Slit-lamp based assessment of peripheral versus central regions of the human corneal endothelium.

PURPOSE: To compare the central and peripheral (< 2 mm from the limbus) endothelial regions of the human cornea of young contact lens (CL) neophytes, in terms of endothelial cell density (ECD), polymorphism through cell hexagonality (HEX), and polymegethism through the coefficient of variation of cell areas (CoV). MATERIALS AND METHODS: In vivo, central, temporal, and nasal ECDs, HEXs, and CoVs were determined for thirty healthy right eyes (age: 22-30 years) using a Takagi 700 G L LED slit-lamp biomicroscope equipped with the centre-dot EndoKer© for the analysis of endothelium images. RESULTS: The mean central ECD (2586 ±â€¯233 mm-2) was lower than peripheral ECDs (nasal 2733 ±â€¯225 mm-2, temporal 2674 ±â€¯260 mm-2) (p = 0.03 and p = 0.02, respectively). The mean central HEX (53.0 ±â€¯4.6%) was lower than peripheral HEXs (nasal 57.2 ±â€¯4.3%, temporal 57.1 ±â€¯4.5%) (both p < 0.005), while the mean central CoV (28.2 ±â€¯2.5%) was higher than peripheral CoVs (nasal 25.8 ±â€¯2.2%, temporal 26.1 ±â€¯2.2%) (both p ≤ 0.001). CONCLUSIONS: In young healthy non-CL wearers, the peripheral corneal endothelium displays a higher ECD and significantly better cell regularity (higher HEX and lower CoV) compared to the central cornea. A clinical evaluation of the corneal endothelium requires, therefore, a full characterization of the different areas.

Cost-Benefit and Cost-Utility Analysis of Amphotericin B Supplementation of Corneal Storage Media With Endothelial Keratoplasty-Prepared Tissue.

PURPOSE: To determine the cost-effectiveness of amphotericin B supplementation, we analyzed both current costs to treat postendothelial keratoplasty (EK) fungal infections and potential costs associated with amphotericin B supplementation. METHODS: We collected 19 US cases of post-EK fungal eye infections from the published literature and assessed the associated costs from the literature. A survey of surgeons was also conducted with questions regarding their experiences in managing these infections. RESULTS: We estimated that the costs to diagnose, manage, and treat post-EK fungal keratitis and post-EK fungal endophthalmitis are USD $21,113 and $34,850, respectively. The largest portion of the costs can be attributed to the need for additional surgical management, which is required in 79% of the cases. We estimated the total cost of amphotericin B supplementation to be $44.39 per graft with use of conventional amphotericin B and conservative assumptions regarding supplementation processes. Cost-effectiveness analysis demonstrated that amphotericin B supplementation is cost-effective at $100,000 per quality-adjusted life-year level only if amphotericin B supplementation can prevent more than 69.62% of post-EK fungal infections, assuming the incidence of post-EK fungal infection remains at the level it was between 2012 and 2017. CONCLUSIONS: We found that amphotericin B supplementation can be cost-effective under conservative assumptions if it is moderately effective in preventing post-EK fungal infections.

Human Corneal Endothelial Cell Assessment From Tissues Preserved in Serum-Based and Synthetic Storage Media.

PURPOSE: To assess the difference between endothelial cells from tissues preserved in media supplemented with fetal bovine serum (FBS) and recombinant human serum albumin (rHSA). METHODS: In a donor-matched study, 48 tissues were preserved for 28 days at 31°C in Cornea Max and Cornea Syn supplemented with FBS and rHSA, respectively. Endothelial cells were visualized by 2 masked observers before and after preservation. Endothelial cell density (ECD) and the number of iatrogenic folds were counted manually. Alizarin red staining and tight junction protein (Zonula Occludens-1) were used to assess cell morphology (hexagonality and polymorphism). Intraobserver and interobserver cell counts were recorded and analyzed. Wilcoxon and one-way analysis of variance tests were used, where P < 0.05 was deemed statistically significantly different. RESULTS: Significant amount of iatrogenic folds were observed in the tissues supplemented with FBS compared with rHSA postpreservation (P = 0.0007). Approximately 69% and 71% hexagonal cells (P = 0.0303) and 29% and 26% polymorphic cells (P = 0.0234) were observed in the FBS and rHSA groups, respectively. Postpreservation, operator 1 counted 1766 cells/mm in FBS and 1864 cells/mm in rHSA. Operator 2 counted 1702 cells/mm in FBS and 1858 cells/mm in rHSA. ECD counts from FBS (interoperator) were statistically significant (P = 0.0429). However, significance was not observed in the ECD counts (interoperator) from the rHSA-preserved tissues (P = 0.8738). CONCLUSIONS: rHSA-supplemented media allow better visualization of the corneal endothelial cells. This reduces the rate of discard observed due to counting errors. Use of rHSA improves the current standard of care and reduces the use of animal-derived products.

