Human Cadaver Eyes Research Articles

POSTERIOR HYALOID DETACHMENT AND INTERNAL LIMITING MEMBRANE PEELING ASSISTED BY ANTHOCYANINS FROM ACAI FRUIT (EUTERPE OLERACEA) AND 10 OTHER NATURAL VITAL DYES

The purpose of this study was to determine whether natural dyes facilitate posterior hyaloid detachment (posterior vitreous detachment [PVD]) and retinal internal limiting membrane (ILM) peeling in human eyes. Open-sky vitrectomy with posterior hyaloid and ILM removal was performed in 86 human cadaveric eyes. After core vitrectomy, 11 different dyes were injected into the vitreous cavity to aid hyaloid detachment and ILM removal. The dyes were allowed to settle on the macula for 5 minutes after PVD and were removed by mechanical aspiration. Intraocular forceps were used for ILM peeling, which was confirmed by light microscopy of the peeled tissue. Acai fruit (Euterpe oleracea) extract and 10 additional dyes from plants or animal sources were tested: pomegranate (Punica granatum), logwood (Haematoxylum campechianum), chlorophyll extract from alfalfa (Medicago sativa), cochineal (Dactylopius coccus), hibiscus (Hibiscus rosa-sinensis), indigo (Indigofera tinctoria), paprika (Capiscum annuum), turmeric (Curcuma longa), old fustic (Maclura tinctoria), and grape (Vitis vinifera). The dyes facilitated PVD and ILM peeling. Acai fruit (E. oleracea) extract, logwood (H. campechianum), cochineal (D. coccus), and old fustic (M. tinctoria) facilitated PVD in all cases; dye-assisted PVD was compared with triamcinolone-assisted PVD performed previously in a comparative model. Acai fruit (E. oleracea) extract, cochineal (D. coccus), and chlorophyll extract from alfalfa (M. sativa) showed the best capability for ILM staining; dye-assisted ILM removal was compared with the ILM peeling guided by indocyanine green staining performed previously in a comparative model. Light microscopy confirmed the ILM removal in all cases. Anthocyanin dye of the acai fruit (E. oleracea) and the dyes from cochineal (D. coccus) and chlorophyll extract from alfalfa (M. sativa) resulted in the best capability for posterior hyaloid and ILM staining in human cadaveric eyes and may be a useful tool for vitreoretinal surgery.

Quality of corneal lamellar cuts quantified using atomic force microscopy

To quantify the cut quality of lamellar dissections made with the femtosecond laser using atomic force microscopy (AFM). Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida, USA. Experimental study. Experiments were performed on 3 pairs of human cadaver eyes. The cornea was thinned to physiologic levels by placing the globe, cornea side down, in 25% dextran for 24 hours. The eyes were reinflated to normal pressures by injecting a balanced salt solution into the vitreous cavity. The eyes were placed in a holder, the epithelium was removed, and the eyes were cut with a Visumax femtosecond laser. The energy level was 180 nJ for the right eye and 340 nJ for the left eye of each pair. The cut depths were 200 μm, 300 μm, and 400 μm, with the cut depth maintained for both eyes of each pair. A 12.0 mm trephination was then performed. The anterior portion of the lamellar surface was placed in a balanced salt solution and imaged with AFM. As a control, the posterior surface was placed in 2% formalin and imaged with environmental scanning electron microscopy (SEM). Four quantitative parameters (root-mean-square deviation, average deviation, skewness, kurtosis) were calculated from the AFM images. From AFM, the 300 μm low-energy cuts were the smoothest. Similar results were seen qualitatively in the environmental SEM images. Atomic force microscopy provided quantitative information on the quality of lamellar dissections made using a femtosecond laser, which is useful in optimizing patient outcomes in refractive and lamellar keratoplasty surgeries.

