Corneal Cultures Research Articles

Antimicrobial resistance trends in bacterial keratitis over 5 years in Sydney, Australia.

Antimicrobial resistance (AMR) patterns in bacterial keratitis may fluctuate in a geographic location over time. To investigate any change in AMR patterns in Sydney, Australia. Retrospective case series. All patients with microbial keratitis who underwent a corneal scrape and culture from 2012 to 2016 at the Sydney Eye Hospital. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry identified organisms. The Calibrated Dichotomous Susceptibility method determined antibiotic susceptibilities. Isolated organisms and antibiotic susceptibilities. There were 1084 corneal scrapes from 957 patients. The mean age was 54 years (range 18-100) and 52% were male. Cultures were positive in 711 of 1084 scrapes (66%), with 884 organisms identified. Of the bacteria isolated, 685 of 884 (78%) were Gram-positive and 199 of 884 (22%) were Gram-negative. Overall, the most common bacteria were coagulase-negative Staphylococci (CoNS) (405/884, 46%). Methicillin-resistance was detected in 7% of Staphylococcus aureus isolates (7/103). Methicillin-resistance in CoNS (ie, also cefalotin resistance) was reported in 19% of isolates and ciprofloxacin 8%. For methicillin-sensitive S aureus (MSSA), 5% of isolates were resistant to ciprofloxacin. For Corynebacterium spp., 34% of isolates were resistant to chloramphenicol and 9% to ciprofloxacin. The most common Gram-negative bacteria was Pseudomonas aeruginosa (109/199, 55%). One case was resistant to ciprofloxacin. Coagulase-negative staphylococcal species were the most frequently suspected of causing bacterial keratitis. Increased resistance to cefalotin was identified for CoNS and to ciprofloxacin for Corynebacterium spp., MSSA and P aeruginosa compared to a previous study in Sydney in 2002 to 2003.

Vibrio cholerae non-O1 - the first reported case of keratitis in a healthy patient

BackgroundVibrio cholerae non-O1 is a virulent pathogen that causes significant morbidity and mortality in humans. Herein, we report a case of corneal ulcer caused by this pathogen.Case presentationA 59-year-old fisherman with no systemic history was struck in the right eye by a marine shrimp and developed keratitis. Corneal scrapping culture revealed the presence of the V. cholerae non-O1, and its identification was confirmed by Analytical Profile Index 20E system and polymerase chain reaction. He was successfully treated with topical levofloxacin (0.3%) and fortified amikacin (12.5 mg/mL) for 2 weeks. The visual acuity recovered to 20/25 after treatment without complications.ConclusionsThis is the first case report of keratitis caused by V. cholerae non-O1 strain. Ocular injury by marine creatures and contaminated seawater can contribute to severe corneal ulcer. Early diagnosis can be achieved by meticulous history taking and a comprehensive laboratory workup. Simultaneously, an effective antibiotic therapy can lead to a positive outcome.

Prevalence of equine ulcerative keratomycosis in Colorado and association of environmental factors: A retrospective and descriptive study (2002–2017)

SummaryEquine ulcerative keratomycosis is relatively commonly presented to teaching hospitals in North America, with a prevalence from 24 to 86%. Horses in Colorado may have lower risk due to the dry mountain climate, but data are lacking. This study aimed to determine the prevalence of ulcerative keratomycosis amongst horses with ulcerative keratitis presented to Colorado State University Veterinary Teaching Hospital (CSU‐VTH) ophthalmology service and to evaluate environmental factors (season, temperature, humidity, wind speed and elevation) associated with ulcerative keratomycosis in Colorado. A database search identified horses with ulcerative keratitis presented to the ophthalmology service at CSU‐VTH from January 2002 to August 2017. Sixty‐one horses met the inclusion criteria of a corneal cytology and/or culture or histopathology at the time of diagnosis; cases lacking that were excluded. Environmental factors at the boarding sites, including season, temperature, humidity, wind speed and elevation and clinical outcomes for fungal cases, were recorded. Prevalence of fungal infection amongst equine ulcerative keratitis was 16.4% (10/61), suggesting that CSU‐VTH has considerably fewer ulcerative keratomycosis cases than other veterinary teaching hospitals in North America. Spring (50%, 5/10) and fall (40%, 4/10) had the highest prevalence of fungal ulcerative keratitis in Colorado. Only one case was reported in summer (10%, 1/10); no horses were positive (0%, 0/10) in winter. Only wind speed seemed to influence the development of ulcerative keratomycosis with higher wind speeds associated with greater rates of fungal involvement (P = 0.047). Other environmental factors did not show a detectable association (all P‐values >0.05). Outcomes were variable. It was concluded that horses from the area of CSU‐VTH appear to be at lower risk for ulcerative keratomycosis than from the areas around other North American veterinary teaching hospitals that have reported data. Most horses with keratomycosis in this area present in the spring and fall.

