An untargeted metabolomics approach to study changes of the medium during human cornea culture.

INTRODUCTION: Two main approaches (organ culture and hypothermia) for the preservation and storage of human donor corneas are globally adopted for corneal preservation before the transplant. Hypothermia is a hypothermic storage which slows down cellular metabolism while organ culture, a corneal culture performed at 28-37 °C, maintains an active corneal metabolism. Researchers, till now, have just studied the impact of organ culture on human cornea after manipulating and disrupting tissues. OBJECTIVES: The aim of the current work was to optimize an analytical procedure which can be useful for discovering biomarkers capable of predicting tissue health status. For the first time, this research proposed a preliminary metabolomics study on medium for organ culture without manipulating and disrupting the valuable human tissues which could be still used for transplantation. METHODS: In particular, the present research proposed a method for investigating changes in the medium, over a storage period of 20 days, in presence and absence of a human donor cornea. An untargeted metabolomics approach using UHPLC-QTOF was developed to deeply investigate the differences on metabolites and metabolic pathways and the influence of the presence of the cornea inside the medium. RESULTS: Differences in the expression of some compounds emerged from this preliminary metabolomics approach, in particular in medium maintained for 10 and 20 days in presence but also in the absence of cornea. A total of 173 metabolites have been annotated and 36 pathways were enriched by pathway analysis. CONCLUSION: The results revealed a valuable untargeted metabolomics approach which can be applied in organ culture metabolomics.

Ex vivo evaluation of retinal cytotoxicity after the use of multiple medical devices in pars plana vitrectomy in porcine eyes.

This study aimed to evaluate viability of retinal cells after the use of multiple intraoperative devices, namely a vitreal dye (triamcinolone acetonide,TA), a ERM/ILM dye (solution of trypan blue 0.15% and brilliant blue 0.025%), and two intraocular tamponades, namely perfluoro-n-octane, (PFO) and silicone oil (SO 1000 cSt), with minimal and maximal removal of their residues, during a simulated pars plana vitrectomy (PPV) in porcine eyes ex-vivo. The in vitro cytotoxicity of each of these compounds was verified on ARPE-19 cells by direct tests according to the ISO 10993-5 (2009). Pars plana vitrectomy was performed on 25 enucleated porcine eyes divided in five groups according to the following conditions: Group A) No surgery control: eye bulbs were kept at room temperature for 40 min; Group B) Sham surgery: PPV with the sole use of BSS for 40 min; Group C) Cytotoxic control: PPV with BSS infusion (20 min) followed by intravitreal injection of 1H-PFO (contact time: 20 min); Group D) Surgery with residues: PPV with BSS infusion and sequential intravitreal injection of TA, ERM/ILM dye, PFO and SO, with minimal removal of each compound after a specified contact-time (overall duration: 40 min); Group E) Surgery with minimal residues: PPV performed as in group D, but with maximal removal of each compound (overall duration: 40 min). All the experimental procedures were performed at room temperature. Immediately after surgery, the retina was extracted from each eye bulb and samples of 3-mm diameter were prepared. Retinal viability was determined for each sample by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay. A cell viability <70% was considered the cytotoxicity threshold. Kruskal-Wallis test was used to evaluate the differences in retinal viability between groups. No cytotoxicity was detected in retinal samples in groups A, B and E. Samples from eye bulbs that had undergone surgery with minimal removal of residues (group D) and cytotoxic controls (group C) showed high retinal cytotoxicity. The tested conditions indicated that the combined use of TA, ERM/ILM dye, PFO and SO during PPV does not affect retinal cells viability if all the devices are properly removed, whereas the cytotoxicity detected in group D may suggest that the presence and accumulation of the residues of the compounds used intraoperatively could negatively impact retinal viability due to a cumulative and/or synergistic cytotoxic effect between them, supporting the crucial role of an optimal removal of the intraoperative medical devices to ensure a safe vitrectomy to the patient.

Safety of silicone oils as intraocular medical device: An in vitro cytotoxicity study.

