were 54.51 (±1.02) diopters (D) and these values steeply declined over the next 3 months to 44.95 (±0.90) D. Thereafter, K‐values gradually decreased to reach a plateau by 12–15 months of age of 39.90 (±0.42) D. Because K‐values still appeared to be slightly diminishing at this point, six other > 2‐year‐old cats were evaluated by keratometry and were found to have K‐values of 38.99 (±0.81). Two to four diopters of astigmatism was common in young kittens whereas adult cats had a low mean degree of astigmatism (< 1 D). A formula that predicted keratometry values in diopters (K) as a function of age in weeks (w) was established as follows: K = 39.83 + 26.87 exp(?0.074 w). The central cornea increased in thickness primarily during the first 4 months of life with 9 week‐old kittens having values of 0.379 (±0.012) mm; 16‐week‐old kittens, 0.548 (±0.021) mm and 67 week‐old cats, 0.567 (±0.012) mm. Conclusions The maturation process of the feline cornea proceeds over the first 1–2 years of life to attain an adult status that is characterized by a roughly spherical state of approximately 39 D corneal curvature, substantially flatter than the human cornea, and a central thickness similar to the human cornea. Research studies of the refractive or optical properties of the cornea in which cats are used as experimental animals should be conducted on animals greater than 18 months of age.