Characterizing the impact of 2D and 3D culture conditions on the therapeutic effects of human mesenchymal stem cell secretome on corneal wound healing in vitro and ex vivo

The therapeutic effects of secreted factors (secretome) produced by bone marrow-derived human mesenchymal stem cells (MSCs) were evaluated as a function of their growth in 2D culture conditions and on 3D electrospun fiber scaffolds.Electrospun fiber scaffolds composed of polycaprolactone and gelatin were fabricated to provide a 3D microenvironment for MSCs, and their mechanical properties were optimized to be similar to corneal tissue. The secretome produced by the MSCs cultured on 3D fiber matrices versus 2D culture dishes were analyzed using a Luminex immunoassay, and the secretome of MSCs cultured on the 3D versus 2D substrates showed substantial compositional differences. Concentrations of factors such as HGF and ICAM-1 were increased over 5 times in 3D cultures compared to 2D cultures. In vitro proliferation and scratch-based wound healing assays were performed to compare the effects of the secretome on corneal fibroblast cells (CFCs) when delivered synchronously from co-cultured MSCs through a trans-well co-culture system versus asynchronously after harvesting the factors separately and adding them to the media. Cell viability of CFCs was sustained for 6 days when co-cultured with MSCs seeded on the fibers but decreased with time under other conditions. Scratch assays showed 95% closure at 48 h when CFCs were co-cultured with MSCs seeded on fibers, while the control group only exhibited 50% closure at 48 h. Electrospun fibers seeded with MSCs were then applied to a rabbit corneal organ culture system, and MSCs seeded on fibers promoted faster epithelialization and less scarring. Corneas were fixed and stained for alpha smooth muscle actin (α-SMA), and then analyzed by confocal microscopy. Immunostaining showed that expression of α-SMA was lower in corneas treated with MSCs seeded on fibers, suggesting suppression of myofibroblastic transformation.MSCs cultured on electrospun fibers facilitate wound healing in CFCs and on explanted corneas through differential secretome profiles compared to MSCs cultured on 2D substrates. Future work is merited to further understand the nature and basis of these differences and their effects in animal models. Statement of significancePrevious studies have shown that the secretome of bone marrow-derived mesenchymal stem cells (MSC) is promotes corneal wound healing by facilitating improved wound closure rates and reduction of scarring and neovascularization. The present research is significant because it provides evidence for the modulation of the secretome as a function of the MSC culture environment. This leads to differential expression of therapeutic factors secreted, which can impact corneal epithelial and stromal healing after severe injury.In addition, this article shows that co-continuous delivery of the MSC secretome improves cell migration and proliferation over aliquoted delivery, and that MSCs grown on three-dimensional electrospun fiber constructs may provide a favorable microenvironment for cultured MSCs and as a carrier to deliver their secreted factors to the ocular surface.