This study aimed to assess the cytotoxic effect of low molecular weight components (LMWC) and conventional silicone oils (SOs) 1000 cSt with different degree of purification (raw, intermediate, and purified) using in vitro cytotoxicity tests. Direct contact cytotoxicity tests were performed in BALB 3T3 and human retinal pigment epithelial cells (ARPE-19) using quantitative and qualitative evaluation according to the ISO 10993-5 (2009) standards. Conventional SOs 1000 cSt in form of raw, intermediate (intermediate product obtained during distillation process), and purified SO (final product after distillation) and a concentrate of LMWC (including siloxane chains with molecular weight up to 1557 g/mol) were directly applied to 100% of cell layer area for 24 h. Cell viability was quantified using 3-(4,5-dimethylthiazole-2-yl)-2,5-28 diphenyltetrazolium bromide (MTT) and neutral red uptake assays in ARPE-19 and BALB3T3, respectively. All tested samples, including the concentrate of LMWC, resulted to be not cytotoxic according to ISO 10993-5 in both qualitative and quantitative evaluations. However, the cellular viability was significantly higher in the intermediate and purified SO compared with the raw SO in ARPE-19 cells. No reduction in cell viability was detected by LMWC. The absence of cytotoxicity was observed for all tested samples in both BALB3T3 and ARPE-19 after 24 h of application. A direct cytotoxic effect is not likely to be involved in the potential complications related to SO and LMWC. Long-term potential adverse effects of SO could be related to the raw material and to different concentrations of LMWC.

A new approach to extend the storage of donor corneas after 28 days of corneal culture in an Italian eye bank.

This study aimed to evaluate the possibility to extend the storage of unused organ-cultured donor corneas. After 28 days of corneal culture in TISSUE-C (AL.CHI.MI.A. S.R.L., Italy) and 5-day storage in transport/deswelling medium CARRY-C (AL.CHI.MI.A. S.R.L., Italy), 25 corneas that were deemed suitable for transplantation were transferred in fresh TISSUE-C at 31 °C for additional 7 days and then in fresh CARRY-C at room temperature for 24 h. Tissues were assessed for endothelial cell density (ECD), endothelial mortality and morphology after the standard and the extended corneal storage. In addition, the effect of donor age < 85 years and ≥ 85 years on corneal characteristics was assessed. After the extended storage, 6 out of 25 tested corneas (24%) showed ECD values below the acceptance limit (< 2000 cells/mm2). 19 corneas (76%) were still suitable for transplantation and showed a 5.9% loss in ECD, which was not statistically significant (P = 0.0949) compared to standard storage period. The two donor age groups did not show statistically significant differences in any tested parameter, although a trend for lower ECD and higher mortality in Descemet's folds after standard storage was observed in the ≥ 85 age donor group. Thus, the attempt of the current study to provide new sight-restorative options for unused tissues and increasing the availability of corneas in case of shortage gave encouraging results. Although a higher vulnerability of corneas from very old donors could not be statistically demonstrated in the present study, higher sample size could be required for prolonging the shelf life of these tissues.

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

BACKGROUND: 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. METHODS: 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 1 day after transplantation. The TISSUE-C and CARRY-C media were also tested after removing antimicrobial and antifungal agents using a dedicated device. RESULTS: 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 (S viridans, S haemolyticus and E faecalis) and one CARRY-C (S cerevisiae and P acnes). CONCLUSION: 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.

Formulation optimization and evaluation of oromucosal in situ gel loaded with silver nanoparticles prepared by green biosynthesis.

