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.     

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.     

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.     

Immunohistochemical study of matrix metalloproteinases‐2 and ‐9, macrophage inflammatory protein‐2 and tissue inhibitors of matrix metalloproteinases‐1 and ‐2 in normal,purulonecrotic and fungal infected equine corneas

Objective Determine the effects of matrix metalloproteinases (MMPs)‐2, ‐9, macrophage inflammatory protein‐2 (MIP‐2), tissue inhibitors of matrix metalloproteinase (TIMP)‐1 and ‐2 by immunohistochemical expression in fungal affected and purulonecrotic corneas. Procedure Paraffin‐embedded equine corneal samples; normal (n = 9), fungal affected (FA; n = 26), and purulonecrotic without fungi (PN; n = 41) were evaluated immunohistochemically for MMP‐2, ‐9, MIP‐2, TIMP‐1 and ‐2. The number of immunoreactive inflammatory cells was counted and statistics analyzed. Western blot was performed to detect MMP‐2, MMP‐9, TIMP‐1 and TIMP‐2 proteins. Results Matrix metalloproteinases‐2, ‐9, MIP‐2, TIMP‐1 and ‐2 immunoreactivity was identified in corneal epithelium of normal corneas, and in corneal epithelium, inflammatory cells, keratocytes, and vascular endothelial cells of both FA and PN samples. Inflammatory cell immunoreactivity was significantly higher in FA and PN samples than in the normal corneas. There was positive correlation between MMP‐2 and MIP‐2, MMP‐9 and MIP‐2, and MMP‐9 and TIMP‐1 in inflammatory cell immunoreactivity in FA samples. There was positive correlation between MMP‐9 and MIP‐2, MMP‐9 and TIMP‐2, MIP‐2 and TIMP‐1, and MIP‐2 and TIMP‐2 in inflammatory cell immunoreactivity in PN samples. Western blot confirmed the presence of all four proteins in equine corneal samples. Conclusion Increased immunoreactivity of MMP‐2 and ‐9 in FA and PN samples is indirectly related to MIP‐2 through its role in neutrophil chemo‐attraction. Tissue inhibitors of matrix metalloproteinase‐1 and TIMP‐2 are up‐regulated in equine purulonecrotic and fungal keratitis secondary to MMP‐2 and MMP‐9 expression. The correlation between MMPs ‐2 and ‐9, MIP‐2, TIMPs ‐1 and ‐2 suggests that these proteins play a specific role in the pathogenesis of equine fungal keratitis.     

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.     

Analysis of a commercial dimethyl‐sulfoxide‐stabilized frozen canine platelet concentrate by turbidimetric aggregometry

Objectives – To assess platelet function of a commercial dimethyl‐sulfoxide (DMSO)‐stabilized frozen platelet concentrate (PC) using turbidimetric aggregometry. Design – In vitro analysis. Setting – Research laboratory in a school of veterinary medicine. Animals – Five units of frozen PC in 6% DMSO were studied. Fresh platelet‐rich plasma (PRP), with and without 6% DMSO, from 6 healthy dogs were used as controls. Interventions – Turbidimetric platelet aggregation was measured after initiation of platelet aggregation by addition of adenosine diphosphate (ADP), collagen, or thrombin at concentrations of 30 μM, 20 μg/mL, and 0.5 U/mL, respectively. Measures were performed at thaw and repeated 2 hours after thaw for the frozen PC. Measurements and Main Results – Compared with PRP, the frozen PC showed decreased aggregation in response to thrombin (amplitude of 84% versus 25%, P=0.01), and collagen (amplitude of 13% versus 3%, P=0.05) but not ADP (6.5% versus 18%, P=0.2). Compared with frozen PC at thaw, the frozen PC at 2 hours after thaw showed decreased aggregation in response to thrombin, collagen, and ADP (P<0.05). There was no difference in aggregation between PRP in 6% DMSO and frozen PC. Conclusions – These in vitro data suggest there is a decrease in platelet response to agonists associated with the freeze‐thaw process in the commercially available 6% DMSO canine frozen PC.     

