Cadaver Eyes Research Articles

A new device to noninvasively estimate the intraocular pressure produced during ocular compression.

PurposeTo describe a noninvasive instrument that estimates intraocular pressure during episodes of external globe compression and to demonstrate the accuracy and reliability of this device by comparing it to the intraocular pressures simultaneously and manometrically measured in cannulated eyes.MethodsA thin fluid-filled bladder was constructed from flexible and inelastic plastic sheeting and was connected to a pressure transducer with high pressure tubing. The output of the pressure transducer was sent to an amplifier and recorded. This device was validated by measuring induced pressure in the fluid-filled bladder while digital pressure was applied to one surface, and the other surface was placed directly against a human cadaver eye or in vivo pig eye. The human cadaver and in vivo pig eyes were each cannulated to provide a manometric intraocular pressure control.ResultsThe measurements obtained with the newly described device were within ~5% of simultaneously measured manometric intraocular pressures in both a human cadaver and in vivo pig eye model for a pressure range of ~15–100 mmHg.ConclusionThis novel noninvasive device is useful for estimating the intraocular pressure transients induced during any form of external globe compression; this is a clinical setting where no other devices can be used to estimate intraocular pressure.

Intraocular pressure measurement inside the anterior chamber: a new technical solution and results

To develop a new manometric device for intravital measurement of intraocular pressure (IOP) in the anterior chamber and to assess tonometric data reliability, including post-radial keratotomy (RK) measurements. The experiment was conducted in 2 isolated cadaver eyes, while the clinical study enrolled 20 patients (21 eyes) scheduled for cataract phacoemulsification surgery. Of them, 10 patients (10 eyes) with immature cataract and mild to moderate myopia constituted the control group. The study group consisted of the other 10 patients (11 eyes) with immature cataract, who had undergone RK more than 15 years earlier. The following tonometry methods were used: dynamic bi-directional corneal applanation (ORA, Reichert, USA), dynamic contour tonometry (Pascal tonometer, Zeimer, Switzerland), and rebound tonometry (ICare Pro, Tiolat, Finland). An original device was developed for intravital manometric measurements. Manometric data obtained during the experiment matched the preset pressure in the anterior eye chamber. The median manometry results in the control and study groups were similar and equaled 21.5 and 21.0 mmHg, respectively. Preoperative tonometry readings ranged from 14.9 to 16.5 mmHg in the control group and from 19.7 to 23.3 mmHg - in the study group (with the exception of midperipheral rebound tonometry that showed 15.8 mmHg). The developed device can well be used in experimental research. Midperipheral rebound tonometry was found to be the most informative method for post-RK IOP assessment. Manometry results in the study group mismatched tonometry readings in the controls, which might be due to the specifics of equipment calibration and requires further investigation.

Comparison of Manual, Femtosecond Laser, and Precision Pulse Capsulotomy Edge Tear Strength in Paired Human Cadaver Eyes

To compare the anterior lens capsulotomy edge tear strength created by manual continuous curvilinear capsulorhexis (CCC), femtosecond laser capsulotomy (FSLC), and a new automated precision pulse capsulotomy (PPC) device. A 3-arm study in paired human cadaver eyes. A total of 44 eye specimens from 22 donors in the United States. Capsulotomy was performed in all eye specimens using manual CCC, a femtosecond laser (LenSx, Alcon, Fort Worth, TX), or an automated PPC device (Zepto, Mynosys Inc., Fremont, CA). The first study arm consisted of 8 pairs of eyes in which 1 eye received PPC and the fellow eye received FSLC. The second study arm consisted of 8 pairs of eyes, with 1 eye receiving PPC and the fellow eye receiving manual CCC. The third study arm consisted of 6 pairs of eyes, with 1 eye receiving a manual CCC and the fellow eye receiving FSLC. After phacoemulsification, 2 capsulotomy edge retractors attached to force transducers were used to stretch the capsulotomy edge of each eye and to measure the resisting force until the capsulotomy edge was torn. Capsulotomy edge tear strength in millinewtons. The PPC edge tear strength was greater than that of FSLC for all 8 pairs of eyes by an average factor of 3.1-fold (PPC mean 73.3±24.9 mN vs. femtosecond laser mean 26.1±6.8 mN; P= 0.012, Wilcoxon matched-pairs, signed-ranks test). The PPC tear strength was greater than that of manual CCC for all 8 pairs of eyes by an average factor of 4.1-fold (PPC mean 95±35.2 mN vs. manual CCC mean 29.1±23.1 mN; P= 0.012, Wilcoxon matched-pairs signed-ranks test). There was no significant difference in the tear strength of capsulotomies produced by manual CCC (mean 21.3±4.9 mN) and FSLC (mean 24.5±11.4 mN) (P= 0.75, Wilcoxon matched-pairs signed-ranks test). The strength of the PPC capsulotomy edge was significantly stronger than that produced by femtosecond laser or manual CCC.