Assessment of Corneal Endothelium during Continued Organ Culture of Pre-Stripped Human Donor Tissue for DMEK Surgery.

PURPOSE: To measure corneal endothelial cell (EC) quality and quantity following Descemet membrane (DM) stripping of human donor corneas and continued storage in organ culture medium containing dextran. METHODS: DM stripping was performed in 30 organ cultured, corneoscleral discs. Corneas were divided into 3 groups of 10 corneas each. Baseline mean EC density (cells/mm2) was 2,372 (SD ± 259) in group 1, 2,540 (SD ± 266) in group 2, and 2,665 (SD ± 263) in group 3. Following subtotal DM stripping, culture was continued at 31°C for 24 hours (group 1), 72 hours (group 2), and 120 hours (group 3), respectively. EC density was measured before stripping and at the end of culture. At the end of culture, corneal EC morphology was graded using a scoring system and EC viability was measured by detection of adenosine triphosphate. RESULTS: At the end of culture, mean EC density was 2,159 (SD ± 293) in group 1, 1,946 (SD ± 182) in group 2, and 2,047 (SD ± 225) in group 3. This constitutes an EC loss of 9,1% (SD ± 5,3%) in group 1, 23,0 % (SD ± 6,5%) in group 2, and 22,7% (SD ± 9,1%) in group 3 (p < 0.001). After completion of follow-up, all groups contained corneas with EC counts < 2,000 cells/mm2. Cell morphology scores did not differ between the three experimental groups. EC viability measurements showed a tendency toward lower readings with extended length of culture. CONCLUSIONS: Corneal EC loss does occur following DM stripping and continued organ culture. EC loss increases with storage past 24 hours, but donor corneas may fall below 2,000 cells/mm2 independently of storage duration. The use of eye bank prepared donor lamellae for Descemet Membrane Endothelial Keratoplasty (DMEK) may increase patient safety by offering standardized quality control before tissue release.

Evaluation and Quality Assessment of Prestripped, Preloaded Descemet Membrane Endothelial Keratoplasty Grafts.

PURPOSE: To determine graft quality and feasibility of Descemet membrane endothelial keratoplasty (DMEK) grafts that are prestripped and preloaded into injectors by eye bank technicians before shipping to surgeons. METHODS: DMEK grafts (n = 31) were prepared from donor corneas and preloaded into Straiko Modified Jones tubes and set inside viewing chambers filled with 20 mL of Optisol-GS. Preloaded grafts were evaluated using specular microscopy and slit-lamp biomicroscopy. Endothelial cell loss (ECL) was captured by vital dye staining and quantified using FIJI. A subset of preloaded tissues was subjected to a shipping validation and 5-day storage assay. Fourteen additional DMEK grafts (not preloaded) were examined to quantify damage resulting from prestripping alone. RESULTS: Specular microscopy was able to be performed for all preloaded tissues. Average ECL for preloaded tissues quantified by vital dye staining and FIJI after overnight storage was 16.8% ± 5.9%, and differed from slit-lamp ECL estimation by an average of 5.3% ± 3.6%. The average damage caused by prestripping alone was 9.3% ± 5.9%, and it was significantly less than that of preloaded tissues (P < 0.01). Average ECL for preloaded tissues subjected to round-trip shipping events was 18.5% ± 12.4%, and ECL for tissues stored at 4°C for 5 days after preloading was 13.1% ± 9.5%. CONCLUSIONS: It is possible to prepare, evaluate, and ship DMEK grafts loaded inside a glass carrier and viewing chamber. The ability to evaluate tissues after processing allows for adherence to the Eye Bank Association of America Medical Standards, and for surgeons to receive the most accurate tissue information.

