The Intraocular Lens as a Drug Delivery Device: In Vitro Screening of Pharmacologic Substances for the Prophylaxis of Posterior Capsule Opacification.

Purpose: Numerous pharmacologic substances have been proposed for preventing posterior capsule opacification (PCO). The following trial was to compare those drugs to find more suitable options. IOL should then be modified by the pharmaceuticals as a drug-delivery device. Methods: A systematic literature search was performed to identify published substances. FHL-124 was used to determine cell proliferation and toxicity using a dye reduction test (XTT). Prescreened substances showing a reduction on cell growth without being toxic were soaked into an IOL. Those IOL were tested for their effect on PCO in an anterior-segment model and the human ex vivo capsular bag model. Toxicity on a corneal endothelial cell line (CEC-SV40) was determined. Release kinetics of methotrexate from the IOL was measured. Toxicity testing in both cell lines was done in serum-free conditions. All growth assays were exposed to 10% fetal calf serum (FCS)-supplemented medium. Results: The substances inhibited cell growth at the following EC50: caffeic acid phenethyl ester 1.6 ± 0.9 nM, disulfiram 359 ± 33 nM, methotrexate 98.0 ± 29.7 nM, rapamycin 70.2 ± 14.0 pM, and retinoic acid 1.1 ± 0.12 nM. All but disulfiram showed an effect in the anterior segment model when soaked into an IOL. Long-term inhibitory effects in the human capsular bag model were observed for caffeic acid phenethyl ester and methotrexate IOLs. Only methotrexate and disulfiram did not show any toxicity on endothelial cells. Methotrexate was released constantly from the hydrophilic IOL for 2 weeks. Conclusions: We could identify caffeic acid phenethyl ester and methotrexate in vitro as potential candidates for IOL modification for PCO prophylaxis.

Comparison of the performance and safety of 2 ophthalmic viscosurgical devices in cataract surgery.

PURPOSE: To compare the clinical performance and safety of 2 ophthalmic viscosurgical devices (OVDs)-Twinvisc (OVD 1) and Duovisc (OVD 2)-in cataract surgery. SETTING: European multicenter study. DESIGN: Prospective randomized controlled study. METHODS: Patients with cataract had phacoemulsification and intraocular lens implantation in 1 eye. They were randomly assigned to receive OVD 1 or OVD 2. Preoperative and postoperative examinations over 3 months included mean intraocular pressure (IOP), incidence of IOP peaks (≥30 mm Hg and ≥24 mm Hg), endothelial cell count (ECC), corneal thickness, and intraocular inflammation. A subjective evaluation of the OVDs was performed. RESULTS: The study comprised 220 patients. The incidence of IOP peaks and the mean IOP were not statistically significantly different between the 2 groups at any of the follow-up visits. At 6 hours, the incidence of IOP spikes 30 mm Hg or higher was 6.5% and 7.2% in the OVD 1 and the OVD 2 groups, respectively (P = .846). For the IOP spikes 24 mm Hg or higher, the incidence was 16.8% and 25.2%, respectively (P = .128). Three months postoperatively there was no statistically significant difference in ECC and pachymetry between the 2 groups. Mild inflammation was noticed up to 7 days postoperatively after which it resolved in both groups. Subjectively, the OVD 2 was easier to use, whereas the OVD 1 had better cohesive and dispersive properties. CONCLUSIONS: Both OVDs have similar performance and safety profiles in phacoemulsification cataract surgery. No clinically relevant differences were found between the 2 devices regarding transient IOP spikes, mean IOP, corneal endothelium injury, or inflammation.

Ex vivo expansion of bovine corneal endothelial cells in xeno-free medium supplemented with platelet releasate.