The Effect of Riboflavin–UV-A Treatment on Corneal Limbal Epithelial Cells—A Study on Human Cadaver Eyes

To determine the effect of riboflavin-UV-A treatment on the corneal limbal epithelial cells during a corneal collagen cross-linking (CXL) procedure. Thirty freshly enucleated human cadaveric eyeballs were subjected to a CXL procedure, mimicking the clinical protocol. During the UV-A exposure, one half of the limbus (sector A) was left unprotected, whereas the other half (sector B) was covered by a metal shield. Limbal biopsies from both sectors before and after the procedure were analyzed. Each strip of tissue was divided into 3 segments, for cell count of viable cells, for cultivation on human amniotic membrane (HAM), and for stem cell and differentiated corneal epithelial cell marker studies using reverse transcriptase-polymerase chain reaction. Compared with the cell count before CXL, there was a statistically significant drop in the mean number of viable cells after CXL in sector A but not in sector B. Biopsies from both sectors before CXL and from sector B after CXL showed good growth on HAM. Biopsies from sector A after CXL showed no growth on HAM. The putative stem cell marker ABCG2 was absent in all samples and p63 was absent in 3 of 10 samples taken from sector A after CXL. All markers were present in all samples from sector B after CXL. Riboflavin-UV-A treatment can result in damage to limbal epithelial cells, particularly the stem cells. Covering the limbal region with a metal shield effectively prevents this damage.

Quantitative measurement of wound architecture in microincision cataract surgery

To evaluate whether changes in internal wound architecture after phacoemulsification can be measured quantitatively by analysis of scanning electron micrographs. Baylor College of Medicine, Houston, Texas, USA. Experimental study. Two comparative studies in human cadaver eyes were performed using coaxial small-incision cataract surgery (SICS), bimanual microincision cataract surgery (MICS), coaxial MICS, and a variety of phacoemulsification tips (MicroSurgical Technologies, Microphaco Tapered, Microtip Turbosonics, and Microphaco Mackool). After surgery, the cornea and scleral rims were harvested and digital scanning electron micrographs were taken. The internal corneal wound was analyzed using measurement software to determine the area of endothelial cell loss and length of Descemet membrane tears. Quantifiable differences were observed between combinations of techniques and tip designs. The mean area of endothelial cell loss was 2.93 ± 0.31 mm(2) (SD) after coaxial MICS (n = 4) and 2.85 ± 0.54 mm(2) after bimanual MICS (n = 4). However, after normalizing for differences in tip or sleeve diameter, the area of endothelial cell loss ranged from 1.4-fold to 1.7-fold less with coaxial SICS than with bimanual MICS with Microphaco Tapered, Microtip Turbosonics, and Microphaco Mackool tips. The mean total length of tearing was 1.38 ± 0.38 mm for bimanual MICS (n = 4) and 0.84 ± 0.61 mm for coaxial MICS (n = 4). The length and area of corneal wounds could be quantitated with accuracy and the measurements could be used to make quantifiable comparisons of phacoemulsification techniques and tips. Dr. Dimalanta is an employee of Alcon Research, Ltd. No other author has a financial or proprietary interest in any material or method mentioned.

USE OF LUTEIN AND ZEAXANTHIN ALONE OR COMBINED WITH BRILLIANT BLUE TO IDENTIFY INTRAOCULAR STRUCTURES INTRAOPERATIVELY

To determine whether a natural dye solution based on lutein and zeaxanthin alone or combined with Brilliant Blue stains and facilitates peeling of intraocular membranes in human eyes. In this study of 60 cadaveric eyes, open-sky vitrectomy including posterior hyaloid detachment was performed. Different lutein and zeaxanthin concentrations (0.01-20%) were tested alone or combined with different Brilliant Blue concentrations (0.0125-0.025%) in the corneal endothelium, corneal epithelium, anterior and posterior capsule, vitreous cavity through the macula including the posterior hyaloid, and internal limiting membrane. The various dye solutions were in contact with the intraocular membranes for <1 minute and then were removed by mechanical aspiration or membrane peeling initiated and completed with intraocular forceps. The specimens were examined by light and electron transmission microscopy. Contact between lutein and zeaxanthin and the retinal, lens, and vitreous surface resulted in orange and greenish staining of the intraocular membranes, which facilitated surgical steps in all eyes. Lutein and zeaxanthin alone was useful for vitreous identification and lutein and zeaxanthin combined with Brilliant Blue had strong affinity for internal limiting membrane and anterior capsule. Light microscopy confirmed internal limiting membrane removal in all eyes tested. No dye solutions remained in the eyes after the membrane removal. A natural dye solution based on lutein and zeaxanthin alone or combined with Brilliant Blue efficiently stained the anterior capsule, vitreous, and internal limiting membrane in human cadaveric eyes and may be a useful tool for vitreoretinal or cataract surgery.