Treating oral diseases remains challenging as API is quickly washed out of the application site by saliva turnover and mouth movements. In situ gels are a class of application forms that present sol-gel transition's ability as a response to stimuli. Their tunable properties are provided using smart polymers responsible for stimuli sensitivity, often providing mucoadhesivity. In this study, antimicrobial in situ gels of thermosensitive and pH-sensitive polymers loaded with silver nanoparticles were prepared and evaluated. The nanoparticles were prepared by green synthesis using Agrimonia eupatoria L. extract. According to the data analysis, the in situ gel with the most promising profile contained 15 % of Pluronic® F-127, 0.25 % of methylcellulose, and 0.1 % of Noveon® AA-1. Pluronic® F-127 and methylcellulose significantly increased the viscosity of in situ gels at 37 °C and shear rates similar to speaking and swallowing. At 20 °C, a behavior close to a Newtonian fluid was observed while being easily injectable (injection force 13.455 ±â€¯1.973 N). The viscosity of the formulation increased with temperature and reached 2962.77 ±â€¯63.37 mPa·s (37 °C). A temperature increase led to increased adhesiveness and rigidity of the formulation. The critical sol-gel transition temperature at physiological pH was 32.65 ±â€¯0.35 °C. 96.77 ±â€¯3.26 % of Ag NPs were released by erosion and dissolution of the gel after 40 min. The determination of MIC showed effect against E. coli and S. aureus (0.0625 mM and 0.5000 mM, respectively). The relative inhibition zone diameter of the in situ gel was 73.32 ±â€¯11.06 % compared to gentamicin sulfate. This work discusses the optimization of the formulation of novel antibacterial in situ gel for oromucosal delivery, analyses the impact of the concentration of excipients on the dependent variables, and suggests appropriate evaluation of the formulation in terms of its indication. This study offers a promising dosage form for local treatment of oral diseases.

Selective ILM Staining and Safety of Two Vital Dyes During a Human-Like Pars Plana Vitrectomy Ex Vivo in Porcine Eyes.

PURPOSE: An ideal dye for intraocular use should effectively stain the target tissue while being easy to apply and remove. Additionally, it should not have any adverse effects resulting from prolonged contact with the retinal tissue. Recently, concerns have been raised about the safety of some vital dyes during surgical procedures as they may cross the internal limiting membrane and deposit on the retina. In this study, we aimed to investigate whether commercially available vital dyes, VIEW-ILM® and TWIN® (AL.CHI.MI.A. S.r.l., Ponte San Nicolò, Padova, Italy), have the potential to cross the internal limiting membrane during vitreoretinal surgery and deposit on the retina. Furthermore, we evaluated their safety in vitro and in vivo. METHODS: A human-like pars plana vitrectomy was performed on porcine eyes ex vivo, with VIEW-ILM® or TWIN® used to stain the internal limiting membrane either with or without subsequent internal limiting membrane peeling. The two dyes were then extracted from retinal punches with or without internal limiting membrane, and quantified using high performance liquid chromatography. Safety was evaluated through in vitro cytotoxicity tests and in vivo skin sensitization and irritation tests according to ISO standards. RESULTS: High performance liquid chromatography analyses demonstrated that VIEW-ILM® and TWIN® effectively stained the internal limiting membrane without crossing the membrane. No residual dyes were found in the retinal layers after internal limiting membrane removal. Furthermore, both in vitro and in vivo safety tests confirmed the absence of cytotoxicity, skin sensitization, and irritation. CONCLUSION: The results of this study support the safety and efficacy of VIEW-ILM® and TWIN® for internal limiting membrane staining. The experimental protocol described in this study could be utilized to gain a comprehensive understanding of the characteristics of vital dyes.

Assessment of performance and safety of Corneal Chamber hypothermic storage medium and PSS-L corneal rinsing solution in human and porcine corneas.