The role of corneal endothelial morphology in graft assessment and prediction of endothelial cell loss during organ culture of human donor corneas.

PURPOSE: Endothelial assessment is crucial in the release of corneas for grafting. We retrospectively analysed the role of endothelial morphology parameters in predicting endothelial cell loss during organ culture. METHODS: Human donor corneas were cultured in minimal essential medium with 2% fetal calf serum and antibiotics. Initial endothelial morphology was assessed microscopically using score parameters polymegethism (POL), pleomorphism (PLE), granulation (GRA), vacuolization (VAC), segmentation of cell membranes (SEG), Descemet's folds (DF), trypan blue-positive cells (TBPC) and endothelial cell-free areas (ECFA). Some corneas were primarily rejected based on endothelial assessment. Endothelial cell density (ECD) was assessed at the beginning (I-ECD) and end of culture. Corneas were then placed in dehydration medium (as above + 5% dextran 500). In a subgroup, ECD was reassessed after dehydration. Endothelial cell loss during culture (ECL@Culture) and culture+dehydration (ECL-Culture&Dehydration) were calculated. Data were given as mean ± SD and analysed using multiple linear and logistic regression. Odds ratios (OR) and 95% confidence intervals (CI) were calculated. RESULT: I-ECD was 2812 ± 360/mm2 (n = 2356). The decision to reject a cornea due to endothelial assessment was associated negatively with I-ECD (OR = 0.77/100 cells, CI 0.7-0.82) and positively with ECFA (OR = 2.7, CI 1.69-4.35), SEG (OR =1.3, CI 1.01-1.68) and donor age (OR = 1.26/decade, CI 1.33-1.41). ECL@Culture was 153 ± 201/mm2 (n = 1277), ECL@Culture&Dehydration was 169 ± 183/mm2 (n = 918). ECL@Culture was associated positively with donor age, I-ECD, GRA and TBPC, and negatively with PLE, and DF. ECL@Culture&Dehydration was associated positively with age, sex, initial ECD, POL, PLE, VAC and TBPC. CONCLUSION: Morphological parameters displayed associations with the exclusion of corneas from culture and with endothelial cell loss. Appropriate parameter selection for screening purposes may help improve graft quality.

Generation and Feasibility Assessment of a New Vehicle for Cell-Based Therapy for Treating Corneal Endothelial Dysfunction.

The corneal endothelium maintains corneal transparency by its pump and barrier functions; consequently, its decompensation due to any pathological reason causes severe vision loss due to corneal haziness. Corneal transplantation is the only therapeutic choice for treating corneal endothelial dysfunction, but associated problems, such as a shortages of donor corneas, the difficulty of the surgical procedure, and graft failure, still need to be resolved. Regenerative medicine is attractive to researchers as a means of providing innovative therapies for corneal endothelial dysfunction, as it now does for other diseases. We previously demonstrated the successful regeneration of corneal endothelium in animal models by injecting cultured corneal endothelial cells (CECs) in combination with a Rho kinase (ROCK) inhibitor. The purpose of the present study was to optimize the vehicle for clinical use in cell-based therapy. Our screening of cell culture media revealed that RELAR medium promoted CEC adhesion. We then modified RELAR medium by removing hormones, growth factors, and potentially toxic materials to generate a cell therapy vehicle (CTV) composed of amino acid, salts, glucose, and vitamins. Injection of CECs in CTV enabled efficient engraftment and regeneration of the corneal endothelium in the rabbit corneal endothelial dysfunction model, with restoration of a transparent cornea. The CECs retained >85% viability after a 24 hour preservation as a cell suspension in CTV at 4°C and maintained their potency to regenerate the corneal endothelium in vivo. The vehicle developed here is clinically applicable for cell-based therapy aimed at treating the corneal endothelium. Our strategy involves the generation of vehicle from a culture medium appropriate for a given cell type by removing materials that are not favorable for clinical use.

