Comparative observation of freeze–thaw‐induced damage in pig,rabbit, and human corneal stroma

Objective Although variations exist between species with respect to outcomes after cryopreservation, little is known about the differences in the susceptibility of the corneal stroma to cryoinjury. We performed this study to investigate freeze–thaw‐induced damage in keratocytes and collagen in rabbit, pig, and human corneas. Animals studied Rabbit, pig, and human. Procedures We prepared 250‐μm‐thick anterior stroma from rabbit, pig, and human corneas after scraping off the epithelium and endothelium. Each 250‐μm‐thick corneal stroma without epithelium was placed in a 50‐mL tube, frozen with liquid N₂ for 15 min and taken out to thaw rapidly at 37 °C. This procedure of rapid freezing and thawing was repeated three times. Differences between the species with respect to cells and collagen structures were examined using hematoxylin–eosin (H&E) staining, terminal deoxynucleotidyl transferase‐mediated nick end labeling (TUNEL) assay, and transmission electron microscopy (TEM). We orthotopically transplanted the pig and rabbit corneal transplants after the triple freeze–thaw cycle into rabbit eyes and evaluated graft survival. Results On gross examination, rabbit corneas became opaque after the triple freeze–thaw procedure, while pig and human corneas remained transparent. Histologically, keratocytes were apoptotic on TUNEL assay and TEM in rabbit, pig, and human corneas. Collagen fibrils were fragmented and the arrangement of collagen fibrils was severely disturbed in rabbit corneas on H&E staining and TEM; collagen was well preserved in pig and human corneas. Rabbit corneal stroma underwent autolysis after transplantation, whereas the pig corneal stroma remained clear for 1 month. Conclusions Our study showed that rabbit corneal stroma was more susceptible to freeze–thaw injury than pig and human corneas.     

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.     

Isolation and cultivation of equine corneal keratocytes, fibroblasts and myofibroblasts

Objective  To establish an in vitro model for the investigation of equine corneal wound healing. To accomplish this goal, a protocol to isolate and culture equine corneal keratocytes, fibroblasts and myofibroblasts was developed.
Animal material  Equine corneal buttons were aseptically harvested from healthy research horses undergoing humane euthanasia for reasons unrelated to this study. Slit-lamp biomicroscopy was performed prior to euthanasia by a board-certified veterinary ophthalmologist to ensure that all samples were harvested from horses free of anterior segment disease.
Procedure  Equine corneal stroma was isolated using mechanical techniques and stromal sub-sections were then cultured. Customized media at different culture conditions was used to promote growth and differentiation of corneal stromal cells into keratocytes, fibroblasts and myofibroblasts.
Results  Cell culture techniques were successfully used to establish a method for the isolation and culture of equine corneal keratocytes, fibroblasts and myofibroblasts. Immunohistochemical staining for alpha-smooth muscle and F-actin was used to definitively differentiate the three cell types.
Conclusion  Equine corneal stromal keratocytes, fibroblasts and myofibroblasts can be predictably isolated and cultured in vitro using this protocol.     

Ultrastructure features of camel cornea--collagen fibril and proteoglycans

Introduction The uniform distribution of collagen fibrils and proteoglycans maintain the transparency of normal cornea. We describe the ultrastructural features of camel cornea including collagen fibrils and proteoglycans (PGs). Methods Camel corneas (of 6‐, 8‐, and 10‐month‐old animals) were fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer and processed for electron microscopy. The ‘AnalySIS LS Professional’ program was used to analyze the collagen fibril diameter. Results The camel cornea consists of four layers: the epithelium (227 μm), stroma (388 μm), Descemet’s membrane (DM), and endothelium. The epithelium constituted 36% of the camel cornea, whereas corneal stroma constituted 62% of the corneal thickness (629 μm). The PGs in the posterior stroma were significantly larger in number and size compared with the anterior and middle stroma. The collagen fibril diameter was 25 nm and interfibrillar spacing 40 nm. Fibrillar structures are present throughout the DM. Conclusion The structure of the camel cornea is very different from human and other animals. The unique structure of the cornea might be an adaptation to help the camel to survive in a hot and dry climate. The camel cornea may also be a good model to study the effect of hot and dry climates on the cornea.     