Gene Expression Profiles and Retinal Potential of Stem/Progenitor Cells Derived from Human Iris and Ciliary Pigment Epithelium

The aim of our study was to isolate and characterize the properties of neurospheres and differentiated cellular progeny derived from iris and ciliary pigment epithelial (IPE and CPE) cells of human cadaveric eyes. In this study we investigated the gene expression profiles of the stem/progenitor cells and the differentiated progeny derived from IPE and CPE cells, as the changes underlying differentiation of the stem/progenitor derived from the IPE and CPE cells from human cadaveric eye are essentially unknown. The IPE and CPE cells from human cadaver eyes were cultured in the presence of mitogens to generate neurospheres (NS) and the growth characteristics were evaluated. The Neurospheres (NS) were plated under conditions inducing differentiation and their potential was analyzed by RT-PCR, immunocytochemistry, calcium imaging studies and microarray studies to measure the changes involved in the process of differentiation. The IPE and CPE cells can generate NS containing progenitor cells in the presence of mitogens and were capable of producing different retinal cell types as demonstrated by RT-PCR and immunocytochemistry. The cluster analyses of the differentially expressed genes show the dynamic changes that occur during the course of IPE and CPE neurospheres differentiating into retinal progeny. The study gives clues towards the changes that occur during differentiation from NS into retinal progeny. In the present study we have demonstrated the expansion and maintenance of SCs from IPE and CPE of cadaveric eyes. These cells maintain their self-renewal properties and the ability to differentiate along retinal cell lineages and hence could be a practical source of donor cells for ex-vivo stem cell therapy during retinal degeneration.

Evaluation of human sclera after femtosecond laser ablation using two photon and confocal microscopy

Glaucoma is the second-leading cause of blindness worldwide and is often associated with elevated intraocular pressure (IOP). Partial thickness intrascleral channels can be created with a femtosecond laser operating at a wavelength of 1700 nm. Such channels have the potential to increase outflow facility and reduce elevated IOP. Analysis of the dimensions and location of these channels is important in understanding their effects. We describe the application of two-photon microscopy and confocal microscopy for noninvasive imaging of the femtosecond laser created partial-thickness scleral channels in human cadaver eyes. High-resolution images, hundreds of microns deep in the sclera, were obtained to allow determination of the shape and dimension of such channels. This demonstrates that concept of integrating femtosecond laser surgery, and two-photon and confocal imaging has the future potential for image-guided high-precision surgery in transparent and translucent tissue.

Femtosecond Lenticule Extraction (FLEx): Clinical Results, Interface Evaluation, and Intraocular Pressure Variation

To characterize the clinical profile of femtosecond lenticule extraction (FLEx) correlated with ultrastructural analysis of the corneal interface and in vivo real-time intraocular pressure (IOP). Prospective clinical case series with experimental studies; consecutive patients underwent FLEx at a single tertiary center over 10 months with postsurgical follow-up of 3 months. The patients were divided into three groups according to spherical equivalence (SE) (A, < -5.0 diopters [D]; B, ≥ -5.00 D and < -9.00 D; and C, ≥ -9.0 D). Twelve human cadaveric eyes analyzed using scanning electron microscopy after receiving FLEx; 40 rabbit eyes received FLEx with in vivo IOP measurements. The main outcome measures were refractive outcomes from study subjects; with corneal interface and IOP in experimental studies. Thirty-three subjects (22 females, 66.7%) underwent FLEx in both eyes (66 eyes). Mean age was 32 years (range, 21 to 46 years). Preoperative mean SE was -5.77 ± 2.04 D with astigmatism of -1.03 ± 0.72 D. There was a slight hyperopic shift (mean SE 0.14 ± 0.53 D); 94% achieved uncorrected visual acuity ≥20/25 3 months postoperatively. Refractive stability was achieved within 1 month (P < 0.001). Ultrastructurally, the smoothness of the corneal interface was independent of ablation depth (mean irregularity scores A, B, C: 8.8 ± 0.6, 10.3 ± 0.4, 8.7 ± 0.6, respectively; P = 0.88). The increase in IOP during FLEx was similar to that in femtosecond (FS)-LASIK, albeit a twofold duration of raised IOP in FLEx (P < 0.001). These results suggest that FLEx is predictable and effective in treating myopia and myopic astigmatism. Experimental studies support the early clinical results and safety of this procedure.