Clinical-grade ex vivo expansion of corneal endothelial cells can increase the availability of corneal tissues for transplantation and treatment of corneal blindness. However, these cells have very limited proliferative capacity. Successful propagation has required so far to use very complex growth media supplemented with fetal bovine serum and other xenocomponents. We hypothesized that human platelet releasates rich in multiple growth factors, and in particular neurotrophins, could potentially be a useful supplement for ex vivo expansion of corneal endothelium cells due to their neural crest origin. Platelet releasates were prepared by calcium salt activation of apheresis platelet concentrates, subjected or not to complement inactivation by heat treatment at 56°C for 30 minutes. Platelet releasates were characterized for their content in proteins and were found to contain high amount of growth factors including platelet-derived growth factor-AB (30.56 to 39.08 ng/ml) and brain-derived neurotrophic factor (30.57 to 37.11 ng/ml) neurotrophins. We compared the growth and viability of corneal endothelium cells in DMEM-F12 medium supplemented with different combinations of components, including 2.5%∼10% of the platelet releasates. Corneal endothelium cells expanded in platelet releasates exhibited good adhesion and a typical hexagonal morphology. Their growth and viability were enhanced when using the complement-inactivated platelet releasate at a concentration of 10%. Immunostaining and Western blots showed that CECs maintained the expressions of four important membrane markers: Na-K ATPase α1, zona occludens-1, phospho-connexin 43 and N-cadherin. In conclusion, our study provides the first proof-of-concept that human platelet releasates can be used for ex vivo expansion of corneal endothelium cells. These findings open a new paradigm for ex vivo propagation protocols of corneal endothelium cells in compliance with good tissue culture practices and regulatory recommendations to limit the use of xenogenic materials.

Effect of amniotic fluid on the in vitro culture of human corneal endothelial cells.

The present study was designed to evaluate the effects of human amniotic fluid (HAF) on the growth of human corneal endothelial cells (HCECs) and to establish an in vitro method for expanding HCECs. HCECs were cultured in DMEM-F12 supplemented with 20% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using either FBS- or HAF-containing media. Cell proliferation and cell death ELISA assays were performed to determine the effect of HAF on cell growth and viability. The identity of the cells cultured in 20% HAF was determined using immunocytochemistry (ICC) and real-time reverse transcription polymerase chain reaction (RT-PCR) techniques to evaluate the expression of factors that are characteristic of HCECs, including Ki-67, Vimentin, Na+/K+-ATPase and ZO-1. HCEC primary cultures were successfully established using 20% HAF-containing medium, and these cultures demonstrated rapid cell proliferation according to the cell proliferation and death ELISA assay results. The ICC and real time RT-PCR results indicated that there was a higher expression of Na+/K+-ATPase and ZO-1 in the 20% HAF cell cultures compared with the control (20% FBS) (P < 0.05). The 20% HAF-containing medium exhibited a greater stimulatory effect on HCEC growth and could represent a potential enriched supplement for HCEC regeneration studies.

Excimer laser mushroom penetrating keratoplasty: new technique.

PURPOSE: To evaluate retrospectively the outcomes of 15 consecutive mushroom-shaped penetrating keratoplasties performed by using excimer laser for both the recipient bed and the fresh donor corneas. METHODS: Fifteen eyes of 14 patients who underwent excimer laser mushroom-shaped penetrating keratoplasty from October 13, 2010, to October 14, 2011, were included in our retrospective study. Eight were men and 6 were women, with a mean age of 31.45 ± 6.52 (range 27-65) years. Eleven (73.3%) had postinfective central deep corneal scar; 4 (26.7%) had severe keratoconus with Descemet opacity. RESULTS: The mean follow-up was 11.9 ± 2.7 months. The mean preoperative best-corrected visual acuity (BCVA) was 0.15 ± 0.16; the postoperative BCVA was 0.69 ± 0.24 after 12 months with a mean refractive astigmatism of 1.8 ± 1.1 D. The mean preoperative endothelial cell count of the donor corneas was 2297.0 ± 189.7 cells/mm²; after 12 months, it was 1906.5 ± 165.8 with a decrease of 17.0%. No intraoperative complications occurred. CONCLUSIONS: Our results showed that excimer laser mushroom penetrating keratoplasty is safe. Furthermore, it does not appear to influence the visual outcomes of the penetrating keratoplasty surgery. This technique is useful for those who use an excimer laser.

Long-term cultivation of human corneal endothelial cells by telomerase expression.