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.     

Rat allogeneic mesenchymal stem cells did not prolong the survival of corneal xenograft in a pig-to-rat model

Objective  Recent reports have demonstrated that mesenchymal stem cells (MSCs) can modulate/suppress immunologic responses through interactions with different immune cells. We performed this study in order to investigate the immunomodulatory effects of MSCs in corneal xenotransplantation.
Animals studied  Pig and rat.
Procedures  We orthotopically transplanted pig corneas into rats and topically applied allogeneic rat MSCs to the corneas for 2 h immediately after transplantation. Graft survival was clinically assessed using slit-lamp biomicroscopy and the median survival time (MST) was calculated. The rejected grafts were histologically examined using antibodies against CD4, CD8, CD161, and CD68. The expression of IL-2, IL-6, IL-10, and IFN-γ was also evaluated in the rejected grafts using an enzyme-linked immunosorbent assay.
Results  The survival of corneal xenografts was not significantly prolonged by MSC application (MST 10.5 days) compared with the controls (MST 9.67 days) ( P  = 0.4189). Histologically, the rejected grafts in both groups were massively infiltrated with neutrophils and macrophages. Some CD8⁺ T cells and rare NK cells were found in the rejected grafts. The levels of IL-6 and IL-10 were significantly increased in the rejected grafts from MSC-treated rats compared with the grafts from MSC-untreated rats. However, the levels of IL-2 and IFN-γ were not different between the two groups.
Conclusions  Topical application of allogeneic rat MSCs was ineffective in prolonging corneal xenograft survival in a pig-to-rat model.     

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.     

Clinical study of cryosurgery efficacy in the treatment of skin and subcutaneous tumors in dogs and cats

Objective— To evaluate the efficacy of cryosurgery for treatment of skin and subcutaneous tumors in dogs and cats.
Study Design— Prospective study.
Animals— Dogs (n=20), cats (10).
Methods— Cutaneous or subcutaneous tumors were treated by liquid nitrogen cryosurgical spray (1 cm from target tissue at 90° until a 5-mm halo of frozen tissue was achieved) for 15–60 seconds. Malignant lesions had 3 freeze–thaw cycles benign tumors, 2 cycles. The second or third freeze cycle was performed after complete thaw of the preceding freeze. Wounds healed by second intention. Follow-up was weekly for 1 month and then twice monthly until wounds healed, and final outcome was determined by telephone interview of owners.
Results— Tumor size ranged from 0.3 to 11 cm diameter with 28 (60%) being 0.3–1 cm; 8 (17%) 1.1–3 cm, and 11 (23%) >3.4 cm. Complications included edema, erythema and for extremity lesions, pain and lameness. Treated lesions (n=47) had an overall remission of 98% (mean follow-up, 345±172.02 days [range, 150–750 days]). One malignant peripheral nerve sheath tumor recurred 7 months after cryosurgical treatment.
Conclusion— Cryosurgery is an efficient method for treatment of skin and subcutaneous tumors in dogs and cats.
Clinical Relevance— Cryosurgical ablation is an effective means of treating small cutaneous or subcutaneous tumors in dogs and cats, especially in older animals where wound closure or cosmetic outcome might limit surgical excision alone.     

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.     

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.     