Precision Pulse Capsulotomy: Preclinical Safety and Performance of a New Capsulotomy Technology

To assess the preclinical safety and performance of a new precision pulse capsulotomy (PPC) method. Human cadaver eye studies and surgical, slit-lamp, and histopathologic evaluation in a consecutive series of 20 live rabbits. Human cadaver eyes and New Zealand white rabbits. Precision pulse capsulotomy uses a highly focused, fast, multipulse, low-energy discharge to produce a perfectly round anterior capsulotomy instantaneously and simultaneously along all 360°. Capsulotomies are performed using a disposable handpiece with a soft collapsible tip and circular nitinol cutting element. Miyake-Apple imaging and scanning electron microscopy (SEM) of PPC were conducted in human cadaver eyes. Surgical, postoperative slit-lamp, and histopathologic assessments of PPC were performed in 20 live rabbits and were compared with manual continuous curvilinear capsulorrhexis (CCC) in the fellow eye. Anterior chamber (AC) thermocouple temperature measurements were evaluated in a subset of rabbit eyes. Capsulotomy edge circularity, SEM morphologic features and zonular movement with PPC in human cadaver eyes. Anterior chamber temperature during PPC and grading of ocular inflammation, corneal endothelial damage, anterior capsular opacification (ACO), and posterior capsular opacification (PCO). Miyake-Apple imaging showed minimal zonular stress, and thermocouple measurements demonstrated negligible AC temperature changes during PPC. Precision pulse capsulotomy produced round, complete capsulotomies in all 20 rabbit eyes, leading to successful in-the-bag intraocular lens (IOL) implantation. Slit-lamp examinations at 3 days and 1, 2, and 4 weeks after surgery showed no significant differences between PPC and CCC in corneal edema, AC inflammatory reaction, capsular fibrosis, ACO, and PCO. Postmortem studies showed no difference in the corneal endothelium between PPC and CCC eyes. All IOLs were well centered in PPC eyes, and histopathologic analysis showed no greater inflammatory infiltrates. Precision pulse capsulotomy is a new method to automate consistent creation of a perfectly circular anterior capsulotomy with a disposable handheld instrument that can be used in the normal phacoemulsification surgical sequence. Compared with CCC in fellow rabbit eyes, PPC was equally safe and showed no greater zonular stress compared with CCC in human cadaver eyes. Human cadaver eye SEM showed amuchsmoother capsulotomy edge compared to those produced by femtosecond laser.

RPE Cell and Sheet Properties in Normal and Diseased Eyes.