Age-Related Changes in Venous Endothelial Phenotype at Human Retinal Artery–Vein Crossing Points

To investigate the venous endothelial phenotype at retinal artery-vein (AV) crossings and age-related changes in human cadaveric eyes. Eighteen human donor eyes free of known ocular diseases were divided into two groups according to age (≤30 and >50 years). The central retinal artery was cannulated and perfused with oxygenated Ringer's solution with 1% bovine serum albumin. The perfusate solutions were switched to fixative, membrane permeabilizing solution, and selected labeling solutions for microfilament F-actin and nucleic acid in vascular endothelial cells and smooth muscle cells. The eyes were then immersion fixed and the retinas flat mounted. The venous endothelial cells were examined by confocal microscopy at the AV, pre-AV, and post-AV crossing regions. There was no significant difference between the younger and older groups in endothelial cell length upstream or downstream from an AV crossing. At the AV crossing, the venous endothelial cells were shorter in the younger group and longer in the older group compared with those upstream or downstream from the AV crossing. Stress fibers were not frequently observed in the endothelial cells of younger donors. However, the older group had numerous stress fibers in their endothelia at AV crossing points. Age-related phenotype changes in venous endothelial cells have been identified in the region of AV crossings providing supportive evidence for the hypothesis of age-related and site-specific changes in the vascular endothelial cells as an important factor contributing to the pathogenesis of branch retinal vein occlusion.

Assessment of a single-piece hydrophilic acrylic IOL for piggyback sulcus fixation in pseudophakic cadaver eyes

To evaluate a single-piece hydrophilic acrylic intraocular lens (IOL) designed for sulcus fixation in a piggyback configuration in postmortem pseudophakic human eyes. John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, USA. Experimental study. Pseudophakic human cadaver eyes were imaged by high-frequency ultrasound (Artemis) to assess the overall position of the primary IOL and the sulcus diameter. The piggyback IOL (Sulcoflex) was then injected into the ciliary sulcus of these eyes. After fixation in formalin, they were reevaluated by high-frequency ultrasound for assessment of IOL fixation, fit, centration, tilt, and clearance from the primary IOL and intraocular structures and analyzed after sectioning. Data could be obtained from 11 eyes, all in which the primary IOL was located in the capsular bag. Different foldable IOLs and different degrees of Soemmerring ring formation were represented. The piggyback IOL could be injected and positioned in the ciliary sulcus and had overall appropriate centration and minimum or no tilt. Clearance between the 2 IOLs ranged from 232 to 779 μm, mostly depending on the thickness of the primary IOL. Direct assessment of the sulcus-fixated haptics showed no disturbances to the ciliary processes. The new IOL has large optic and overall diameters, smooth and undulating haptics, and a convex-concave optic profile. Results show that these characteristics minimize the possibility of interaction with the primary IOL and uveal tissues, decreasing the likelihood of optical aberrations and pigmentary dispersion.

Effect of Instrument Orientation on the Accuracy of Intraocular Pressure Measurements in Human Cadaveric Eyes

To determine the effects of probe orientation on the accuracy of intraocular pressure (IOP) measurements obtained with pneumatonometry (Model 30 Classic Pneumatonometer, Reichert Ophthalmic Instruments, Depew, NY) and with a handheld electronic tonometer (Tono-Pen XL, Reichert Ophthalmic Instruments, Depew, NY). Six enucleated human eyes were obtained fewer than 24 hours postmortem. IOP was maintained at 10, 20, and 30 mm Hg, sequentially, via liquid column manometry. At each IOP setpoint, the eyes were positioned to mimic a sitting, supine, and prone patient. Pneumatonometry was performed in the sitting and supine orientations. Tono-Pen measurements were performed in the sitting, supine, and prone orientations. Accuracy was analyzed using multifactor repeated measures analysis of variance, and one-sample t tests. At all IOP setpoints, for both instruments, probe orientation had no significant effect on the IOP measurement (pneumatonometer P=0.58; Tono-Pen P=0.85). At all 3 setpoints (10, 20, and 30 mm Hg) the pneumatonometer overestimated IOP (P<0.0001; P<0.0001; P=0.005, respectively). The Tono-Pen overestimated IOP at the 10 mm Hg setpoint (P<0.0001), but underestimated IOP at the 20 and 30 mm Hg setpoints (P=0.03; P<0.0001, respectively). Under experimental conditions, probe orientation had no significant effect on IOP measurements for either instrument, suggesting that both can be used without correction in the tested orientations. In enucleated human cadaveric eyes, the pneumatonometer overestimated IOP at all setpoints. The handheld electronic tonometer overestimated IOP at 10 mm Hg, but underestimated IOP at the higher setpoints. It is unknown if these findings are generalizable to human eyes in vivo.