The effect of assisted reproductive technology on ocular assessments.

PURPOSE: The aim was to study the various ocular assessments in women undergoing assisted reproductive techniques (ART). METHODS: A total of 117 women with infertility were enrolled for study and the ART protocol was followed. The outcome measures were intraocular pressure (IOP), central corneal thickness, corneal endothelial cell counts, Schirmer I test done at baseline (V0), post-oral contraceptive (V1), post-GnRH agonist induction phase (V2), post-conception (V3), third trimester of pregnancy (V4) and three months post-partum (V5). Schirmer I test (without topical anaesthesia) less than 10 mm in at least one eye was considered a criterion for dry eye. RESULTS: Out of 117 women enrolled for in vitro fertilisation (IVF), only 48 patients conceived. Sixteen women had abortions and remaining 32 women, who had childbirth were followed until three months post-partum. Baseline mean IOP showed a slight decrement and corneal pachymetry and corneal endothelial cell counts showed slight increment from phases V1 to V4 of ART but statistically they were not significant (p > 0.05). At three months post-partum (V5) mean IOP, corneal pachymetry, corneal endothelial cell counts become closer to baseline in both eyes. The number of patients with at least one eye with dryness (Schirmer I less than 10 mm) significantly increased at the third trimester (p = 0.02) and three months post-partum (p = 0.035), whereas in the rest of the phases, it was comparable to baseline (p > 0.05). CONCLUSION: The ocular physiological changes (IOP, corneal pachymetry and corneal endothelial cell counts) seen in various phases of ART were non-specific. The ART appears to reduce tear secretions as measured by Schirmer I; however, further research would be required to determine the impact of ART on the tear film and whether ART is associated with symptoms and signs of dry eye.

Quality assessment of corneal storage media and their components.

BACKGROUND: To keep the loss of endothelial cell density in donor corneas to a minimum, a storage medium which is adjusted to their nutritional needs is necessary. Different media, used either serum-supplemented or serum-free, are available. The quality of medium- and serum-batches as well as support of endothelial cell viability by the medium are to be tested with a quality assured screening system that allows routine examination. METHODS: A screening system was developed which is based on cell-culture tests with the well-established human corneal endothelial cell line HCEC-12, and therefore can be performed without the need for donor corneas. The cells are plated at a defined density in cell-culture dishes, and are cultured for a defined period of time in the test media. Evaluation is carried out by assaying cell count, activity of cell metabolism (resazurin conversion), and determining the number of apoptotic and necrotic cells (combined vital staining with YO-PRO®-1/propidium iodide and subsequent flow cytometry). RESULTS: Human corneal endothelial cells that are cultured in a medium which is adjusted to their nutritional needs achieve higher cell numbers and show a higher metabolic rate. Simultaneously, the percentage of apoptotic and necrotic cells is lower. The screening system developed in this study allows for easy and reliable detection of slightest differences between different media, different processing steps for same media, and different supplements, as well as different serum batches. CONCLUSIONS: The differentiated results show that the screening system is sensitive enough to show even minor quality differences. Therefore, it is more suitable than the hitherto commonly used growth assay with primary, mostly porcine, corneal endothelial cells.

Assessment of central corneal thickness and corneal endothelial morphology using ultrasound pachymetry, non-contact specular microscopy, and Confoscan 4 confocal microscopy.