Previous studies of human retinal pigment epithelium (RPE) morphology found spatial differences in density: a high density of cells in the macula, decreasing peripherally. Because the RPE sheet is not perfectly regular, we anticipate that there will be differences between conditions and when and where damage is most likely to begin. The purpose of this study is to establish relationships among RPE morphometrics in age, cell location, and disease of normal human and AMD eyes that highlight irregularities reflecting damage. Cadaveric eyes from 11 normal and 3 age-related macular degeneration (AMD) human donors ranging from 29 to 82 years of age were used. Borders of RPE cells were identified with phalloidin. RPE segmentation and analysis were conducted with CellProfiler. Exploration of spatial point patterns was conducted using the "spatstat" package of R. In the normal human eye, with increasing age, cell size increased, and cells lost their regular hexagonal shape. Cell density was higher in the macula versus periphery. AMD resulted in greater variability in size and shape of the RPE cell. Spatial point analysis revealed an ordered distribution of cells in normal and high spatial disorder in AMD eyes. Morphometrics of the RPE cell readily discriminate among young vs. old and normal vs. diseased in the human eye. The normal RPE sheet is organized in a regular array of cells, but AMD exhibited strong spatial irregularity. These findings reflect on the robust recovery of the RPE sheet after wounding and the circumstances under which it cannot recover.

A novel method for teaching key steps of strabismus surgery in the wet lab

The surgical laboratory environment plays an important role in modern surgical education. Learning how to complete the key steps of strabismus surgery prior to actual surgery may enhance efficiency and sensitize the surgeon to potential complications. Cadaveric extraocular muscle is difficult to manipulate and specimens are not readily available; therefore, an alternative is needed to teach strabismus surgery techniques. We developed a wet lab to practice the key steps of strabismus surgery using commercially available bacon as an extraocular muscle substitute and cadaveric pig eyes for conjunctival and scleral tissue in a framework of peer teaching, expert supervision, and validated written feedback.

Biomimetic Accommodating Intraocular Lens Using a Valved Deformable Liquid Balloon.

Presbyopia is a common age-related condition that prevents people from focusing on near objects. The etiology of presbyopia continues to be debated, but the end effect of all postulated mechanisms is the lack of deformation of the human lens. Using our understanding of the biomechanical properties of the natural human lens, we created a unique accommodating intraocular lens. Although this lens can be used for lenticular disease such as myopia and hyperoperopia, this study addresses the needs of cataract patients with presbyopia. The lens was implanted into presbyopic human cadaver eyes. Focal length of the lens was measured with simulated muscle contraction. Lens dimensions were measured using artificial tissue and a finite-element analysis (FEA) to simulate accommodation. Lens power was measured at various fill volumes. Accelerated soak testing for an equivalent of 7.4years was performed and lens weight and optical transmittance were measured. Previously presbyopic human eyes were able to accommodate between 2.0 and 7.4 diopters after lens implantation. FEA and lens measurements demonstrated a change in curvature of the anterior and posterior portions of the lens during accommodation. After accelerated aging, lens weight remained unchanged and optical transmission was 96%. Lens power increased with fill volume. A deformable liquid lens reversed presbyopia, can be individualized by optically adjusting for each patient, is stable for long periods of time, and is compatible with minimally invasive surgical techniques. A deformable liquid-filled lens can significantly improve accommodation over the presbyopic natural lens.

Comparing consistency of clear corneal incisions using a traditional diamond keratome and a newly designed diamond keratome.

PurposeTo compare the consistency of incision architecture utilizing a traditional diamond keratome and a newly designed diamond keratome.MethodsWe used a traditional diamond keratome and newly designed diamond keratome to create clear corneal incisions in human cadaveric donor eyes. Three surgeons with varying levels of experience made 30 incisions with each keratome; and the wound architecture was measured including incision lengths, epithelial and endothelial widths, and the central epithelial incision’s deviation from a straight line entrance.ResultsThe mean absolute difference in right and left incision lengths (traditional: 0.182 ± 0.158 mm and new 0.088±0.077 mm [P<0.003]), mean absolute difference in epithelial and endothelial incision width (traditional: 0.181±0.144 mm and new 0.080±0.092 mm [P<0.002]), endothelial incision central deviation from a straight line (traditional: 0.128±0.242 mm and new −0.046±0.124 mm [P<0.001]) were all significantly more consistent with the newly designed diamond keratome than with the traditional diamond keratome.ConclusionThe newly designed diamond keratome creates a more consistent clear corneal incision than a traditional diamond keratome across a variety of surgical skill levels and does not require advanced levels of training to achieve this consistency.