Effect of bovine freeze-dried amniotic membrane (Amnisite-BA™) on uncomplicated canine corneal erosion

Objective   To evaluate the effectiveness of bovine freeze-dried amniotic membrane (FD-AM) (Amnisite-BA™) in the surgical treatment of corneal ulceration in dogs.
Animals studied   Eight normal Shih-tzu dogs.
Procedures   The corneas of 16 eyes were scored with an 8.0-mm trephine under general anesthetic and 100% ethanol was applied to remove a standardized button of corneal epithelium. The eyes were treated as described below and the corneas were evaluated 48 h later. The dogs were divided into four treatment groups: (i) control, (ii) amniotic membrane transplantation (AMT), (iii) nictitating membrane flap and (iv) contact lens. The proportion of the corneal wound that healed was calculated and all eyes were enucleated. Histological sections of cornea were assessed with the proliferating cell nuclear antigen (PCNA) assay.
Results   The proportion of corneas healed in the different treatment groups was (i) 38.02%, (ii) 89.15%, (iii) 52.31%, and (iv) 60.56%. Epithelial healing was significantly increased in the AMT group (ii) ( P  = 0.001) while groups (iii) and (iv) were not significantly different from the control group ( P  = 0.537 and P  = 0.198, respectively). The number of PCNA positive cells was (i) 275.00, (ii) 740.50, (iii) 285.75 and (iv) 420.59, these varying compared with the control group with statistical significance of (ii) P  = 0.002, (iii) P  = 0.999, and (iv) P  = 0.467. The greatest healing rate and epithelial cell proliferation was achieved with AMT compared to the other treatment regimes.
Conclusions  The results of this study show that FD-AM transplantation is an effective treatment for enhancing canine corneal wound healing and suggest that the approach will provide superior results compared to conventional treatments for the condition.     

Inhibition of collagenase breakdown of equine corneas by tetanus antitoxin,equine serum and acetylcysteine

Objective To determine whether tetanus antitoxin, equine serum, and acetylcysteine, which are currently used in the treatment of equine corneal ulcer, inhibit the digestion of equine corneal collagen when exposed to collagenase in vitro. Animals studied Corneas from 40 adult horses. Procedures Sections of equine corneas were incubated with saline, a solution of bacterial collagenase in saline, bacterial collagenase in saline plus equine tetanus antitoxin, bacterial collagenase in saline plus equine serum, or bacterial collagenase in saline plus acetylcysteine. Each one of the collagenase inhibitors was tested at different concentrations. The degree of corneal collagen digestion was determined by concentrations of hydroxyproline released into the incubation media and/or by weight loss of the cornea. Results Corneas exposed to collagenase released a significant (0.05 level) large amount of hydroxyproline (43.1 ± 2.3 µg/mL/100 mg cornea/5 h) and decreased cornea weight by up to 89%. Blood serum (200 µL/mL), purified albumin or globulin fractions of serum, tetanus antitoxin (120 units/mL), and acetylcysteine (20 mg/mL) when used at the highest concentrations blocked collagenase digestive activity by approximately 50%. Dilution of inhibitors decreased corneal protection and linearly increased corneal weight loss. Purified equine serum albumin and globulin fractions were equally effective in protecting corneas. Conclusions This experiment indicates that tetanus antitoxin, serum and acetylcysteine equally protected corneas from collagenase digestion, in vitro. However, a clinical trial is needed to establish relative therapeutic value.     

Lamellar keratoplasty with a graft of lyophilized acellular porcine corneal stroma in the rabbit

Objective To evaluate the efficacy of lamellar keratoplasty in the rabbit using a graft of lyophilized acellular porcine corneal stroma (APCS). Animal studied Twelve adult 2–2.5 kg Zealand white rabbits were studied. Procedure The cell components of the porcine cornea were removed by the means of enzymatic digestion, freezing, and thawing and then APCS was lyophilized. The 6.5 mm diameter APCS was implanted on a 6.0‐mm diameter keratectomy wound each of 12 rabbits. The postoperative clinical and histological evaluations were performed in the early, intermediate, and late periods. Results All corneal wounds healed. Ten of the 12 grafts of APCS were integrated completely with the receptive cornea except two grafts scraped partially off by the eyelid. The blepharospasm, ocular discharge, and edema of the cornea were marked 1 week after transplantation. New vessels invaded the graft after week 2 and regressed after week 8. The cornea became transparent gradually. The histological evaluation showed that the epithelium on the graft stratified normally post surgery. The keratocytes of the recipient grew into the graft and were proliferative at week 4. The inflammatory cells and new vessels were observed before week 8. The fibrosis in the graft was revealed at week 4 and lessened at week 8. The histological structure of the cornea after surgery was similar to the normal cornea at week 32. Conclusions APCS can recover the integrity of the rabbit's cornea and become transparent in vivo. APCS is an effective graft for lamellar keratoplasty in the rabbit.     