The objective of this study was to explore the potential role of human telomerase reverse transcriptase (TERT) in extending the proliferative lifespan of human corneal endothelial cells (HCECs) under long-term cultivation. A primary culture was initiated with a pure population of HCECs in DMEM/F12 media containing 10% fetal bovine serum and other various supplements. TERT gene was successfully transfected into normal HCECs. A stable HCECs cell line (TERT-HCECs) that expressed TERT was established. The cells could be subcultured for 36 passages. Within this line of cells, TERT not only extended proliferative lifespan and inhibited apoptosis but also enhanced the cell line remaining the normal characteristics similar to HCECs. There were no significantly differences in the expression of the pump function related proteins voltage dependent anion channel 3 (VDAC3), sodium bicarbonate cotransporter member 4 (SLC4A4), chloride channel protein 3 (CLCN3), Na(+)/K(+)-ATPase α1, and ZO-1 in the cell line TERT-HCECs and primary HCECs. TERT-HCECs formed a monolayer cell sheet, maintained similar cell junction formation and pump function with primary HCECs. Karyotype analysis exhibited normal chromosomal numbers. The soft agar colony assay and tumor formation in nude mice assay showed no malignant alterations in TERT-HCECs. Our findings indicated that we had established a cell line with its similar phenotype and properties to primary HCECs. Further study of the TERT-HCECs may be valuable in studying the function of the cells in vivo.

Safety of moxifloxacin and voriconazole in corneal storage media on porcine corneal endothelial cells.

PURPOSE: The aim of this study was to evaluate the safety of moxifloxacin and voriconazole as a supplement in corneal storage media for porcine corneal endothelial cells. METHODS: Twenty-eight eyes were divided into 4 groups. In the control group (the C group), corneal buttons were stored for 3 days in Optisol-GS. In the treatment group, the corneal buttons were preserved for 3 days in Optisol-GS mixed with 250 microg/mL moxifloxacin (M group), 100 microg/mL voriconazole (V group), and 250 microg/mL moxifloxacin plus 100 microg/mL voriconazole (MV group). We evaluated the samples via specular microscopy before and after 3 days of preservation. The mean changes of endothelial cell counts were compared among the 4 groups. Scanning electron microscopy was conducted after 3 days of preservation. RESULTS: Before the preservation, the endothelial cell counts did not differ among the 4 groups (P > 0.05). After 3 days of preservation, the endothelial cell count in the MV group was the lowest among the 4 groups (P < 0.05). After 3 days of preservation, the rate of corneal endothelial cell loss in the M and V groups did not differ significantly from the control group (P > 0.05). The rate of endothelial cell loss in the MV group was significantly higher than that of the control group (P < 0.05). Scanning electron microscopy revealed a normal mosaic pattern for the C, V, and F groups, but hexagonality was not preserved in the MV group. CONCLUSION: Preservation in Optisol-GS mixed with moxifloxacin (250 microg/mL) plus voriconazole (100 microg/mL) induced significant toxicity on the endothelial cells in porcine corneas, when compared with the control group.

[Research and development for treating devastating corneal diseases].