Human adult retinal pigment epithelial cells as potential cell source for retina recovery

The retinal pigment epithelium (RPE), as well as the neural retina, develops from the neuroectoderm and plays a key role in photoreceptor functions. Several degenerative eye diseases, e.g., macular degeneration or retinitis pigmentosa, associated with an impaired RPE function cause the loss of the photoreceptor and partial or complete blindness. Cultured RPE cells obtained from human cadaver eyes could be a valuable source for transplantation to cure retinal degenerative diseases. The paper describes RPE cell isolation, maintenance in culture, and immunohistochemical characteristics of dedifferentiated cells. It was found that RPE cells from human adults exhibit neural cell properties in vitro.

Femtosecond laser capsulotomy

To evaluate a femtosecond laser system to create the capsulotomy. Porcine and cadaver eye studies were performed at OptiMedica Corp., Santa Clara, California, USA; the human trial was performed at the Centro Laser, Santo Domingo, Dominican Republic. Experimental and clinical study. Capsulotomies performed by an optical coherence tomography-guided femtosecond laser were evaluated in porcine and human cadaver eyes. Subsequently, the procedure was performed in 39 patients as part of a prospective randomized study of femtosecond laser-assisted cataract surgery. The accuracy of the capsulotomy size, shape, and centration were quantified and capsulotomy strength was assessed in the porcine eyes. Laser-created capsulotomies were significantly more precise in size and shape than manually created capsulorhexes. In the patient eyes, the deviation from the intended diameter of the resected capsule disk was 29 μm ± 26 (SD) for the laser technique and 337 ± 258 μm for the manual technique. The mean deviation from circularity was 6% and 20%, respectively. The center of the laser capsulotomies was within 77 ± 47 μm of the intended position. All capsulotomies were complete, with no radial nicks or tears. The strength of laser capsulotomies (porcine subgroup) decreased with increasing pulse energy: 152 ± 21 mN for 3 μJ, 121 ± 16 mN for 6 μJ, and 113 ± 23 mN for 10 μJ. The strength of the manual capsulorhexes was 65 ± 21 mN. The femtosecond laser produced capsulotomies that were more precise, accurate, reproducible, and stronger than those created with the conventional manual technique.

Ex Vivo Optical Coherence Tomography Imaging of Collector Channels with a Scanning Endoscopic Probe

To achieve high-fidelity optical coherence tomography (OCT) imaging of ex vivo collector channels (CCs) exiting Schlemm's canal (SC) using a paired-angle rotating scanning endoscopic probe. An endoscopic probe was developed to guide an OCT laser beam onto human cadaver eye tissue samples to detect CCs. The prototype probe consisted of two gradient-index (GRIN) lenses that were housed in two stainless steel needles, respectively. The probe scanned the laser beam across a fan shape area by rotating the two GRIN lenses. The authors built a swept source OCT system to provide the depth scans. Human cadaver eye tissue was prepared for imaging. OCT images were acquired while the wall of SC was scanned. After successfully locating the opening of a CC on the SC wall from the OCT images, the authors applied scanning electron microscopy (SEM) to image the sample for comparison. The prototype probe focused the laser beam to a working distance of approximately 1.4 mm (in air), with spot sizes ranging from 12 to 14 μm. The fan shape scan area had a radius of 3 mm and an arc angle of approximately 40°. Acquired OCT images clearly showed a CC opening on the wall of SC with the channel going into the sclera, from which quantitative measurements were made. Results from OCT and SEM show good agreement with each other. The resolving power of the scanning endoscopic probe is sufficient to locate CCs and to observe their shape.