Effects of growth factors (EGF,PDGF-BB and TGF-beta 1) on cultured equine epithelial cells and keratocytes: implications for wound healing

Objective The physiologic mechanisms involving growth factors, including PDGF‐BB, EGF, and TGF‐β1, as potent mediators of fibroblasts and epithelial cells in corneal wound healing remain unknown. The goal of this study was to determine culture methods for equine epithelial cells and keratocytes and to investigate how exogenous growth factors influence proliferation of both cell types. Procedures Cell cultures were established from healthy corneas harvested from horses immediately following euthanasia and maintained using standard tissue culture protocols. To determine the effects of PDGF‐BB, EGF, TGF‐β1, keratocytes (1 × 10⁵/well) and epithelial cells (2 × 10⁵/well) were each cultured in 12 well plates and exposed separately to the growth factors. The cells were exposed to concentrations of EGF between 0 and 50 ng/mL; PDGF‐BB between 0 and 75 ng/mL; and TGF‐β1 between 0 and 10 ng/mL. Cell proliferation was measured using ³H‐thymidine assay and differences in growth determined using anova and Tukey's HSD test (P < 0.05). Results Epithelial cell and keratocyte cultures were successfully established. EGF maximally stimulated keratocyte and epithelial cells at 25 ng/mL and 5 ng/mL, respectively. PDGF‐BB maximally stimulated keratocytes and epithelial cells at 50 ng/mL and 5 ng/mL, respectively. TGF‐β1 inhibited keratocytes at 5 ng/mL and 10 ng/mL, and epithelial cells at 1 ng/mL and 2 ng/mL. Conclusions Methods were established to maintain epithelial cells and keratocytes in vitro. PDGF‐BB and EGF stimulate, while TGF‐β1 inhibits the proliferation of epithelial cells and keratocytes. These growth factors may play a role in maintenance and repair of the equine cornea.     

Radiotherapy for canine chronic superficial keratitis using soft X-rays (15 kV)

Objective  To evaluate the effect of soft X-ray therapy in the treatment of refractory chronic superficial keratitis (CSK).
Animals studied  Thirteen dogs with severe CSK, that had been refractory to prior medical and/or surgical therapy were treated with soft X-ray therapy.
Procedures  Both corneas of each dog were irradiated with soft X-rays (15 kV), to a total dose of 30 Gy, administered as two fractions over 48–96 h. Treatment was carried out under deep sedation in all dogs. Three dogs were treated by superficial lamellar keratectomy 48 h prior to radiotherapy. Changes in the extent of corneal pigmentation, pigment density and corneal vascularization were documented using a semi-quantitative grading scheme, schematic drawings and clinical photographs.
Results  Only minor, transient adverse effects of treatment, such as photophobia, epiphora and blepharitis were noted. Overall the effect of soft X-rays on the course of the keratitis was superior when compared to the effect of Sr-90 irradiation that had been determined in a previous study.
Conclusion  Soft X-ray irradiation combined with keratectomy is a safe and effective new treatment option for severe and advanced CSK with significant visual impairment due to corneal pathology.     