Diagnostic value of fungal fluorescence staining on corneal scrapings for fungal keratitis

Objective: To analyze the sensitivity and specificity of fungal fluorescent staining in the diagnosis of fungal keratitis, and to compare it with conventional fungal culture, in vivo confocal microscopy (IVCM) and Giemsa staining. To explore its value of clinical application. Methods: Prospective case-control study. A total of 105 consecutive patients (105 eyes) diagnosed with infectious keratitis at Beijing Tongren Hospital from August 2017 to April 2018 were included. Patients with infectious keratitis were divided into fungal keratitis (FK) group and non-fungal keratitis (NFK) group by slit lamp microscopy, corneal in vivo confocal microscopy (IVCM) examination, and the results of Giemsa staining, fluorescent staining and pathogenic culture of corneal scraping from ulcer. The sensitivity and specificity of the above-mentioned examination methods for the diagnosis of fungal keratitis were analyzed. The receiver operating characteristic curve (ROC curve) and Area Under Curve (AUC) values were calculated to determine the diagnostic value of fungal fluorescent staining for fungal keratitis. Results: Among the 105 patients with infectious keratitis, 66 were fungal keratitis, 39 were non-fungal keratitis (29 cases of bacterial keratitis and 10 cases of acanthamoeba keratitis). Isolation from fungal keratitis were mainly Fusarium spp. (43.5%), followed by Alternaria spp. (21.7%) and Aspergillus spp. (19.6%). After fluorescent staining of the ulcer smear, the background of tissue demonstrated homogeneous black or weak blue fluorescence. The cell wall of fungi showed bright blue-violet to blue fluorescence, and the morphology, structure and hyphal density were easily recognized. The sensitivity of different methods for the diagnosis of corneal fungal infection were smear fluorescence staining (97.0%), IVCM (87.9%) , Giemsa staining (86.7%), and fungal culture (69.7%); the specificity of fungal culture was the highest (100%), followed by IVCM and Giemsa staining (94.9%), and fluorescent staining (87.2%). The ascending order of AUC values was: fungal culture (0.848) <Giemsa staining (0.906) <IVCM (0.914) <fluorescence staining (0.921). Conclusion: Fungal fluorescent staining is a rapid and sensitive screening method under microscope with high sensitivity and specificity for the diagnosis of fungal keratitis. It is especially suitable for the diagnosis of patients with low load of hypha or after antifungal therapy. (Chin J Ophthalmol, 2019, 55:601-608).

Feline bacterial keratitis: Clinical features, bacterial isolates, and in vitro antimicrobial susceptibility patterns.

To describe clinical characteristics, bacterial isolates, and in vitro antimicrobial susceptibility patterns for cats with bacterial keratitis. Eighty one cats with bacterial keratitis. Medical records of cats with a clinical diagnosis of bacterial keratitis, confirmed by corneal culture, were reviewed from June 2004 to July 2017. Animal signalment, bacterial isolates, aerobic bacteria in vitro antimicrobial susceptibility test results, and pertinent clinical features were recorded. Percentages of susceptible aerobic bacterial isolates were statistically compared among selected antimicrobials. There were 102 aerobic bacterial isolates recovered from corneal samples of the 81 cats presented with bacterial keratitis. The most frequent bacteria isolated were Staphylococcus species, which constituted 55% (56/102) of total isolates. All aerobic bacterial isolates grouped together had the highest percentage of susceptibility to ofloxacin (100%), ciprofloxacin (94%), chloramphenicol (93%), doxycycline (92%), ticarcillin (90%), gentamicin (89%), moxifloxacin (89%), tobramycin (86%), neomycin (85%), amikacin (84%), and cefazolin (84%). The same isolates had the lowest percentage of susceptibility to polymyxin B (2%), bacitracin (15%), and clindamycin (31%). When analyzed separately, the isolated Staphylococcus species had the highest percentage of susceptibility to ofloxacin (100%), tobramycin (93%), and neomycin (85%) and the lowest percentage of susceptibility to polymyxin B (3%) and bacitracin (13%). Members of the Staphylococcus genus were the most frequent bacteria isolated from cases of feline bacterial keratitis. On the basis of in vitro susceptibility testing and mechanism of action, ofloxacin, ciprofloxacin, ticarcillin, gentamicin, or moxifloxacin are recommended for initial antimicrobial therapy of suspected bacterial keratitis in cats.

Culturing Discarded Peripheral Human Corneal Endothelial Cells From the Tissues Deemed for Preloaded DMEK Transplants.