Erufosine, a phosphoinositide-3-kinase inhibitor, to mitigate posterior capsule opacification in the human capsular bag model

To determine whether erufosine alone or erufosine-loaded intraocular lenses (IOLs) can inhibit growth of human lens epithelial cells after a single administration in the human capsular bag model. Laboratory for Cell Biology, Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. Experimental study. Sixteen human cadaver eyes had sham cataract surgery. The capsular bag was transferred into cell culture. The tissue was exposed to the half maximum inhibitory concentrations of erufosine alone for 72 hours; solvent-only tissue served as a control. Erufosine is a potent inhibitor of phosphoinositide-3-kinase, a downstream kinase with major implications in posterior capsule opacification (PCO) pathogenesis. The IOLs were soaked with erufosine and implanted in the capsular bags; unsoaked IOLs served as controls. For both settings, the time until confluence of the capsular bag was measured. Cell growth was observed and photodocumented. Erufosine as a single therapeutic agent increased the time until confluence of the capsular bag, but not significantly compared with the control. When IOLs were soaked with erufosine, a long-term prophylactic effect was observed in this organ model for PCO, which is known to closely reflect the clinical situation. Erufosine-soaked IOLs effectively inhibited PCO formation as seen in long-term organ culture and might become of clinical relevance. Drs. Kampik and Eibl-Lindner are inventors of IOLs treated with alkylphosphocholines for pharmacological after-cataract prophylaxis, patent international application PCT/EP2010/051490. No other author has a financial or proprietary interest in any material or method mentioned.

Differences in Unfolded Protein Response Pathway Activation in the Lenses of Three Types of Cataracts.

PurposeTo investigate the activation of three unfolded protein response (UPR) pathways in the lenses of age-related, high myopia-related and congenital cataracts.Methods and MaterialsLens specimens were collected from patients during small incision cataract surgery. Lenses from young cadaver eyes were collected as normal controls. Real-time PCR and Western blotting were performed to detect the expression of GRP78, p-eIF2α, spliced XBP1, ATF6, ATF4 and p-IRE1α in the lenses of normal human subjects and patients with age-related, myopia-related or congenital cataracts.ResultsIn the lenses of the age-related and high myopia-related cataract groups, the protein levels of ATF6, p-eIF2α and p-IRE1α and the gene expression levels of spliced XBP1, GRP78, ATF6 and ATF4 were greatly increased. Additionally, in the congenital cataract group, the protein levels of p-eIF2α and p-IRE1α and the gene expression levels of spliced XBP1, GRP78 and ATF4 were greatly increased. However, the protein and gene expression levels of ATF6 were not up-regulated in the congenital cataract group compared with the normal control group.ConclusionsThe UPR is activated via different pathways in the lenses of age-related, high myopia-related and congenital cataracts. UPR activation via distinct pathways might play important roles in cataractogenesis mechanisms in different types of cataracts.

Intraocular Lens Fragmentation Using Femtosecond Laser: An In Vitro Study.

To transect intraocular lenses (IOLs) using a femtosecond laser in cadaveric human eyes. To determine the optimal in vitro settings, to detect and characterize gasses or particles generated during this process. A femtosecond laser was used to transect hydrophobic and hydrophilic acrylic lenses. The settings required to enable easy separation of the lens fragment were determined. The gasses and particles generated were analysed using gas chromatography mass spectrometer (GC-MS) and total organic carbon analyzer (TOC), respectively. In vitro the IOL fragments easily separated at the lowest commercially available energy setting of 1 μJ, 8-μm spot, and 2-μm line separation. No particles were detected in the 0.5- to 900-μm range. No significant gasses or other organic breakdown by products were detected at this setting. At much higher energy levels 12 μJ (4 × 6 μm spot and line separation) significant pyrolytic products were detected, which could be harmful to the eye. In cadaveric explanted IOL capsule complex the laser pulses could be applied through the capsule to the IOL and successfully fragment the IOL. IOL transection is feasible with femtosecond lasers. Further in vivo animal studies are required to confirm safety. In clinical practice there are a number of large intraocular lenses that can be difficult to explant. This in-vitro study examines the possibility of transecting the lasers quickly using femtosecond lasers. If in-vivo studies are successful, then this innovation could help ophthalmic surgeons in IOL explantation.