PURPOSE: To prove the safety and performance of the hypothermic corneal storage medium "Corneal Chamber" and the rinsing solution "PSS-L" in support of the new Conformité Européenne (CE) certification process in accordance with the Medical Device Regulation. METHODS: Fifteen (n=15) human donor corneas and 11 (n=11) porcine corneas were evaluated for the following parameters: endothelial cell density (ECD) and mortality, percentage of hexagonal cells (HEX%), coefficient of cellular area variation (CV%) and corneal transparency at Day 0 and after 14±1 days of storage in Corneal Chamber medium at 2-8°C. Then, the same parameters were assessed after rinsing of corneas in PSS-L for 1 min at room temperature. Evaluation of gentamicin sulfate carryover after corneal storage and PSS-L rinsing was performed by ultra-high performance liquid chromatography analysis on human corneas homogenates. RESULTS: Human and porcine corneas stored in Corneal Chamber medium showed a good overall quality of the tissue according to the quality parameters evaluated. In particular, mean ECD, HEX% and CV% did not show statistically significant changes at the end of storage and endothelial mortality increased to 3.1±3.3 and 7.8±3.5% in human and porcine corneas, respectively. Tissue rinsing with PSS-L did not affect the quality parameters evaluated before and gentamicin sulfate residues were absent in human corneas. CONCLUSIONS: Corneal preservation in Corneal Chamber medium at 2-8°C for 14 days and the corneal rinse with PSS-L are safe and effective procedures allowing the preservation of the corneal quality parameters as well as the complete elimination of gentamicin sulfate from the tissues before transplantation.Cite Now.

Clinical Features and Treatment Outcomes of Carbapenem-Resistant Pseudomonas aeruginosa Keratitis.

Importance: Evaluation of the microbiological diagnostic profile of multidrug-resistant Pseudomonas aeruginosa keratitis and potential management with rose bengal-photodynamic antimicrobial therapy (RB-PDAT) is important. Objective: To document the disease progression of carbapenemase-resistant P aeruginosa keratitis after an artificial tear contamination outbreak. Design, Setting, and Participants: This retrospective observation case series included 9 patients 40 years or older who presented at Bascom Palmer Eye Institute and had positive test results for multidrug-resistant P aeruginosa keratitis between January 1, 2022, and October 31, 2023. Main Outcomes and Measures: Evaluation of type III secretion phenotype, carbapenemase-resistance genes blaGES and blaVIM susceptibility to antibiotics, and in vitro and in vivo outcomes of RB-PDAT against multidrug-resistant P aeruginosa keratitis. Results: Among the 9 patients included in the analysis (5 women and 4 men; mean [SD] age, 73.4 [14.0] years), all samples tested positive for exoU and carbapenemase-resistant blaVIM and blaGES genes. Additionally, isolates were resistant to carbapenems as indicated by minimum inhibitory concentration testing. In vitro efficacy of RB-PDAT indicated its potential application for treating recalcitrant cases. These cases highlight the rapid progression and challenging management of multidrug-resistant P aeruginosa. Two patients were treated with RB-PDAT as an adjuvant to antibiotic therapy and had improved visual outcomes. Conclusions and Relevance: This case series highlights the concerning progression in resistance and virulence of P aeruginosa and emphasizes the need to explore alternative therapies like RB-PDAT that have broad coverage and no known antibiotic resistance. The findings support further investigation into the potential effects of RB-PDAT for other multidrug-resistant microbes.

A suitable and efficient procedure for the removal of decontaminating antibiotics from tissue allografts.

The presence of residual antibiotics in tissue allografts after decontamination with antibiotic cocktails may result in widely documented adverse effects in predisposed subjects. Moreover, antibiotic residues may mask contaminating microorganisms, resulting in falsely negative sterility tests, with potential risk of post-surgical infections. The objective of the present study was to define a rinsing procedure capable of eliminating antibiotic residues from cardiovascular, bone and skin tissues after decontamination with BASE.128. Different washing patterns, employing BASE medium, were applied. The presence of antibiotic residues in tissue homogenates was assessed by agar diffusion test at different stages of tissue processing. To test whether antibiotic residues can result in falsely negative microbiological analysis, we induced a superficial tissue contamination with known inoculum concentration. By employing four different porcine tissues, we here report direct evidence that the presence of even limited amounts of antibiotics in decontaminated tissues interferes with sterility testing. This has implications in terms of increased risk of infections in allograft recipients. To minimize this risk, we developed a procedure for extensive removal of antibiotics from allografts, allowing for subsequent detection of microbial contaminations that may occur during transportation, storage or processing prior to allograft transplantation. Our study emphasizes the importance of validating all processes and analytical methods in tissue banking, in order to warrant tissue safety. This will minimize the risks of post-surgical infections as well as antibiotic-induced anaphylaxis in predisposed patients.