To investigate if the peripheral corneal endothelium that is discarded after the preparation of preloaded Descemet membrane endothelial keratoplasty (DMEK) grafts for transplantation could be successfully used for corneal endothelial cell culture. Complete Descemet membrane-endothelial complex (11.00 mm) was peeled from research-grade tissues (n = 15). The periphery (2.75 mm) of clinical-grade tissues (n = 15) deemed for preloaded DMEK transplants was gently peeled and preserved for 48 hours in tissue culture media, followed by centrifugation at 1000 rpm for 5 minutes. After enzymatic digestion, the cells from each group were plated in 2 different wells of an 8-well chamber slide. Media were refreshed and the confluence rate was monitored every alternate day. Live/dead staining and the expression of ZO-1, Tag1A3, Tag2A12, and Ki-67 markers were used to assess the viability, morphology, tight-junctions, cell area, and number of proliferative cells. The Wilcoxon and Student's t test were applied, where P < 0.05 was deemed statistically significant. Average endothelial cell density at confluence was 2,352 cells/mm2 from complete endothelium and 2,510 cells/mm from peripheral endothelium (P = 0.0351). The confluence rate (%), hexagonality (%), polymorphism (%), cell area (μm), and Ki-67 positivity (%) did not differ between both groups (P > 0.05). All the antibodies were expressed in both groups at confluence. The discarded peripheral endothelial cells obtained after preparing a preloaded DMEK graft for clinical application has a huge reservoir of healthy endothelial cells having proliferative potential. Using these discarded tissue pieces from donor tissues will significantly increase the primary source of healthy donor endothelial cells for regenerative treatments, which are otherwise difficult to obtain.

Compressed collagen intermixed with cornea-derived decellularized extracellular matrix providing mechanical and biochemical niches for corneal stroma analogue

Compressed collagen is a promising scaffold for corneal stroma analogue due to its facile incorporation of keratocytes while mimicking the mechanical niche of a native cornea with dense collagen fibrillar structures. However, it does not offer the sufficient biochemical niche crucial for in vivo-like quiescent keratocyte phenotype. In this study, we engineered a scaffold for a corneal stroma analogue that mimics both the mechanical and biochemical niches of the corneal stroma by introducing cornea-derived decellularized extracellular matrix (Co-dECM) to the collagen compression process. The compressed collagen intermixed with Co-dECM (COLEM; Co-dECM content, <50 wt%) maintained a uniform structure and showed an elastic modulus and tensile strength on the order of 100 kPa, which is comparable with that of conventional compressed collagen. The COLEM with the 50 wt% Co-dECM content was found to possess 2-fold higher amount of the glycosaminoglycans as compared to the compressed collagen. The biochemical components of Co-dECM in the COLEM were verified to significantly promote the expression of quiescent keratocyte-specific genes, i.e., KERA and ALDH3A1, while improving the optical transmittance of the COLEM by reducing the diameter of collagen fibrils. The ability of the COLEM to construct multicellular in vitro corneal tissue was demonstrated by an additional corneal epithelial cell culture. The results support the hypothesis that COLEM has strong potential use in the development of corneal equivalent for in vitro models and tissue transplantation.

Veterinary ocular microbiome: Lessons learned beyond the culture.

Ocular pathogens cause many painful and vision-threatening diseases such as infectious keratitis, uveitis, and endophthalmitis. While virulent pathogens and pathobionts play important roles in disease pathogenesis, the scientific community has long assumed disruption of the ocular surface occurs prior to microbial colonization and subsequent infection. While nonpathogenic bacteria are often detected in corneal and conjunctival cultures from healthy eyes, cultures also frequently fail to yield growth of common ocular pathogens or nonpathogenic bacteria. This prompts the following question: Is the ocular surface populated by a stable microbial population that cannot be detected using standard culture techniques? The study of the microbiome has recently become a widespread focus in physician and veterinary medicine. Research suggests a pivotal symbiotic relationship with these microbes to maintain healthy host tissues, and when altered is associated with various disease states ("dysbiosis"). The microbiota that lives within and on mammalian bodies have long been known to influence health and susceptibility to infection. However, limitations of traditional culture methods have resulted in an incomplete understanding of what many now call the "forgotten organ," that is, the microbiome. With the introduction of high-throughput sequencing, physician ophthalmology has recognized an ocular surface with much more diverse microbial communities than suspected based on traditional culture. This article reviews the salient features of the ocular surface microbiome and highlights important future applications following the advent of molecular techniques for microbial identification, including characterizing ocular surface microbiomes in our veterinary species and their potential role in management of infectious and inflammatory ocular diseases.