Morphological analysis of cell distribution and network structure via gap junctions in swine corneal stroma

Because of corneal transplantation limitations, there is a need for cornea-specific regenerative medicine. The development of such regenerative medicine has been delayed because of the complex and unique structure of the corneal stroma. Few studies have explored the corneal stroma cell distribution and cell types in vivo. This study investigated regional differences in morphological characteristics and distributions of corneal keratocytes and immunocompetent cells in the corneal stroma to clarify their functions and structural characteristics. The porcine eyeballs were subjected to light microscopy, transmission electron microscopy, scanning electron microscopy, and immunofluorescence staining analyses. Corneal cells were primarily located in the limbus, rather than the center of the cornea; the long keratocyte diameter was largest on the epithelial side of the corneal limbus, while the short diameter was largest on the endothelial side of the central cornea. Moreover, there were significantly more corneal cells on the epithelial side than on the endothelial side in both the central and limbus areas. Gap junctions between cells in the corneal stroma were present on the surfaces of cytoplasmic processes. Many cytoplasmic processes were scattered throughout the corneal stroma; they were connected both vertically and horizontally, forming an intercellular network. Additionally, immunocompetent cells on the epithelial side suggested to participate in this network via gap junctions. The morphology of keratocytes and immunocompetent cells on the epithelial side suggests that they play important roles in corneal homeostasis.     

Pathogenesis of corneal lesions caused by Moraxella bovis in gnotobiotic calves

Moraxella bovis was instilled into the conjunctival sac of gnotobiotic calves and corneas were sampled serially after infection. Lesions developed in seven of eight infected calves, but were absent in a noninfected control calf. Histologically, M. bovis was first seen in foci of swollen epithelium and within basal epithelial cells adjacent to ulcers. Corneal ulcers were severe in later stages of infection; fibrin deposits, neutrophils, and bacteria were present in the stromas. Examination of early lesions by scanning electron microscopy showed M. bovis in pits on the surfaces of dark epithelial cells, enmeshed in degenerate epithelial cells and within erosions and an ulcer; in later samples, bacteria were rare. Ultrastructurally, M. bovis was seen in surface pits in superficial epithelial cell processes and within swollen epithelial cells. In stroma, M. bovis was frequently seen among collagen fibrils, within neutrophil phagosomes, and associated with cellular debris. This study demonstrates that a virulent strain of M. bovis can invade bovine corneal epithelial cells and can cause keratitis in the absence of injurious ultraviolet irradiation or other known predisposing environmental factors.     

The effects of moxifloxacin ophthalmic solution 0.5% or gatifloxacin ophthalmic solution 0.3% treatment on corneal wound healing in pigmented rabbits following anterior keratectomy

Purpose These studies examined corneal healing rates, Type‐IV collagen and zonula occludens membrane‐associated protein (ZO‐1) expression, as well as aqueous PGE₂ and IL‐1β concentrations in pigmented rabbits treated with either moxifloxacin 0.5%, gatifloxacin 0.3% or BSS^® following anterior keratectomy. Methods Anterior keratectomy surgery was followed by topical administration with commercial ophthalmic formulations of either moxifloxacin or gatifloxacin or BSS^® (TID for 96 h). Images of the fluorescein‐stained healing corneas were analyzed for wound area. At 48 or 96 h following surgery, aqueous humor samples were collected and analyzed for the inflammatory mediators PGE₂ and IL‐1β using an ELISA. The corneas were subsequently evaluated using both scanning and transmission electron microscopy. In a second parallel study, corneas were evaluated at both 48 and 96 h for Type‐IV collagen and ZO‐1 expression using immunohistochemistry. Results Fluorescein‐stained corneal images at 96 h postsurgery demonstrated that 90% ± 8% re‐epithelialization for moxifloxacin, 81% ± 14% for gatifloxacin, and 88 ± 6% for BSS^® (P > 0.05). PGE₂ levels in the aqueous humor of fluoroquinolone treated eyes were reduced at 48 h compared to BSS^® treated eyes. IL‐1β was undetectable in all samples. No differences in Type‐IV collagen or ZO‐1 expression were observed between any treatment groups. There were no differences between groups in histological appearance or in ultrastructural healing processes. Conclusions These studies demonstrated that the commercial ophthalmic formulations of moxifloxacin and gatifloxacin were similar to each other in their effects on the levels of aqueous humor PGE₂ and rates of corneal wound re‐epithelialization.