Establishing a procedure for dimethyl sulfoxide removal from cardiovascular allografts: a quantitative study.

The success of allotransplants is critically dependent on both tissue viability and efficient removal of potentially toxic cryopreservants. In this study we analysed the dimethyl sulphoxide (Me2SO) content of cardiovascular tissue samples stored in tissue banks and optimized a washing protocol to be used before surgical implant. We compared the Me2SO content of heart valves, arteries and veins and quantitatively determined by HPLC the washing kinetics of each group of tissue samples under strictly controlled conditions using an industrial washing medium (BASE). Our results showed that heart valves and arteries have significantly slower Me2SO release kinetics than veins. Approximately 20 % of the initial content of cryopreservant could still be detected in the valves and arterial tissue after 15 min of continuous washing. Conversely, veins were almost completely cleared of the cryoprotectant under the same conditions. We propose a washing protocol consisting of two sequential washing with BASE for a total of 25 min for valves and arteries and 15 min for veins. In our hands, this protocol reliably ensures the removal of more than 95 % of the initial Me2SO content.

Comment on "Brownian motion with time-dependent friction and single-particle dynamics in liquids".

Recently, Lad, Patel, and Pratap [Phys. Rev. E 105, 064107 (2022)10.1103/PhysRevE.105.064107] revisited a microscopic theory of molecular motion in liquids, proposed by Glass and Rice [Phys. Rev. 176, 239 (1968)10.1103/PhysRev.176.239]. They argued that the friction coefficient for a Brownian particle in a liquid should exponentially depend on time and derived an equation of motion for the particle's velocity autocorrelation function (VAF). The equation was solved numerically and fitted to the results of molecular dynamics simulations on different liquids. We show that this solution, obtained under the condition of zero derivative of the VAF at time t=0, is physically incorrect at long times. This is evidenced by our exact analytical solution for the VAF, not found by Lad et al., and numerically, by using the same method as in the commented work.

Oxidative Stress and Antioxidant-Based Interventional Medicine in Ophthalmology.

The different anatomical compartments of the eye are highly subjected to reactive oxygen species (ROS) generation due to internal factors, such as metabolic high oxygen consumption, as well as environmental factors, including UV light. An antioxidant defense system is endowed in the eye tissues to regulate ROS quantity and activity. When this homeostatic system is overwhelmed, oxidative stress occurs, causing cellular damage, chronic inflammation, and tissue degeneration. It also plays a significant role in the development and progression of various ocular diseases. Understanding the mechanisms underlying oxidative stress in ocular conditions is thus crucial for the development of effective prevention and treatment strategies. To track marketed products based on antioxidant substances as active ingredients, the databases of the European Medicines Agency and the U.S. Food and Drug Administration were consulted. Only a limited number of items were identified, which were either used as therapeutic treatment or during ocular surgery, including antioxidants, synthetical derivatives, or pro-drugs designed to enhance tissue permeation and activity. This review aims to provide an overview of the primary ocular pathologies associated with oxidative stress and of the available pharmacological interventions centered around antioxidant molecules. Such insights are essential for advancing the development of effective prevention and novel treatment approaches.

P15-A112†Descemet's membrane endothelial keratoplasty (DMEK) graft preparation in porcine cornea.