Trends in contact lens microbial keratitis 1999 to 2015: a retrospective clinical review.

Contact lens microbial keratitis (CLMK) is the most common cause of microbial keratitis in our community. Define the trend in rate of CLMK and define patient demographics/culture results that may have a predictive value in patients with CLMK. Retrospective review of clinical records of patients with MK. All patients with positive corneal scraping between 1999 and 2015 at the Princess Alexandra Hospital, Brisbane, Queensland identified through local microbiology database. Trend in CLMK tested with chi-squared test of peak 3 years vs other years and Poisson regression of interrupted time series. Patient characteristics predictive of CLMK were defined by creating a polynomial regression model by stepwise variable selection. Yearly rate of CLMK. Records of 895 episodes of MK were included. The most common: risk factor was contact lens wear (324, 36.2%), isolated organism was Pseudomonas aeruginosa (P. aeruginosa 181, 55.9%) and treatment was monotherapy with a fluoroquinolone 172, 53%). CLMK was most common between 2009 and 2011 (49.5% vs other years 32%, P < 0.001). Poisson regression of the interrupted time series showed there was a significant decrease in the rate over time after 2010 (P < 0.001). Independent factors predictive of CLMK in multivariate regression were young age (15-49 years) and corneal culture positive for P. aeruginosa CONCLUSIONS AND RELEVANCE: The rate of CLMK in our community ranged between 32% and 50% and the rate of disease appears to have peaked during 2009 to 2011 and subsequently declined.

A two-centre validation study of sterility test of corneal storage media with elimination of interfering antimicrobials in compliance with the European Pharmacopoeia.

The purpose of this study was to validate the sterility test of corneal culture and deswelling/transport media using a device for removal of antimicrobials before incubation in BACTEC™ automated system in two Italian Eye Banks. Corneal culture medium, TISSUE-C, and deswelling/transport medium, CARRY-C, were inoculated with 10-100cfu of six European Pharmacopoeia (EP) reference strains and either treated with medical device RESEP for removal of antimicrobials (RESEP+ group) or left untreated (RESEP- group) before injection into the BACTEC Plus bottles. The same steps were repeated in the absence of inocula with tryptone soy broth samples as negative controls, and the inocula were also directly injected in the BACTEC™ bottles as growth controls. All the samples were incubated in BACTEC™ automated system for7days, and the time to detection of microbial growth was recorded automatically. At both the Eye Banks, in the RESEP+ groups, microbial growth was detected in 100% of samples. In the RESEP- group, the method sensitivity ranged from 66.7 ± 21.1 to 88.9 ± 6.4% for TISSUE-C samples while for CARRY-C samples the method sensitivity ranged from 94.5 ± 5.1 to 100%. The method specificity corresponded to 100% for all the groups at both Eye Banks. This two-centre validation study showed that the use of RESEP increased the sensitivity of sterility test using BACTEC™ automated system up to 100% and, consequently, allowed validation of the method for sterility testing of corneal storage and deswelling/transport media according to the EP requirements. The test could not be validated without the use of RESEP.

Factors Affecting Corneal Organ Culture Contamination: A 6-year Study at the New South Wales Tissue Bank.