Investigation on the concentration of riboflavin in sclera tissue

To investigate the permeability and saturation characteristics of riboflavin in sclera tissue, in order to explore a reasonable dosage regimen in sclera cross-linking. Experimental study. Five human cadaver eyes were also included in this study, every eye was cut into seven sclera stripes and then 0.1% riboflavin solution was dripped to the sclera stripes for 5, 10, 15, 20, 25, 30 minutes respectively. After that, the stripes were made into frozen sections and observed by confocal microscope. To calculate the mean fluorescence density and compare with each other. In addition, 36 New Zealand rabbits were randomly divided into group A and B groups. Each group was divided into six subgroups. In group A, 0.1% riboflavin solution was dripped to the sclera of right eye for 5, 10, 15, 20, 25 and 30 minutes respectively. In group B, 0.5% ribof lavin solution was dripped to the sclera of right eye for 5, 10, 15, 20, 25 and 30 minutes respectively. The left eye were given with physiological saline as blank check. Executed the rabbits to take out the eye and then the nose upper quadrant of the equator sclera was taken to made into frozen sections which were observed by two-photon microscope. To calculate the mean fluorescence density and compare the six subgroups in every group respectively, and compare the subgroups with the same riboflavin-applicated time between group A and B respectively. The fluorescence intensity of sclera gradually strengthened with the longer riboflavin-applicated time, but there was no statistical difference between 20 minutes and the longer time groups (P > 0.05). With the same riboflavin-applicated time in rabbit sclera, the fluorescence intensity of 0.5% concentration group was higher than 0.1% concentration group (P < 0.05). The riboflavin reached an saturation level in the sclera of human cadaver eyes and rabbit eyes after 20 minutes application. The higher concentration of riboflavin-applicated, the higher concentration of riboflavin in the rabbit sclera.

Applied Endoscopic Anatomical Evaluation of the Lacrimal Sac.

Dacryocystorhinostomy (DCR), a popular surgical procedure, has been performed using an endoscopic approach over recent years. Excellent anatomical knowledge is required for this endoscopic surgical approach. This study was performed in order to better evaluate the anatomical features of the lacrimal apparatus from cadavers in the Isfahan forensic center as a sample of the Iranian population. DCR was performed using a standard method on 26 cadaver eyes from the forensic center of Isfahan. The lacrimal sac was exposed completely, then the anatomical features of the lacrimal sac and canaliculus were measured using a specified ruler. A total of 26 male cadaveric eyes were used, of which four (16.7%) were probably non-Caucasian. Two (8%) of the eyes needed septoplasty, one (4%) needed uncinectomy, and none needed turbinoplasty. Four (16%) lacrimal sacs were anterior to axilla, one (4%) was posterior and 20 (80%) were at the level of the axilla of the middle turbinate. The mean difference of distance from the nasal sill to the anterior edge of the lacrimal sac (from its mid-height) was 39.04 (±4.92) mm. The mean difference of distance from the nasal sill to the posterior edge of the lacrimal sac (from its mid-height) was 45.50 (±4.47) mm. The mean of width and length of the lacrimal sac was 7.54 (±1.44) mm and 13.16 (±5.37) mm, respectively. The mean difference of distance from the anterior edge of the lacrimal sac to the posterior edge of the uncinate process was 14.06 (±3.00) mm, while the mean difference of distance from the anterior nasal spine to the anterior edge of the lacrimal sac (from its mid-height) was 37.20 (±5.37) mm.The mean height of the fundus was 3.26 (±1.09) mm. The mean difference of distance from the superior punctum to the fundus was 12.70 (±1.45) mm, and from the inferior punctum to the fundus was 11.10 (±2.02) mm. Given the differences between the various studies conducted in order to evaluate the position of the lacrimal sac, studies such as this can help to better identify the position of lacrimal sac during surgery based on ethnic differences. In addition, these studies can help novice surgeons to better navigate in a surgical scenario.