PURPOSE: Descemet's membrane endothelial keratoplasty (DMEK) is a frequently used treatment option for patients with corneal endothelial dysfunction. The aim of this study was to set up a method to prepare porcine DMEK grafts and to simulate DMEK surgery in porcine eye bulbs in order to establish an ex-vivo-model for laboratory investigations on DMEK surgery conditions. METHODS: Ten (n=10) porcine eye bulbs from domestic pigs (Sus scrofa domestica), between 6 and 8 months old, were recovered at a local slaughterhouse, transported on ice and processed within 2 h after death. Porcine eye bulbs were decontaminated by immersion in 10 mL of 5% povidone-iodine and corneas were dissected under aseptic conditions, leaving approximately 2 mm of the scleral rim. DMEK grafts were prepared by means of mechanical stripping technique using specific surgical instruments for DMEK (Moria, France) on fresh corneas (n=2) and on corneas stored in Eusol-C (AL.CHI.MI.A. Srl, Italy) at 4°C for 7 days (n=4) and for 14 days (n=4). Endothelial cell (EC) density was compared before DMEK-preparation (specular and light microscopy on trypan blue stained tissues) and after DMEK-preparation (fluorescence microscopy on Calcein-AM stained tissues). DMEK graft injection was simulated in anterior chamber of fresh porcine eye bulbs. RESULTS: The porcine DMEK grafts preparation resulted to be more challenging compared to human DMEK grafts. Despite similarity between human and porcine corneas, porcine Descemet membrane (DM) firmly adheres to the underlying stroma. DMEK grafts preparation was not successful at day 0; DMEK preparation was possible by mechanical stripping technique on corneas stored in Eusol-C for 7 and 14 days obtaining naturally rolled endo-out porcine DMEK grafts. An EC mortality increase up to 20% was observed on DMEK graft compared to initial whole corneal tissue. DMEK roll injection was successfully simulated in anterior chamber of the porcine eye bulb. CONCLUSION: Naturally rolled DMEK endo-out grafts were successfully prepared by mechanical stripping technique on porcine corneal tissues stored in Eusol-C at 4°C (up to 14 days). DMEK Surgery including the tissue injection in anterior chamber could be simulated. Further studies will be performed to improve ex-vivo-porcine DMEK surgery model.

P14-A117†Assessment of performance and safety of corneal chamber hypothermic storage and PSS-L corneal rinsing in human and porcine corneas.

PURPOSE: To prove the safety and performance of the hypothermic corneal storage medium Corneal Chamber, containing Eusol-C solution (AL.CHI.MI.A. S.r.l.) and of the rinsing solution PSS-L (AL.CHI.MI.A. S.r.l.) in support to the new CE certification process in accordance to the EU 2017/745 Medical Device Regulation METHODS: Fifteen (n=15) human donor corneas unsuitable for transplantation and n=11 porcine corneas were evaluated for the following quality parameters: ECD, HEX%, CV%, endothelial morphology, endothelial mortality and transparency at day 0 and after 14±1 days (day 14) of storage in Corneal Chamber at 2-8°C. Then, corneas were rinsed in PSS-L for 1' at room temperature (RT) and the same parameters were assessed Post Rinsing (Day 14PR). In order to evaluate the antimicrobial carryover after the corneal storage in Corneal Chamber(14 days at 4°C), gentamicin sulphate was quantified in human and porcine corneas homogenates by UHPLC. RESULTS: Human and porcine corneas stored in Corneal Chamber at 2-8°C for 14 days showed a good overall quality of the tissue according to quality parameters evaluated. In particular, mean ECD, HEX% and CV% did not show statistically significant changes at the end of storage and endothelial mortality increased of 3.1±3.3% in human corneas and 7.8±3.5% in porcine corneas. Slight variations in endothelial morphology score and corneal transparency were observed. Rinsing with PSS-L did not negatively affect the quality parameters evaluated before and after rinsing and gentamicin sulfate residues were completely removed. CONCLUSION: The storage of corneal tissues in Corneal Chamber at 2-8°C for 14 days and the corneal rinse with 30 ml of PSS-L at RT for 1 min are safe and effective procedures allowing the preservation of the corneal quality parameters including ECD, endothelial mortality, endothelial morphology, HEX%, CV%, and corneal transparency and the elimination of gentamicin sulfate from the tissues before transplantation.