BACKGROUND: The aim was to compare the repeatability, reproducibility and inherent precision of ultrasound pachymetry (USP), noncontact specular microscopy (SP-2000P) and the Confoscan 4 confocal microscope (z-ring CS4) in measuring endothelial cell density (ECD), coefficient of variation of cell size (CV), and central corneal thickness (CCT) in normal eyes. METHODS: In this prospective study, one eye was selected from each of 30 subjects for the measurements of ECD, CV and CCT, which were taken by two observers. Results were analyzed statistically by repeated-measures analysis of variance (ANOVA) for intra-observer repeatability, inter-observer reproducibility, unpaired t-test, paired t-test, and Bland-Altman analyses to determine limits of agreement (LOA) between the three instruments. RESULTS: Mean ECD, measured by SP-2000P and z-ring CS4, were 3115.50 ± 279.70 cells/mm(2) and 3167.50 ± 264.75 cells/mm(2), respectively (observer 1), and 3192.63 ± 249.42 cells/mm(2) (z-ring, observer 2). Mean CV measurements were 27.12 ± 2.51 and 27.10 ± 2.41 (SP-2000P and z-ring CS4, respectively; observer 1), and 27.17 ± 2.25 (z-ring, observer 2). Mean CCT values were 555.11 ± 35.83 µm (USP), 535.82 ± 41.10 µm (SP-2000P) and 552.57 ± 36.83 µm (z-ring CS4), and 554.97 ± 36.34 µm (z-ring CS4, observer 2). However, pairwise tests in all cases there was good repeatability and reproducibility as shown by inter-observer and intra-observer analysis of variance for each of the instruments. CONCLUSIONS: The SP-2000P and the z-ring CS4 can be used interchangeably to measure ECD and CV. For CCT, the sample size was too small to test for differences of the CCT measurements between the three instruments.

Impact of surgeon acceptance parameters on cost and availability of corneal donor tissue for transplantation.

OBJECTIVE: To examine the effects of more restrictive donor corneal parameters on the cost and availability of transplantable tissue. METHODS: Corneal tissue data from the Midwest Eye-Banks were collected from 2008 through 2011. Endothelial cell density (ECD) and donor age were arbitrarily restricted in a statistical model based on donor tissue availability. A hypothetical baseline corneal donor tissue fee of $3000 was used for the model. RESULTS: Overall, 19,990 tissues were recovered from 10,668 donors and met Food and Drug Administration and Eye Bank Association of America donor eligibility criteria and current age and ECD criteria for surgical use for corneal transplantation. The mean corneal ECD of screened corneas was 2694 ± 338 cells per square millimeter (range, 2000-4694 cells/mm2). The average age of the recovered donor corneas eligible for surgery was 55.6 ± 14.4 years. Donors aged 51 to 75 years contributed 70.5% of the surgical tissue. In this model, a minimum ECD restriction of 2300, 2500, or 2800 cells per square millimeter would reduce the corneal tissue availability to 87.7%, 70.6%, or 36.5% of current levels, respectively. If donor age were restricted to ≤ 70, ≤ 65, or ≤ 60 years, the percentage of corneal tissue available would decrease to 89.5%, 74.3%, or 57.5% of current levels, respectively. CONCLUSIONS: Tissue criteria restrictions would affect corneal surgeons and eye banks. Restrictions on donor age and ECD would decrease the availability of surgically suitable tissue and increase the costs of cornea transplant tissue.

Measurement area and repeatability of semiautomated assessment of corneal endothelium in the Topcon specular microscope SP-2000P and IMAGEnet system.

PURPOSE: To investigate the repeatability of the semiautomatic assessment of corneal endothelial cells and its association with the measurement area in the Topcon SP-2000P microscope and IMAGEnet system. METHODS: Specular microscopic images of 86 healthy subjects were captured and analyzed using the Topcon SP-2000P microscope and IMAGEnet system. The same images were analyzed twice, on separate days, by the same examiner using the built-in measurement tool of the IMAGEnet system. The measurement areas were defined with a frame mounted on a computer screen. Four different-sized measurement areas were chosen for the semiautomatic measurements: box A (5.4 × 13.9 cm(2)), box B (4 × 10 cm(2)), box C (4 × 7 cm(2)), and box D (2 × 5 cm(2)). Average cell size (ACS), endothelial cell density (ECD), coefficient of variance, and hexagonality were measured. Repeatability was assessed based on the limit of agreement (LOA). RESULTS: The means of ACS, ECD, and hexagonality were not statistically different across 4 measurement areas (analysis of variance, P > 0.05). The mean differences (bias) were modest for ACS (range, -1.9∼3.9 µm(2)), ECD (range, -27.2∼14.6 cells per square millimeter), coefficient of variance (range, -0.14∼1.00), and hexagonality (range, -1.3%∼6.8%). Limits of agreement (mean difference ± 1.96× SD) were greater in the measurements with smaller areas: limit of agreement values for ECD were 14.6 ± 99.6, -3.8 ± 101.1, -27.2 ± 179, and -15.8 ± 488 cells per square millimeter for boxes A, B, C, and D, respectively. Similar trends were found in the repeatability of ACS and hexagonality. CONCLUSIONS: Repeatability is improved when larger measurement areas are chosen.