Cyclosporine A prevents ex vivo PCO formation through induction of autophagy-mediated cell death

The purpose of this study was to determine the Cyclosporine A (CsA) dose and minimum drug delivery time needed to prevent posterior capsule opacification (PCO) in an ex vivo canine model and evaluate the mechanism of CsA-induced cell death. Canine lens epithelial cells (LEC) were treated with CsA and changes in cell migration, proliferation, and density were monitored over time. CsA-treated LEC underwent transmission electron microscopy (TEM), immunofluorescence, and immunoblotting in the presence or absence of autophagy inhibitors to evaluate the mechanism of cell death. Lens capsules were harvested from canine cadaver eyes for an ex vivo model of PCO. Lens capsules were treated with CsA for 1, 2, 3, 4, 5, 6, or 7 days, and subsequently maintained in culture for a total of 28 days in the absence of drug. CsA reduced LEC viability in a dose dependent manner. Morphologically, CsA-treated LEC were swollen, had intact nuclei, lacked peripheral chromatin condensation, and demonstrated prominent vacuolization; TEM revealed autophagosomes. LC3-II protein expression and acridine orange fluorescence increased in CsA-treated cells. A small non-significant induction of cleaved caspase-3 was observed in CsA-treated LEC. Lens capsules treated with 5, 6, or 7 days of 10 μg/mL CsA showed a significant decrease in ex vivo PCO formation; 6 days of drug delivery prevented PCO. This study finds that morphologic changes, formation of acidic vesicles, and increased expression of LC3-II supports the hypothesis that CsA mediates LEC death via autophagy; this is a novel finding in the lens. Induction of CsA-induced apoptosis was minimal. Six days of intracapsular CsA drug delivery prevented ex vivo PCO formation.

Quantitative proteomics analysis by iTRAQ in human nuclear cataracts of different ages and normal lens nuclei.

The goal of this study was to quantitatively identify the differentially expressed proteins in nuclear cataracts of different ages and normal lens nuclei in humans. Forty-eight human lens nucleus samples with hardness grades III, IV were obtained during cataract surgery by extracapsular cataract extraction. Seven normal transparent human lens nuclei were obtained from fresh normal cadaver eyes during corneal transplantation surgery. Lens nuclei were divided into seven groups according to age and optic axis: Group A (average age 80.8 ± 1.2 years), Group B (average age 57.0 ± 4.0 years), Group C average age 80.3 ± 4.5 years), Group D (average age 56.9 ± 4.2 years), Group E (average age 78.1 ± 2.5 years), Group F (average age 57.6 ± 3.3 years) and Group G (seven normal transparent human lenses from normal cadaver eyes, average age 34.7 ± 4.2 years). Water-soluble, water-insoluble, and water-insoluble-urea-soluble protein fractions were extracted from samples. The three-part protein fractions from the individual lenses were combined to form the total proteins of each sample. The proteomic profiles of each group were further analyzed using 8-plex iTRAQ labeling combined with 2D-LC-MS/MS. The data were analyzed with the ProteinPilot software for peptide matching, protein identification, and quantification. Differentially expressed proteins were validated by Western blotting. We employed biological and technical replicates and selected the intersection of the two results, which included 80 proteins. Nine proteins were differentially expressed among the 80 proteins identified using proteomic techniques. In age-related nuclear cataracts (ARNC), the expression levels of fatty acid-binding protein and pterin-4-alpha-carbinolamine dehydratase were upregulated, whereas the levels of alpha-crystallin B chain (CRYAB), GSH synthetase, phakinin, gamma-crystallin C, phosphoglycerate kinase 1, betaine-homocysteine S-methyltransferase 1 (BHMT1), and spectrin beta chain were downregulated. These proteins may be associated with abnormal protein aggregation and oxidative stress. GSH synthetase and CRYAB expression levels in the nuclear cataract decreased with age. The mass spectrometric analysis results were consistent with the Western blot validation. The results indicate that CRYAB and GSH synthetase may be involved in ARNC pathogenesis. iTRAQ combined with 2D-LC-MS/MS provides new methods for future studies of pathological mechanisms and protective drug development for ARNC.