Expression of matrix metalloproteinases, type IV collagen, and interleukin-10 in rabbits treated with morphine after lamellar keratectomy

Purpose To study the effects of topical administration of 1% morphine on corneal analgesia in rabbits submitted to lamellar keratectomy and to assess the expression of matrix metalloproteinase‐1, metalloproteinase‐2, metalloproteinase‐9 (MMPs), type IV collagen, and interleukin‐10 (IL‐10) during the treatment. Methods Morphine group (MG) received 50 μL of topical 1% morphine four times daily, while the control group received saline instead. Corneal touch threshold (CTT) and the wound area were assessed until corneal healing. Corneal samples were processed for routine histology, immunohistochemistry, zymography, and ELISA. Results Following keratectomy, CTT increased significantly from 6 to 96 h time points. Mean corneal re‐epithelization rate and scores of leukocyte infiltration did not differ significantly between treatment groups. Immunolabeling pattern for MMP‐1, MMP‐9, and type IV collagen was similar in both treatment groups. In the MG, zymography indicated significantly higher levels of active MMP‐2 on days 6 and 12; and in the latent MMP‐9, on days 3 and 6, and in the active MMP‐9, on day 6. Latent MMP‐2 and MMP‐9, and active MMP‐9 decreased to values close to those of healthy corneas on day 12, but levels of active MMP‐2 remained significantly elevated in the MG. IL‐10 levels measured on days 1–6 were reduced as compared to those of healthy corneal tissue and returned to levels close to those of healthy corneas on day 12. Conclusion Topical morphine promoted corneal analgesia for up to 4 days and did not delay corneal re‐epithelization. The re‐establishment of MMPs and IL‐10 to levels close to baseline values at the end of the study and the expression of type IV collagen in both groups reinforce that, with caution, 1% morphine can be used after lamellar keratectomy in rabbits.     

A cross‐linked hyaluronan gel accelerates healing of corneal epithelial abrasion and alkali burn injuries in rabbits

Objective To evaluate the efficacy of a chemically modified and cross‐linked derivative of hyaluronan (CMHA‐SX) for treatment of corneal epithelial abrasion and standardized alkali burn injuries. Animals Twelve female New Zealand white rabbits in two groups were used. Procedures Bilateral 6‐mm diameter corneal epithelial abrasions were made in each of six rabbits in one group and 6‐mm standardized alkali burn injuries were made in the second group. A 1% CMHA‐SX formulation was applied topically four times per day in right eye of each rabbit for 1 week, and phosphate buffered saline (PBS) was placed in left (control) eye of each rabbit. The wound size was determined by staining with 1% fluorescein and photographed at the slit lamp with a digital camera at 0, 1, 2, 3 days postoperatively in the first group and 0, 1, 2, 3, 7, 12 days in the second group. Rabbit corneas were collected for histological examination on day 7 in the first group and day 12 in the second group. Results Closure of corneal wound in the abrasion model was complete in the CMHA‐SX treated eye by 48 h. The wound closure rate and thickness of the central corneal epithelium in the CMHA‐SX treated group was greater than in control eyes for both the abrasion and alkali burn injuries. Moreover, the CMHA‐SX treated cornea exhibited better epithelial and stromal organization than the untreated control cornea. Conclusions Chemically modified and cross‐linked derivative of hyaluronan improved corneal wound healing and could be useful for treating noninfectious corneal injuries.     

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.     

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.     

In vivo confocal microscopy of equine fungal keratitis

Objective To describe in vivo corneal confocal microscopy of horses with fungal keratitis and correlate findings with clinical, histopathological, and microbiological evaluations of clinical cases and an ex vivo experimental equine fungal keratitis model. Animals studied A total of 12 horses with naturally‐acquired fungal keratitis and ex vivo equine corneas experimentally infected with clinical fungal isolates. Procedures Horses with naturally‐acquired fungal keratitis were examined with a modified Heidelberg Retina Tomograph II and Rostock Cornea Module. Confocal microscopy images of clinical isolates of Aspergillus fumigatus, Fusarium solani, and Candida albicans were obtained by examination of in vitro cultures and experimentally infected ex vivo equine corneas. Results Non‐specific in vivo corneal confocal microscopic findings in horses with fungal keratitis included leukocyte infiltrates, activated keratocytes, anterior stromal dendritic cell infiltrates, and vascularization. Linear, branching, hyper‐reflective structures that were 2–6 μm in width and 200 to >400 μm in length were detected in all horses with filamentous fungal keratitis. Round to oval hyper‐reflective structures that were 2–8 μm in diameter were detected in a horse with yeast fungal keratitis. The in vivo confocal microscopic appearance of the organisms was consistent with fungal morphologies observed during examination of in vitro cultures and infected ex vivo equine corneas. Conclusions In vivo corneal confocal microscopy is a rapid and non‐invasive method of diagnosing fungal keratitis in the horse. This imaging technique is useful for both ulcerative and non‐ulcerative fungal keratitis, and is particularly advantageous for confirming the presence of fungi in deep corneal stromal lesions.     