To report the rate of microbial contamination and analyze possible risk factors for contamination of banked corneas stored using the organ culture method. Data from the New South Wales Tissue Banks incorporating the Lions NSW Eye Bank, between September 1, 2011, and November 30, 2017, were reviewed retrospectively. All corneas collected during this period and stored in organ culture storage media were tested for microbial contamination. The influence of potential factors on the rate of contamination was analyzed using the χ test and logistic regression using generalized estimating equations. A total of 4410 corneas were included in this study, of which 110 were medium culture positive, representing a microbial contamination rate of 2.5%. The main contaminants were Candida species followed by Staphylococcus species. Corneal tissue collected in summer and autumn had a significantly higher contamination rate (P = 0.006). All other factors studied were not shown to have a statistically significant association with contamination after accounting for within-pair correlation and confounders. A relatively low contamination rate of 2.5% observed in our study reflects the stringent laboratory protocols, strict donor selection criteria, and high level of experience among staff at the Lions NSW Eye Bank. Our study demonstrated that the season of collection had a strong association with the rate of organ culture contamination. Because Candida species contributed the largest percentage of contamination, specific measures to reduce and eliminate fungal proliferation should be considered by eye banks particularly in warm seasons.

Medication Burden for Patients With Bacterial Keratitis.

To understand medication use and patient burden for treatment of bacterial keratitis (BK). A retrospective study was conducted examining medical records of adult patients with BK in an academic cornea practice. Data collected included medications used in the treatment of BK, dosing of medications, and the number and total duration of clinical encounters. Costs of medications were estimated using the average wholesale pharmacy price. Linear regression analysis was used to investigate associations of medication use with patient demographics and corneal culture results and reported with beta estimates (β) and 95% confidence intervals (95% CIs). Forty-eight patients with BK (56% female) were studied. Patients were treated for a median of 54 days with 10 visits, 5 unique medications, 587 drops, and 7 prescriptions. The estimated median medication cost was $933 (interquartile range: $457-$1422) US dollars. Positive bacterial growth was significantly associated with more visits (β: 6.16, 95% CI: 1.75-10.6, P = 0.007), more days of treatment (β: 86.8, 95% CI: 10.8-163, P = 0.026), more prescribed medications (β: 2.86, 95% CI: 1.04-4.67, P = 0.003), and more doses of medications (β: 796, 95% CI: 818-1412, P = 0.012) compared with patients who did not undergo corneal scraping. Patients were prescribed 132 more drops of medication for every 10 years of older age (β: 132, 95% CI: 18.2-246, P = 0.024). Sex and income were not associated with medication burden or treatment length. Older patients and those with positive cultures incur the most medication burden in treatment of BK. Providers should be aware of medication usage and cost burden as it may affect compliance with treatment.

Deregulation of Acanthamoeba castellanii steroidogenesis is amoebicidal and protects cultured corneal cells from Ac attack

Human infections by amoeba represent rare diseases, yet they are an emerging health problem that can develop into rapid death, as in cases involving the spinal cord or amoeba that penetrate the brain. Our recent papers highlighted in J. Lipid Res, 2017 and ASBMB Today magazine, 2018 focus on development of new sterol methyltransferase (SMT) inhibitors to prevent steroidogenesis in the parasitic amoeba Acanthamoeba castellanii (Ac). Now we show, Ac cultured on unexplored sterol C24‐methyltransferase (SMT) inhibitors 25‐azalanosterol and 25‐thialanosterol iodine salt are highly potent anti‐amoeba drugs exhibiting IC50 25 nM and 15 nM, respectively and a minimum amoebicidal concentration of 1 μM. Both analogs inhibited the cloned 24‐AcSMT and 28‐AcSMT yielding Ki values of 13 nM/37nM and 9nM/16nM, respectively and blocked Ac steroidogenesis at the target enzyme. Against human epithelial kidney cell growth these drugs had no effect to 40 μM or effected cholesterol biosynthesis (selective index greater than 103); in mice at 50 mg/kg, 3H‐analog could rapidly cross the blood brain barrier with minimal effect on liver cholesterol biosynthesis. Against human corneal epithelial cultures infected with Ac, the drugs protected the eye at doses of 500 nM. These new amoeba sterol biosynthesis inhibitors are currently under investigation for their therapeutic potential.Support or Funding InformationThis research was funded through a grant from the National Institutes of Health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Bacterial Investigation and Antibiogram in Corneal Ulcers in Dogs