Antioxidant Nutraceutical Strategies in the Prevention of Oxidative Stress Related Eye Diseases.

This review aims to discuss the delicate balance between the physiological production of reactive oxygen species and the role of antioxidant nutraceutical molecules in managing radicals in the complex anatomical structure of the eye. Many molecules and enzymes with reducing and antioxidant potential are present in different parts of the eye. Some of these, such as glutathione, N-acetylcysteine, α-lipoic acid, coenzyme Q10, and enzymatic antioxidants, are endogenously produced by the body. Others, such as plant-derived polyphenols and carotenoids, vitamins B2, C, and E, zinc and selenium, and omega-3 polyunsaturated fatty acids, must be obtained through the diet and are considered essential nutrients. When the equilibrium between the production of reactive oxygen species and their scavenging is disrupted, radical generation overwhelms the endogenous antioxidant arsenal, leading to oxidative stress-related eye disorders and aging. Therefore, the roles of antioxidants contained in dietary supplements in preventing oxidative stress-based ocular dysfunctions are also discussed. However, the results of studies investigating the efficacy of antioxidant supplementation have been mixed or inconclusive, indicating a need for future research to highlight the potential of antioxidant molecules and to develop new preventive nutritional strategies.

P20-A129†An innovative wound healing method reveals a donor and post-mortem time-dependent regeneration of corneal epithelium.

PURPOSE: The aim of this study was to establish and optimize a new and reproducible epithelial wound healing model on human corneas. This assay was used to study the kinetics of epithelial regeneration following a chemical injury. METHODS: Thirty (n=30) human corneas unsuitable for transplant were used for the experiments. Corneas were cultured in Storagix medium (FBOV) at 31°C. Epithelial integrity before the beginning of the experiments (pre-wound) was assessed using the vital dyes trypan blue (TB, TB-S 0.25%, AL.CHI.MI.A. srl) and sodium fluorescein (Fluo). 1-heptanol soaked paper disks (6 mm) were applied in the centre of the corneas for 1' to trigger a chemical damage at the epithelial layer. Afterwards, sodium fluorescein and TB stainings were repeated to quantify the damaged area and to monitor healing progression. The damaged area (mm2) was calculated for each time point with Fiji software. Wound healing rate (HR, mm2/die) was calculated for both Fluo (HRF) and TB (HRTB) measurements using the previously described formula:Arithmetical averages (HRFAVG and HRTBAVG) of HRs were calculated and correlated by Pearson correlation coefficient with the following donor's parameters: age, sex, post-mortem time (PMT, time between death and tissue procurement), stromal defects, septicaemia, body temperature, diabetes. RESULTS: The execution of the heptanol wounding is highly reproducible, as highlighted by Fluo and TB staining. The average time for full recovery from wounding was 3,8 ± 0,41 days for Fluo and 3,5 ± 0,63 days for TB. Fluo and TB stainings are interchangeable as they significantly correlate (Pearson correlation coefficient = 0.630; p>0.05). A negative linear correlation was observed between HR and PMT (HRFAVG: corrected R2: 0.243, p = 0.003; HRTBAVG: corrected R2: 0,132, p = 0.028), but not with the other donors' parameters. CONCLUSION: Our wound/healing model might be of great interest for studies of epithelial regeneration kinetics and validation of drugs for the treatment of ocular defects. The inverse correlation between PMT and HR provides valuable insights for scientists investigating the regenerative properties of the corneal epithelium, as well as for eye bank personnel aiming to preserve the regenerative potential of corneal epithelium.

Prospective In Vitro Comparison of Kerasave and Optisol-GS Corneal Storage Solutions.