Storage of Bovine Reproductive Tissues and RNA Extracts on Ice for 24 h or Repeated Freeze–Thaw Cycles do not Affect RNA Integrity

The aims of this study were to test (i) the effect of time of tissue and RNA extracts storage on ice and (ii) the effect of repeated freeze–thaw cycles on RNA integrity and gene expression of bovine reproductive tissues. Fragments of endometrium (ENDO), corpus luteum (CL) and ampulla (AMP) were subdivided and incubated for 0, 1, 3, 6, 12 or 24 h on ice. RNA extracts were incubated on ice for 0, 3, 12 or 24 h, or exposed to 1, 2, 4 or 6 freeze–thaw cycles. RNA integrity number (RIN) was estimated. Expression of progesterone receptor (PGR) and cyclophilin genes from RNA extracts stored on ice for 0 or 24 h, and 1 or 6 freeze–thaw cycles was measured by qPCR. Tissue and RNA extract incubation on ice, and repeated freeze–thaw cycles did not affect RIN values of RNA from ENDO, CL or AMP. Storage on ice or exposure to freeze–thaw cycles did not affect Cq values for PGR or cyclophilin genes . In conclusion, neither generalized RNA degradation nor specific RNA degradation was affected by storage of tissue or RNA extracts on ice for up to 24 h, or by up to 6 freeze–thaw cycles of RNA extracts obtained from bovine ENDO, CL and AMP.     

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.     

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.     

Mitomycin C: a promising agent for the treatment of canine corneal scarring

Objective To evaluate the safety and efficacy of mitomycin C (MMC) in prevention of canine corneal scarring. Methods With an in vitro approach using healthy canine corneas, cultures of primary canine corneal fibroblasts or myofibroblasts were generated. Primary canine corneal fibroblasts were obtained by growing corneal buttons in minimal essential medium supplemented with 10% fetal bovine serum. Canine corneal myofibroblasts were produced by growing cultures in serum‐free medium containing transforming growth factor β1 (1 ng/mL). Trypan blue assay and phase‐contrast microscopy were used to evaluate the toxicity of three doses of MMC (0.002%, 0.02% and 0.04%). Real‐time PCR, immunoblot, and immunocytochemistry techniques were used to determine MMC efficacy to inhibit markers of canine corneal scarring. Results A single 2‐min treatment of 0.02% or less MMC did not alter canine corneal fibroblast or keratocyte phenotype, viability, or growth. The 0.02% dose substantially reduced myofibroblast formation (up to 67%; P < 0.001), as measured by the change in RNA and protein expression of fibrosis biomarkers (α‐smooth muscle actin and F‐actin). Conclusion This in vitro study suggests that a single 2‐min 0.02% MMC treatment to the canine corneal keratocytes is safe and may be useful in decreasing canine corneal fibrous metaplasia. In vivo studies are warranted.     

Normal corneal thickness measurements in pigmented rabbits using spectral‐domain anterior segment optical coherence tomography

Objective  To document the thickness of the central cornea in pigmented rabbits using spectral‐domain anterior segment optical coherence tomography (AS‐OCT). Animals studied  Seventeen pigmented rabbits (6 male, 11 female, both eyes) were involved in this study. Procedures  Thirty‐four eyes from healthy pigmented rabbits underwent a complete ophthalmologic examination, including AS‐OCT. Eight radial scans, 6 mm in length and centered on the cornea, were obtained using the AS‐OCT. Corneal thickness was automatically calculated using pachymetry software. Measurements were displayed as the mean and standard deviation for each of the 17 regions defined by the software. The regions were the center (1 mm radius, area a), the inner ring (2.5 mm radius, area b), the outer ring (3 mm radius, area c), and the eight radial scan lines in eight directions (Superior (1), SN (2), Nasal (3), IN (4), Inferior (5), IT (6), Temporal (7), ST (8)) with an angle of 45° between each consecutive scan line (a, b 1–8, c 1–8). Results  There was no statistically significance difference in corneal thickness between gender, eye, and the eight directions (P = 0.804, P > 0.05, P > 0.05). There was a statistical difference between the thickness in areas a, b, and c (P < 0.05). The corneal thickness increased gradually from the center to the periphery of the 6 mm measured. The center corneal thickness was 387 ± 19.8 μm for OD and 384 ± 20.2 μm for OS. The corneal thicknesses of the thinnest point of the right eyes (OD) and the left eyes (OS) were 376 ± 20.2 and 370 ± 16.8 μm, respectively. There was positive correlation between the thinnest point and area a in both the right eyes (r = 0.892, P < 0.001) and the left eyes (r = 0.832, P < 0.001). Conclusions  This is the first documentation of the rabbit corneal thickness in vivo using the spectral‐domain AS‐OCT. Pigmented rabbit corneas were almost 150 μm thinner than human corneal values. Gender and eye were not associated with any statistical differences in central corneal thickness in this study.     