Optimal use of donor corneal tissue: one cornea for two recipients.

PURPOSE: To evaluate the feasibility, safety, and efficacy of the use of 1 donor cornea for 2 recipients who required anterior lamellar and posterior lamellar keratoplasties, respectively. METHODS: Twelve eyes with anterior corneal stromal pathology and 12 eyes with irreversible endothelial dysfunction were evaluated for transplant surgery at a tertiary eye care referral center. Twelve healthy donor corneas were split into 2 parts, that is, anterior lamellar button (350-µm-thick) and posterior lamellar button (150-µm-thick) using a microkeratome (Moria, Antony, France). The anterior lamellar button was used to perform automated lamellar therapeutic keratoplasty in 12 eyes, and the posterior lamellar button was used to undertake Descemet stripping automated endothelial keratoplasty in 12 eyes. The parameters evaluated were feasibility of the procedure, intraoperative and postoperative complications, if any, and visual outcome. RESULTS: It was possible to use 12 donor tissues for 24 patients as envisaged. No intraoperative or postoperative complications were observed. In the automated lamellar therapeutic keratoplasty group, 83.3% (10 of 12) of patients achieved a best-corrected visual acuity (BCVA) of >20/60, and in the Descemet stripping automated endothelial keratoplasty group 75% (9 of 12) of patients achieved a BCVA of >20/60. CONCLUSIONS: Our study demonstrates that 1 donor corneal tissue can be successfully used for 2 patients as a routine practice with appropriate and optimal case selection. Such techniques may help to reduce the magnitude of corneal blindness in developing countries where there are shortages of donor corneal tissue.

Pan-corneal endothelial viability assessment: application to endothelial grafts predissected by eye banks.

PURPOSE: To present an experimental method for determining the viable cell pool of corneal endothelia and its application to assessing predissected endothelial grafts. METHODS: The endothelial cell density (ECD) of five pairs of human organ cultured corneas was determined using a standard counting method with a calibrated image analysis system. A thin posterior graft (30-50 µm) was manually predissected from a cornea chosen at random. Predissected and control corneas were shipped to the remote center, where standard ECD determination was repeated and was immediately followed by a triple Hoechst/ethidium/calcein labeling coupled with image analysis of the whole graft surface. Numeration of nuclei (H+), dead cells (E+), and total area covered by viable cells (C+) allowed the calculation of viable ECD corresponding to the cell density that the cornea may have after redistribution of viable cells over the whole Descemet surface. RESULTS: The median (range) viable ECD was lower than the standard ECD determined immediately earlier in predissected and control corneas: 1628 (1138-2379) and 2065 (1492-2876) cells/mm(2) (P = 0.043), corresponding to -20% (-1%-38%) and -12% (-3%-26%), respectively (P = 0.08). CONCLUSIONS: Standard counting by eye banks overestimates the actual pool of viable endothelial cells. This may be the main explanation for the initially rapid decrease in ECD universally described in patients after all types of keratoplasty. Early low postoperative ECD may indicate that surgeons graft fewer living cells than the eye banks' ECD let suppose, rather than a massive pre- and postoperative cell death. The novel concept of viable ECD can be useful for assessing all types of corneal processing.

Assessment of attachment factors for primary cultured human corneal endothelial cells.