SU‐E‐T‐204: Real‐Time Monitoring of Age‐Related Macular Degeneration Radiosurgery Using Plastic Scintillation Dosimetry

Purpose: Age‐related macular degeneration (AMD) is the leading cause of vision loss and blindness among those over age 65. In its wet form, blood vessels grow from the choroid, causing macular tension and sometimes retinal displacement. As an alternative to monthly direct‐injection methods, Oraya Therapeutics has developed the IRayTM stereotactic radiosurgery device. This table‐top system has been validated in Monte Carlo simulations, human cadaver eye studies, pre‐clinical animal studies, and a phase 1 clinical trial. A plastic scintillation dosimetry (PSD) system was developed to monitor dose delivery in real‐time and provide measurement of total dose delivered. Methods: The IRayTM uses 100 keV x‐rays to deliver 24 Gy of macular dose, divided into three positions along the inferior pars plana. The PSDˈs small volume (0.5 mm diameter and 2 mm height), allows placement within the machine head. The PSD is fiber‐optically coupled to a photomultiplier tube via a plastic waveguide. Before direct characterization of the IRayTM, the preliminary PSD performance was tested using a portable x‐ray unit, mobile c‐arm fluoroscopy unit, and an ion chamber. Results: In order to avoid buffering issues, real‐time measurements used 250 ms bins, but can be adjustable to 10 ms. A calibration factor (CF) from PSD percent‐depth‐dose to ion chamber percent‐depth‐dose was 1.06 at a depth of 1.5 cm in solid water, the typical macular target depth. Counts‐to‐exposure CF varied less than 1% from 0.862 to 15.26R. Counts‐to‐exposure CF remained linear with R2 of 0.999 up to 16R even with varying dose rate (.002 R/s to 1.0 R/s). Conclusions: The PSD demonstrated linear dose measurements even at high dose and high dose rates. This study demonstrates the feasibility, accuracy, and precision of a PSD for clinical implantation in the IRayTM, a device for treatment of AMD.This research was supported by a grant from Oraya Therapeutics, Inc.

Pathology of 157 human cadaver eyes with round-edged or modern square-edged silicone intraocular lenses: Analyses of capsule bag opacification

To assess the degree of capsular bag opacification in human cadaver eyes with silicone intraocular lenses (IOLs), specifically comparing the differences between round-edged IOLs and modern square-edged IOLs. John A. Moran Eye Center, University of Utah, USA. Experimental study. The eyes were immersed in 10% formalin on enucleation. They had anterior segment scanning with a very-high-frequency ultrasound (Artemis). After the eyes were sectioned at the equator, gross examination of the anterior segment was performed from the posterior aspect to assess the degree of capsular bag opacification, coverage of the IOL edge by the anterior capsule, and IOL fixation. Selected eyes also had histopathologic examination. Eighty-seven eyes with a 3-piece round-edged IOL, 43 with a 3-piece square-edged IOL, 26 with a 1-piece plate IOL, and 1 with an accommodating IOL design were included in the analyses of capsular bag opacification. Comparison between 3-piece round-edged IOLs and square-edged IOLs showed statistically significant differences in central posterior capsule opacification (PCO) (P=.0001687) and peripheral PCO (P<.0001). In eyes with square-edged IOLs, PCO had a tendency to start in areas without capsulorhexis coverage of the optic. Twenty-one of 26 eyes with a silicone plate IOL had a neodymium:YAG posterior capsulotomy for dense PCO. This first study using pseudophakic human cadaver eyes that includes a significant number of modern 3-piece silicone IOLs with square optic edges confirmed the role of this design in the prevention of PCO. No author has a financial or proprietary interest in any material or method mentioned.

Pathology of 219 human cadaver eyes with 1-piece or 3-piece hydrophobic acrylic intraocular lenses: Capsular bag opacification and sites of square-edged barrier breach

To assess capsular bag opacification and sites of initial posterior capsule opacification (PCO) in human cadaver eyes with square-edged 1-piece or 3-piece hydrophobic acrylic intraocular lenses (IOLs). John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, USA. Experimental study. Eyes were immersed in 10% formalin after enucleation and had anterior segment scanning with very-high-frequency ultrasound (Artemis). After the eyes were sectioned at the equator, gross examination of the anterior segment was performed from the posterior aspect to assess capsular bag opacification, anterior capsule coverage of the IOL edge, and IOL fixation. Selected eyes had histopathologic examination. One hundred nineteen eyes with 1-piece IOLs and 100 with 3-piece IOLs were included in the analyses of capsular bag opacification. There was no difference in central (P=.29) or peripheral (P=.76) PCO. In 63 of 84 eyes with a 1-piece IOL and peripheral PCO, the optic-haptic junction was the site of initiation. In eyes with a 3-piece IOL, initial peripheral PCO was observed at nearly the same rate whether there was full 360-degree anterior capsulorhexis overlap of the optic or no overlap (P=.13). In the latter, the site of PCO initiation was in areas lacking capsulorhexis coverage in 46% of eyes. There was no difference in central or peripheral PCO between 1-piece and 3-piece hydrophobic acrylic IOLs. With 1-piece IOLs, PCO tended to start at the optic-haptic junctions. With 3-piece IOLs, full anterior capsule coverage did not produce a statistically significant benefit with respect to PCO prevention.