Corneal ulcers in dogs usually have a traumatic origin. They cause a break in the continuity of underlying corneal stroma and become rapidly contaminated with bacteria. Twenty eight clinical samples were obtained from corneal ulcer affected dogs for bacterial isolation and anti-microbial susceptibility. Bacterial growth was observed in 100% of the samples (n=28) and Staphylococcus spp. was isolated and characterized by culture, gram staining and various biochemical tests. Antibiogram pattern revealed that gatifloxacin and moxifloxacin antibiotics were found to be highly effective in the treatment of corneal ulcers in dogs. Corneal culture and sensitivity testing provided useful information for the diagnosis, determination of appropriate treatment and antimicrobial therapy for corneal diseases in dogs.

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies.

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

Ophthalmic findings in a captive population of Panamanian golden frogs: Atelopus zeteki.

The aim of this study was to describe normal ocular morphology and establish parameters for select diagnostics in a population of Panamanian Golden Frogs (PGF) Atelopus zeteki maintained at the Detroit Zoo. A total of 22 toads free of ocular disease were included in this study. Ophthalmic diagnostic tests included as follows: endodontic absorbent paper point tear test (EAPPTT), assessment of intraocular pressures (IOP), Rose Bengal stain, palpebral fissure width, blink rate and corneal cultures. Histology was performed on stored postmortem samples. Panamanian Golden Frogs have a distinct brow ridge and mobile third eyelid. Biomicroscopy revealed avascular, convex corneas with a shallow anterior chamber. Iris color ranged from yellow to green with dark pigmentation of the peripheral iris. Pupil shape was round. Posterior segment and fundus could not be visualized. Total number of blinks observed over a 10-minute period was one. Mean EAPPTT was 2±1mm and 3±1mm in males and females, respectively (P). Mean IOP using rebound tonometry in horizontal and vertical restraint positions were 21.6±2.28mmHg and 17.45±3.66mmHg, 21.85±2.7mmHg and 17.5±3.93mmHg in males and females, respectively. Mean palpebral fissure width was 5.02±0.46mm and 6.12±0.39mm in males and females, respectively (P). Enterococcus faecalis was isolated from one clinically normal toad. Tear production and blink rate were low and not significantly different between males and females. Intraocular pressure was significantly greater in male toads (P value <0.01). Females had a significantly larger palpebral fissure width when compared to males (P value <0.01) 5819. The ophthalmic parameters established in this study can aid in the evaluation and diagnosis of eye diseases in PGF.

Monitoring the microbial contamination of donor cornea during all preservation phases: A prospective study in the Eye Bank of Rome

In most European eye banks, human donor corneas are microbiologically tested after storage in organ culture conditions, and the tissues that are free of contamination are distributed for transplantation. In this prospective study, 100 donor corneas were tested for microbial contamination after cold storage, corneal culture and corneal deswelling at the Eye Bank of Rome. Samples of cold storage medium (EUSOL-C), corneal culture medium (TISSUE-C) and deswelling medium (CARRY-C) were tested after three, seven and one days of corneal storage, respectively. The CARRY-C medium, used to transport the cornea to the operation theatre, was retested 1day after transplantation. The TISSUE-C and CARRY-C media were also tested after removing antimicrobial and antifungal agents using a dedicated device. We found 67% of the EUSOL-C samples were contaminated mainly by Staphylococcus spp, 14% of TISSUE-C media were contaminated by bacteria and fungi and 3% of CARRY-C media by Staphylococcus spp The analysis performed after removing the antimicrobial and antifungal agents showed growth in three additional TISSUE-C samples (Sviridans, Shaemolyticus and Efaecalis) and one CARRY-C (Scerevisiae and Pacnes). Tissue contamination was unexpectedly high on arrival to the eye bank, indicating the need to review and update decontamination procedures during tissue recovery, and renew training for the recovery teams. Storing donor corneas in organ culture conditions significantly reduced the microorganism burden. Using devices to remove antimicrobial and antifungal agents from samples before testing can increase the sensitivity of the standard microbiological method, and thus help further reduce the risk of microbial transmission.