PURPOSE: To assess the ability of various attachment factors to promote attachment of primary cultured human corneal endothelial cells. MATERIALS AND METHODS: Primary cultured human corneal endothelial cells (HCEC) were incubated for 2 hours in 24-well plates. Wells had been precoated with commercially available cell attachment improvement media (FNC coating mix), human collagen I, human fibronectin, fibronectin/collagen I, or poly-d-lysine. Ratios of cell count before and after rinsing with culture medium and ratios of cells showing morphological signs of spreading to total cells were calculated to measure effectiveness of attachment factors. RESULTS: Incubation of HCEC for 2 hours in wells without precoating of attachment factors led to a rate of 41 +/- 16% (mean +/- SD) of cells showing signs of spreading. FNC coating mix, collagen I, and fibronectin/collagen I increased significantly the percentage of cells showing morphological features of attachment at 2 hours. Total cell loss was highest with poly-d-lysine and no pretreatment with attachment factor. Without the use of any attachment factor, 67 +/- 19% of cells remained after rinsing. The lowest cell loss was observed with FNC coating mix where 108 +/- 5% of cells remained after rinsing. CONCLUSION: Collagen I, collagen I/fibronectin, and FNC coating mix significantly enhance the spreading of human corneal endothelial cells to tissue culture plates after 2 hours. FNC coating mix significantly reduces cell loss due to rinsing. Without the use of any attachment factor, 67% of the cells remained in situ after rinsing.

Ocular injectable formulation assessment for oxidized dextran-based hydrogels.

Initiator-free injectable hydrogels are very interesting for drug and/or cell delivery applications, since they can be administered in a minimally invasive way, and avoid the use of potentially harmful chemical initiators. In the current work, oxidized dextran crosslinked with adipic acid dihydrazide hydrogels were further characterized and tuned to produce formulations, with the aim of producing an injectable formulation for the possible treatment of posterior eye diseases. The gelation rate and the hydrogel dissolution profile were shown to be dependent on the balance between the degree of dextran oxidation, and the concentration of both components. For the in vitro studies, rabbit corneal endothelial cells were seeded on the hydrogels to assess cytotoxicity. Hydrogels prepared with low oxidized dextrans were able to promote cell adhesion and proliferation to confluence in just 24h, while more highly oxidized samples promoted cell adhesion and proliferation, but without achieving confluence. Cell viability studies were performed using MTS assays to verify the non-cytotoxicity of hydrogels and their degradation byproducts, rendering these formulations attractive for further in vivo studies.

Assessment of eye bank-prepared posterior lamellar corneal tissue for endothelial keratoplasty.

OBJECTIVE: To evaluate eye bank-prepared tissue for Descemet's stripping automated endothelial keratoplasty (DSAEK). DESIGN: Experimental study and retrospective case series. PARTICIPANTS: Seventeen human donor corneas and 4 recipient patients undergoing DSAEK surgery. METHODS: Corneal-scleral discs were obtained. Specular microscopy and pachymetry were performed. A designated Tissue Banks International technician used a microkeratome to prepare a flap. Posterior bed thickness was measured. The sectioned tissue was stored, and at 24 and 48 hours, pachymetry was repeated. At 48 hours, specular microscopy was repeated, and endothelial cell viability was assessed with trypan blue. Descemet's stripping automated endothelial keratoplasty was performed in 4 patients using eye bank-prepared posterior lamellar tissue. MAIN OUTCOME MEASURES: Corneal tissue was assessed with the following parameters: corneal thickness measured with ultrasonic pachymetry, cell density counts measured with a keratoanalyzer, and cell viability as observed with trypan blue exclusion. Patient outcomes were measured by changes in visual acuity (VA) and the presence of a clear graft. RESULTS: Donor corneal pachymetry before sectioning averaged 599+/-52 microm. Immediately after sectioning with a microkeratome set at a depth of 300 microm, mean posterior bed thickness was 328+/-95 microm. Thus, the mean cutting depth achieved by the microkeratome when set at 300 micrometers averaged 271+/-83 microm. After storage for 24 hours, the posterior beds measured 352 microm, an average swelling of 24 (7%) microm (P = 0.14). After 48 hours, the posterior beds measured 382 microm, an average swelling of 54 (16%) microm (P = 0.02). Cell counts 48 hours after sectioning decreased by an average of 11% (P = 0.10). Endothelial cell staining confirmed improvement in postsectioning morphology and survival with increased technician experience. All 4 patients receiving eye bank-prepared DSAEK tissue showed uncomplicated postoperative results, with improvement in VA. CONCLUSIONS: The microkeratome cutting depth was moderately accurate. Pachymetry, cell density, and cell viability of sectioned tissue after 48 hours in storage were encouraging overall. Initial clinical results of eye bank-prepared DSAEK tissue showed uncomplicated postoperative courses and improved VA. Additional studies are needed to follow the long-term outcomes in the recipients of these tissues.