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.     

Observations on the ultrastructure of equatorial scleral collagen fibrils in sheep eyes

Objective To investigate collagen fibrils of the equatorial sclera in relation to the age‐related changes in eye size in sheep. Animals studied Lambs and outbred ewes. Procedures Sheep eyes (three lamb and three from adult outbred ewes), presumed disease‐free, were processed for transmission electron microscopy (TEM) immediately postmortem. Tissue blocks from the equatorial region were sectioned across fibril bundles orientated along the equator. Micrographs including at least 500 fibrils were projected at 22 000× magnification for measures of fibril diameters (FDs). Results Lamb eyes were smaller than those of adult ewes but equatorial scleral thickness was only marginally less at 0.232 ± 0.013 vs. 0.254 ± 0.012 mm (P value not significant). Scleral tissue was composed of compacted bundles of collagen fibers that tended to be rounder in outer compared to being flatter in inner regions. In typical (normal) appearing regions, FDs were distinctly larger (68–410 nm) in outer sclera compared to inner sclera (63–281 nm). Outer sclera FDs were bimodal averaging 192 ± 58 nm, compared to unimodal distributions at inner locations averaging 156 ± 48 nm (P < 0.001). Some atypical regions, especially at outer‐mid sclera locations, were also noted where the FD distribution was bimodal but also included numerous microfibrils (<50 nm diameter), with similar appearances being found for both lamb and adult ewe eyes. Conclusions The equatorial sclera is a mixture of rounder versus flatter collagen fiber bundles, the former being more likely to be made up of a mixture of both smaller and larger fibrils, as compared to slightly smaller fibrils.     

The benefits of cooling boar semen in long‐term extenders prior to cryopreservation on sperm quality characteristics

This study investigated the effects of long‐term extenders on post‐thaw sperm quality characteristics following different holding times (HT) of boar semen at 17 and 10°C. Sperm‐rich fractions, collected from five boars, were diluted in Androhep^® Plus (AHP), Androstar^® Plus (ASP), Safecell^® Plus and TRIXcell^® Plus (TCP) extenders. The extended semen samples were held for 2 hr at 17°C (HT 1) and additionally for 24 hr at 10°C (HT 2), after they were evaluated and frozen. CASA sperm motility and motion patterns, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI) and normal apical ridge (NAR) acrosome integrity were assessed in the pre‐freeze and frozen‐thawed semen. The Vybrant Apoptosis Assay Kit was used to analyse the proportions of viable and plasma membrane apoptotic‐like changes in spermatozoa. Results indicated that boar variability, extender and HT significantly affected the sperm quality characteristics, particularly after freezing‐thawing. Differences in the pre‐freeze semen were more marked in the sperm motion patterns between the HTs. Pre‐freeze semen in HT 2 showed significantly higher VCL and VAP, whereas no marked effects were observed in the sperm membrane integrity and viability (YO‐PRO‐1^?/PI^?) among the extenders. Post‐thaw sperm TMOT and PMOT were significantly higher in the AHP and ASP extenders of HT 2 group, whereas VSL, VCL and VAP were markedly lower in the TCP extender. Furthermore, spermatozoa from the AHP‐ and ASP‐extended semen of HT 2 group were characterized by higher MMP, PMI and NAR acrosome integrity following freezing‐thawing. In most of the extenders, the incidence of frozen‐thawed spermatozoa with apoptotic‐like changes was greater in HT 1. The findings of this study indicate that holding of boar semen at 10°C for 24 hr in long‐term preservation extenders modulates post‐thaw sperm quality characteristics in an extender‐dependent manner. These results will further contribute to the improvement in the cryopreservation technology of boar semen.