Characterizing the Impact of 2D and 3D Culture Conditions on the Therapeutic Effects of Mesenchymal Stem Cell Secretome on Corneal Wound Healing &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;ex vivo&lt;/i&gt;

The therapeutic effects of secreted factors (secretome) produced by bone marrow-derived human mesenchymal stem cells (MSCs) were evaluated as a function of their growth in 2D culture conditions and on 3D electrospun fiber scaffolds.Electrospun fiber scaffolds composed of polycaprolactone and gelatin were fabricated to provide a 3D microenvironment for MSCs, and their mechanical properties were optimized to be similar to corneal tissue. The secretome produced by the MSCs cultured on 3D fiber matrices versus 2D culture dishes were analyzed by Luminex immunoassay, and the secretome of MSCs cultured on the 3D versus 2D substrates showed substantial compositional differences. Concentrations of factors such as HGF and ICAM-1 were increased over 5 times in 3D cultures compared to 2D cultures. In vitro proliferation and scratch-based wound healing assays were performed to compare the effects of the secretome on corneal fibroblast cells (CFCs) when delivered synchronously from co-cultured MSCs through a trans-well co-culture system versus asynchronously after harvesting the factors separately and adding them to the media. Cell viability of CFCs was sustained for 6 days when co-cultured with MSCs seeded on the fibers but decreased with time under other conditions. Scratch assays showed 95% closure at 48 hr when CFCs were co-cultured with MSCs seeded on fibers, while the control group only exhibited 50% closure at 48 hr. Electrospun fibers seeded with MSCs were then applied to a wounded rabbit corneal organ culture system, and MSCs seeded on fibers promoted faster epithelialization and less scarring. Corneas were fixed and stained for alpha smooth muscle actin (α-SMA) and keratan sulfate (KS), and then analyzed by confocal microscopy. Immunostaining showed that expression of alpha-SMA was lower in corneas treated with MSCs seeded on fibers, suggesting suppression of myofibroblastic transformation. KS expression was higher in corneas treated with MSCs seeded on fibers, suggesting that keratocytic phenotype was preserved.MSCs cultured on electrospun fibers facilitate wound healing in CFCs and on explanted corneas through differential secretome profiles compared to MSCs cultured on 2D substrates. Future work is merited to further understand the nature and basis of these differences and their effects in animal models.

Examination of Kynurenine Toxicity on Corneal and Conjunctival Epithelium: In vitro and in vivo Studies

Kynurenine (KYN) is a metabolite of tryptophan, proposed for the treatment of corneal diseases. Our goal was to evaluate the effects of KYN on normal human corneal and conjunctival epithelial cells in vitro and the re-epithelization of corneal erosion in rabbits. In our study, we used corneal (10.014 pRSV-T) and conjunctival (HC0597) epithelium cell cultures. KYN was applied at a concentration range of 1–100 µM for 24 and 48 h. We examined the effects on cellular metabolism, viability, interleukin-1β (IL-1β), IL-6, IL-10 secretion, cytoskeleton organization and transwell migration ability. Following a bilateral corneal de-epithelialization, the rabbits received drops containing 1% KYN and a saline solution to the contralateral control eye, 5 times daily. Digital images were analyzed using the EPCO 2000 software. The metabolic activity of cells was slightly decreased by KYN in the corneal but not in the conjunctival epithelium. The viability of both epithelia was improved by KYN; it caused alterations in the secretion of IL-6 and IL-10 but not IL-1β. It had no impact on both epithelia morphology and the organization of the cellular cytoskeleton. KYN stimulated the formation of pseudopodia projections in both epithelia in vitro, which may be important in terms of wound healing. However, there were no differences in the re-epithelization rate in vivo. At the tested concentrations, KYN was not toxic for the corneal and the conjunctival epithelium in vitro and did not affect corneal re-epithelization in rabbits in vivo. Our results suggest that KYN may be taken into consideration for the treatment of ocular disorders.