PURPOSE: The aim of this study was to compare the performance of Kerasave and Optisol-GS for hypothermic corneal storage for 14 days. METHODS: This study was a prospective laboratory investigation. Mate corneas were recovered into Kerasave or Optisol-GS (27 pairs) and stored at 2°C to 8°C for 14 days. Corneas were evaluated by trained eye bank technicians, and study parameters were compared between the initial and final evaluations. Endothelial cell density (ECD), hexagonality (HEX), and coefficient of variation (CV) were evaluated by specular microscopy, and central corneal thickness (CCT) was examined by optical coherence tomography after 1, 3, 7, and 14 days of storage. Corneal transparency was scored using slit lamp examination at days 1 and 14. RESULTS: Average ECD, HEX, and CV for the Kerasave (2653 ± 303 cells/mm 2 , 57 ± 4%, and 36 ± 3%) and Optisol-GS (2623 ± 306 cells/mm 2 , 57 ± 5%, and 36 ± 4%) groups were not significantly different at day 1. There was also no difference at any other study time points (all P > 0.05). ECD did not significantly change from day 1 to day 14 in either group ( P > 0.05), but a statistically significant change in HEX and CV was observed between day 1 and day 14 in both groups ( P < 0.01). Average CCT measured at day 1 for corneas stored in Kerasave was 622 ± 49 µm and those stored in Optisol-GS was 580 ± 35 µm ( P < 0.01). The difference in CCT measurements was not significantly different at day 14 (Kerasave: 674 ± 46 µm vs. Optisol-GS: 647 ± 58 µm, P > 0.05). Corneal transparency was not significantly different between the 2 groups at day 1 or day 14. CONCLUSIONS: The corneal quality and clinically relevant parameters including ECD, endothelial morphometry, and corneal transparency were not different in corneas stored in Kerasave or Optisol-GS for 14 days. The initial difference in CCT between the 2 groups decreased at day 14. These results demonstrated that Kerasave corneal storage solution preserves the corneal endothelium similarly to Optisol-GS.

P12-A107†Porcine cornea ex vivo model as an alternative to human donor tissues for investigating new preservation conditions.

PURPOSE: Considering the growing shortage of corneal tissues for research, the present study aimed to develop and optimize a porcine cornea model with qualitative features comparable to those of human tissues. METHODS: A new decontamination procedure of porcine eye bulbs was set up and its efficacy as well as endothelial mortality were evaluated. Human corneas unsuitable for transplant and porcine corneas were then compared after storage under hypothermic (4-8°C, Eusol-C, AL.CHI.MI.A. S.R.L) or organ-culture (31-35°C, Tissue-C, AL.CHI.MI.A. S.R.L) storage conditions for 14 days. A new method, based on the semi-automatic analysis of Trypan-blue stained endothelial areas by Fiji software, was developed to quantify the whole endothelium viability. Corneas were assessed for central corneal thickness (CCT), corneal transparency, endothelial morphology, and endothelial cell density (ECD) at days 0, 7, and 14 of storage. Portions of lamellar tissues consisting of Descemet's membrane and endothelial cells were prepared for histological investigations. RESULTS: The new decontamination procedure of porcine eye bulbs resulted in 18% versus 89% ('no decontamination' control) of corneas still contaminated after 28 days of storage at 31°C. The decontamination protocol did not affect endothelium viability, as assessed by the new Fiji-based method. ECD (porcine: 3156 ± 144 cells/mm2; human: 2287 ± 152 cells/mm2), CCT (porcine: 1073 ± 151 µm; human: 581 ± 39 µm), transparency (porcine: 88.6 ± 11.0%; human: 76.3 ± 5.4%), and morphology score (porcine: 4.0 ± 0.0; human: 3.2 ± 0.4) measured in the porcine cornea at day 0 were significantly higher than in human corneas. Nonetheless, the qualitative parameters of porcine and human corneas showed comparable trends during the storage under hypothermic (4-8°C) and organ-culture (31-35°C) conditions for 14 days. CONCLUSION: The presented porcine cornea model represents a reliable and alternative model to human donor tissues for preliminary investigations and can be used for testing new media, substances, drugs, or preservation conditions and their impact on corneal tissue quality and safety. Furthermore, the quantitative method to assess whole endothelium mortality can be implemented at eye banks for the evaluation of corneas intended for transplantation.