Assessment of Closure Competency of Sutureless Vitrectomy Sclerotomies After Scleral Hydration

Purpose: To assess the influence that hydration applied on the sclerotomy edges may have on incisional closure resistance after transconjunctival sutureless vitrectomy (TSV).Methods: Experimental, randomized and observer-masked study in which 23-gauge TSV was performed in 80 cadaveric pig eyes. Once each vitrectomy was finished, hydration with balanced salt solution (BSS) was applied on the sclerotomy edges of one of the superior incision sites; no maneuver was performed on the other superior sclerotomy. Intraocular pressure (IOP) was gradually increased by means of the vitrectomy system (Accurus; Alcon Laboratories, TX) until one of the superior sclerotomies opened, allowing internal ocular solution to escape.Results: In 45% of cases (36 of 80 eyes), sclerotomies subjected to hydration allowed intraocular fluid escape (p = 0.43). There were no differences when comparing opening pressure values of hydrated and non-hydrated sclerotomies (p = 0.19).Conclusions: Scleral hydration did not demonstrate increase in the sclerotomy closure resistance in our experimental model. Given the widespread use of sutureless TSV around the world, the results obtained in our research, in spite of being negative, may contribute to the knowledge of the behavior of sutureless sclerotomies.

Ultrastructural Changes in Human Trabecular Meshwork Tissue after Laser Trabeculoplasty.

Purpose. To compare morphologic changes in human trabecular meshwork (TM) after selective laser trabeculoplasty (SLT) and argon laser trabeculoplasty (ALT). Design. Laboratory evaluation of ex vivo human eye TM after laser trabeculoplasty. Methods. Corneoscleral rims from human cadaver eyes were sectioned and treated with varying powers of either SLT or ALT. Specimens were examined using light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Results. TEM of SLT at all powers resulted in disrupted TM cells with cracked and extracellular pigment granules. SEM of SLT samples treated at high power revealed tissue destruction with scrolling of trabecular beams. SEM of ALT-treated tissue showed increasing destruction with exposure to higher power. The presence or absence of “champagne” bubbles during SLT did not alter the histologic findings. Conclusions. SLT-treated human TM revealed disruption of TM cells with cracked, extracellular pigment granules, particularly at higher treatment powers. Tissue scrolling was noted at very high SLT energy levels. ALT-treated tissue showed significant damage to both the superficial and deeper TM tissues in a dose-dependent fashion. Further studies are needed to guide titration of treatment power to maximize the IOP-lowering effect while minimizing both energy delivered and damage to target tissues.

Standardization of Human Corneal Endothelial Cell Isolation and the Use of Denuded Human Amniotic Membrane as a Scaffold for Human Corneal Endothelial cells

Objectives: To standardize the isolation of human corneal endothelial cells (HCECs) and to use the denuded human amniotic membrane (HAM) as a scaffold for isolated HCECs. Methods: Human amniotic membrane was denuded using 1.2 units/ml of Dispase II at 37°C for 60 minutes followed by mechanical scraping. Corneal endothelial and Descemet’s membrane sheets were peeled from human donor cadaveric eyes unfit for surgical use and enzymatically digested with 2 mg/ml of collagenase II solution at 37°C and 5% CO2 for 2 hrs. Isolated cells were resuspended in culture medium with supplements and plated onto uncoated cultureware for four hours to eliminate fibroblasts which adhere more rapidly than endothelial cells. After preplating, the non-adherent cells were seeded onto gelatin coated dishes or onto denuded amniotic membrane in OptiMEM media supplemented with growth factors. The cells were analyzed by microscopy for adherence and polygonal morphology. Results: Microscopy of the denuded amniotic membrane showed no epithelial cell remnants. Enzymatic digestion of cornea left behind acellular Descemet’s sheets with the endothelial cells floating individually or in clusters with preplating aiding in a more fibroblast free endothelial cell isolation. Few isolated cells managed to scaffold onto the amniotic membrane and retain that adhesion during subsequent media replacements. Conclusion: Prolonged Treatment of HAM using the mild enzyme Dispase-II results in denuding the membrane, which serves as a successful scaffold for harvested corneal endothelial cells. This approach may be further explored as a cost effective alternative for endothelial cell proliferation and as an in vitro model for corneal tissue engineering studies.