Reproducibility of endothelial assessment during corneal organ culture: comparison of a computer-assisted analyzer with manual methods.

PURPOSE: To investigate the reproducibility of endothelial assessment of organ-cultured corneas with the computer-assisted Sambacornea analyzer in comparison with manual METHODS: methods. Seven observers of two eye banks determined the endothelial cell density (ECD) of 30 corneas through a grid overlay placed on endothelial photographs using two manual modes, unaided (naked-eye) and pointing (point-out). ECD was measured with the analyzer, first in automated mode, where analysis was completely machine determined, and then in touched-up mode, where the observer selected the analysis zone and corrected poorly drawn cell borders. Interobserver variability of ECD for the different methods was compared. Reproducibility of morphometry parameters was determined for the touched-up mode. RESULTS: Interobserver variability was +/-19.2% (95% confidence interval [CI], 13.0-25.4) and +/-17.6% (95% CI, 11.9-23.3) for the naked-eye and point-out mode, respectively, whereas the touched-up mode gave the least variability of +/-9.6% (95% CI, 6.5-12.7), confirmed by the highest intraclass correlation coefficient of 0.95 (95% CI, 0.91-0.97). Interobserver variability increased with worsening image quality. Manual modes underestimated ECD (naked-eye by a mean 10.7% [SD, 2.9%]; point-out by a mean 6.9% [SD, 2.3%]), whereas the automated mode overestimated ECD by a mean 14.7% (SD, 24.3%). Reproducibility of morphometric parameters by the touched-up mode was acceptable but was influenced by endothelial pleomorphism. CONCLUSIONS: Manual counting shows systematic underestimation of ECD with high interobserver variability. The analyzer in automated mode overestimates ECD and is absolutely unreliable. Detection of cell contours by the specific algorithm, combined with manual correction by a skilled technician, appears to be the most reliable method of ECD and morphometry determination.

A clinical assessment of the anterior eye in arc welders.

BACKGROUND: Corneal irradiation with high doses of ultraviolet-B (UVB) has been shown to damage the corneal endothelium in animals. Human occupational exposure to ultraviolet radiation (UVR) in welding is considered a risk for endothelial damage but the evidence for such an effect is limited. METHODS: External eye photographs and non-contact specular micrographs (Topcon SP2000-P) were obtained from 102 white males aged between 32 and 62 years, 51 being arc welders (with 24 +/- 7 years experience) and 51 office workers. Most welders reported repeated occupational exposure to UVR (that is, welder's 'flashes'). RESULTS: Welders reported a higher level of ocular symptoms and a higher prevalence of pingueculae (47 versus 12 per cent), but only one case with pterygium. The average endothelial cell areas were the same in welders and office workers (398 +/- 55 microm(2) versus 400 +/- 56 microm(2); p = 0.868) as were the endothelial cell density (ECD) values (2,555 +/- 342 cells/mm(2) versus 2,541 +/- 308 cells/mm(2); p = 0.825). ECD decreased with years of welding experience (p < 0.01) but not faster than the decrease in ECD due to age. CONCLUSIONS: Repeated occupational ultraviolet radiation exposure through welding is not associated with any obvious differences in the corneal endothelium. No differences were observed in either ECD or cell polymegethism. Despite the periodic welding flashes, the exposure levels are below those needed to cause damage to the corneal endothelium.