CO2 Laser-assisted Sclerectomy Surgery Part I

To evaluate the safety and performance of a second-generation device for CO2 laser-assisted sclerectomy surgery system in experimental models. Laser-assisted deep sclerectomy using a modified CO2 laser system (OT-134-"IOPtiMate"; IOPtima Ltd, Israel) was performed in 3 experimental models: enucleated pig eyes, human cadaver eyes, and live rabbit eyes. A half-thickness scleral flap was created, and a CO2 laser with a beam-manipulating system was used to achieve deep scleral ablation over the Schlemm canal zone. Aqueous percolation and scleral perforation rates were recorded. Intraocular pressure was monitored in live rabbits up to 21 days postoperatively. The shape and location of the scleral ablation zone, thermal damage, and the healing process were examined by histopathological analysis. Deep scleral ablation and aqueous percolation were repeatedly achieved in all the models. Micro-perforations occurred in 4/18 human eyes (22.2%), in 4/23 rabbit eyes (17.4%), and in none of the 38 porcine eyes. Mean intraocular pressure in the rabbits was significantly decreased (by 6.3±3.6 mm Hg) on the first postoperative day (P<0.0001) and gradually returned to normal. In all but one of the cadaver eyes, effective fluid percolation was achieved. Histology in each case disclosed deep scleral craters with a thin intact sclero-corneal tissue layer at the ablation area. Mild thermal damage, limited to the ablated scleral walls was detected and resolved after 10 days. The results in these experimental studies indicated that CO2 laser-assisted sclerectomy surgery using the OT-134 system is a safe and efficacious procedure for achieving effective fluid percolation.

Human Amniotic Fluid Promotes Retinal Pigmented Epithelial Cells'Trans-Differentiation into Rod Photoreceptors and Retinal Ganglion Cells

To evaluate the effect of human amniotic fluid (HAF) on retinal pigmented epithelial cells growth and trans-differentiation into retinal neurons, retinal pigmented epithelium (RPE) cells were isolated from neonatal human cadaver eye globes and cultured in Dulbecco's modified Eagle's medium-F12 supplemented with 10% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using FBS-containing or HAF-containing media. Amniotic fluid samples were received from pregnant women in the first trimester of gestation. Cell proliferation and death enzyme-linked immunosorbent assays were performed to assess the effect of HAF on RPE cell growth. Trans-differentiation into rod photoreceptors and retinal ganglion cells was also studied using immunocytochemistry and real-time polymerase chain reaction techniques. Primary cultures of RPE cells were successfully established under FBS-containing or HAF-containing media leading to rapid cell growth and proliferation. When RPE cells were moved to in vitro culture system, they began to lose their differentiation markers such as pigmentation and RPE65 marker and trans-differentiated neural-like cells followed by spheroid colonies pertaining to stem/progenitor cells were morphologically detected. Immunocytochemistry (ICC) analysis of HAF-treated cultures showed a considerable expression of Rhodopsin gene (30% Rhodopsin-positive cells) indicating trans-differentiation of RPE cells to rod photoreceptors. Real-time polymerase chain reaction revealed an HAF-dose-dependant expression of Thy-1 gene (RGC marker) and significant promoting effect of HAF on RGCs generation. The data presented here suggest that HAF possesses invaluable stimulatory effect on RPE cells growth and trans-differentiation into retinal neurons. It can be regarded as a newly introduced enriched supplement in serum-free kinds of media used in neuro-retinal regeneration studies.

Fluorophores of lipofuscin granules responsible for human eye fundus autofluorescence

Individual fluorophores and/or their groups contained in a chloroform extract of lipofuscin granules isolated from retinal pigment epithelium of human cadaver eyes were studied by HPLC. Their spectral characteristics were studied, which made it possible to evaluate the contribution of particular fluorophores and/or their groups to the general image of human eye fundus autofluorescence. Many components, being conjugates of all-trans-retinal of different nature, contribute to the total fluorescence spectrum of the chloroform extract. The fluorophore A2E is not predominant.