EGFR inhibitor Gefitinib attenuates posterior capsule opacification in vitro and in the ex vivo human capsular bag model

Posterior capsule opacification (PCO) occurs as a common complication after cataract surgery. Gefitinib is a selective inhibitor of the epidermal growth factor receptor (EGFR) which represents a potential pharmacological target for PCO prevention. In this in vitro study, we assessed the effect and biocompatibility of Gefitinib in PCO prophylaxis. The effect of Gefitinib on the key pathological features of PCO was assessed in vitro. We determined growth in the human capsular bag model, prepared from sixteen cadaver eyes that underwent sham cataract surgery. Furthermore, two lens epithelial cell lines, HLE-B3 and FHL-124, were used to determine concentration-based effects on cell proliferation. In addition, cell-migration, matrix-contraction, and cell spreading were investigated. To exclude toxic concentrations, Gefitinib was assessed for its biocompatibility on six different human ocular cell types from the anterior and posterior segment of the eye. Gefitinib significantly increased the time until confluence of the capsular bag compared to controls (p < 0.001)). In both human lens epithelial cell lines (HLE-B3 and FHL-124), proliferation decreased significantly and as equally strong after incubation with Gefitinib (p < 0.001), as did chemotactic migration (p = 0.004), matrix contraction (p = 0.001), and cell-spreading (p = 0.001). At the IC50 concentration, Gefitinib was well tolerated by six different human ocular cell types of the anterior and posterior segment. The specific EGFR inhibitor Gefitinib might become of clinical relevance in PCO prophylaxis as it attenuated cellular growth and other pathological PCO factors in the ex vivo human capsular bag model and in two human lens epithelial cell lines, while showing good biocompatibility in vitro.

Changes in spectral properties and composition of lipofuscin fluorophores from human-retinal-pigment epithelium with age and pathology.

Fundus autofluorescence mostly originates from bisretinoid fluorophores in lipofuscin granules, which accumulate in retinal-pigment-epithelium cells with age. The dynamics of accumulation, photo-oxidation, and photodegradation of bisretinoids during aging or in the presence of pathology have been insufficiently investigated. Changes in spectral properties and composition of human lipofuscin-granule fluorophores with age and pathology have now been investigated by a high-performance liquid chromatography method using spectrophotometric and fluorescent detectors connected in series. It was found that: (i) N-retinylidene-N-retinylethanolamine (A2E) fluorescence intensity is not predominant in the chloroform extract of human-cadaver-eye retinal pigment epithelium studied; bisretinoid photo-oxidation and photodegradation products have much higher fluorescent properties; (ii) the relative emission maximum in the fluorescence spectrum of suspended retinal-pigment-epithelium cells obtained from an individual human-cadaver eye without pathology is irrespective of donor age and falls within the range 575 ± 15nm; in two cadaver eyes with signs of age-related macular degeneration, emission maxima were shifted by 23-36nm towards the shortwave region; and (iii) the ratio of bisretinoid photo-oxidation and photodegradation products to unoxidized bisretinoids in the chloroform extract of cadaver-eye retinal pigment epithelium increases with donor age, from 0.69 ± 0.03 to 1.32 ± 0.04. The differences in fluorescence properties between chloroform extracts obtained from cadaver eyes with and without signs of age-related macular degeneration could be used to increase the potential of fundus autofluorescence imaging as a noninvasive diagnostic method.