In order to develop new therapeutic modalities for corneal diseases, it is essential to combine cutting-edge translational research based upon liberal original ideas obtained from clinical experience with state-of-the-art basic science and technology. Here, I describe seven important research projects on which our group has been working. 1. Elucidation of the pathogenesis in gelatinous drop-like corneal dystrophy(GDLD). Due to loss of function of the tumor-associated calcium signal transducer 2 (TACSTD2), a responsible gene for this dystrophy, tight-junction-related proteins cease to function, resulting in severe corneal epithelial barrier impairment. As a result, various proteins contained in tear fluid continuously penetrate into the corneal stroma, promoting the development of massive amyloid deposits. 2. The development of cultivated mucosal epithelial transplantation: A landmark surgery, involving the transplantation of cultivated mucosal epithelial cells from in vitro to in vivo, now recognized as the next generation of ocular surface reconstruction. We began performing cultivated allocorneal epithelial transplantations in 1999, and cultivated auto-corneal and auto-oral mucosal epithelial transplantations in 2002. These proved to be very effective in the reconstruction of both the corneal surface and the conjunctival fornix. 3. Elucidation of the pathogenesis of Stevens-Johnson syndrome: Studies have shown that there is a close relationship between corneal epithelial stem cell loss and the associated degree of visual impairment. We discovered that a steroid pulse therapy at the acute phase aimed at minimizing stem cell loss is very effective in restoring visual acuity. This implies that inhibition of the cytokine storm is essential for the treatment of acute-phase Stevens-Johnson syndrome. The innate immunity abnormality seems to be heavily involved at the onset of this devastating disease. 4. Elucidation of the involvement of EP3 and toll like receptor 3 (TLR3) in inflammatory ocular surface reactions : We discovered that EP3, one of the prostanoid receptors expressed by ocular surface epithelium, has a dramatic inhibitory effect on ocular surface inflammation in a mouse model. Since EP3 is also expressed in human ocular surface epithelium, and since abnormality of its single nucleotide polymorphisms (SNPs) is involved in some ocular surface inflammatory diseases, we theorized that an allergic reaction may be negatively regulated by EP3 which is predominantly expressed by the ocular surface epithelium. Our findings show that this is similarly true for TLR3, which, conversely, upregulates ocular surface inflammation. 5. Functional regulation of the ocular surface epithelium: Our findings show that intracellular glutathione (GSH) content in the ocular surface epithelium regulates its intracellular redox state. For instance, the GSH content of the conjunctival epithelium decreases in dry eye diseases, yet recovers after the surgical insertion of a punctal plug. Since various amino acids are also heavily involved in the regulation of cellular functions, we investigated the profile of amino acids contained in tear fluids. Our results indicate that there is a marked difference in amino acid profiles between tear fluids and plasma. Furthermore, we found that several amino acids are up-regulated in inflamed eyes, probably due to an oxidative redox response. 6. The development of new therapeutic modalities for corneal edema: We are developing a new therapeutic modality of cultivated corneal endothelial transplantation using methods based on regenerative medicine. For instance, our findings show that cultivated corneal endothelial sheet transplantation in monkeys maintains corneal transparency for at least four years after transplantation. The supplementation of a Rho kinase (ROCK) inhibitor in the culture media produces an excellent result in culturing human corneal endothelium, maintaining a normal-looking endothelial cell morphology. The use of a ROCK inhibitor, both for cultivated endothelial cell injection into the anterior chamber and for use as a topical application, may prove to be a potential tool for the treatment of corneal endothelial dysfunction. 7. The development of a new type of tear function test : The results of our investigations show that the time-dependent changes of tear film lipid layer (TFLL) spread are compatible with the Voigt model of viscoelasticity, and that the initial velocity of the TFLL spread after a blink decreases in proportion to the decrease in tear volume. Thus, a lipid-layer analysis will become an important tear analysis tool. The above are projects representing the way we believe new treatments for severe corneal diseases are heading.

Intracameral voriconazole: in vitro safety for human ocular cells.

Fungal keratitis is a sight-threatening infection of the cornea. It sometimes leads to loss of the eye. Despite an expanding range of fungal pathogens, there are only few therapeutic agents for its treatment available. Voriconazole is a second-generation synthetic triazole with a broad action against yeasts and molds. The current study investigates the safety of voriconazole for intracameral application in a cell culture model. Endothelial toxicity of voriconazole was evaluated in cultured human corneas. Possible toxic effects of voriconazole (10 microg /mL-10mg/mL) in corneal endothelial cells (CEC), primary human trabecular meshwork cells (TMC), and primary human retinal pigment epithelium (RPE) cells were evaluated after 24h and under conditions of inflammatory stress by treatment with tumor-necrosis-factor alpha (TNF-alpha), lipopolysaccharides (LPS), or interleukin-6 (IL-6) and hydrogen peroxide. Toxicity was evaluated by tetrazolium dye-reduction assay, and cell viability was quantified by a microscopic live-dead assay. No corneal endothelial toxicity could be detected after 30 days of treatment with 250 microg /mL of voriconazole. Concentrations up to 1mg/mL had no influence on CEC, TMC, or RPE cell proliferation, or on cell viability when administered for 24h. Hydrogen peroxide exposure did not increase cellular toxicity of voriconazole at concentrations from 10 to 250 microg /mL. After preincubation with TNF-alpha, LPS, or IL-6 for 24h and subsequent voriconazole treatment for 24h, no significant decrease in proliferation or viability was observed. This study showed no significant toxicity for voriconazole on CEC, TMC, RPE cells, or human corneal endothelium when administered in therapeutic concentrations up to 250 microg /mL.

Cultivation of an immortalized human corneal endothelial cell population and two distinct clonal subpopulations on thermo-responsive carriers.

BACKGROUND: Recently, it was possible to show that human corneal endothelial cells (HCEC) can be cultured on thermo-responsive polymer substrates, and can be harvested as entire cell sheets without losing viability. We sought to study HCEC sheet cultivation on such cell culture carriers under serum-free conditions as the next consequential step in developing methods for generation of corneal endothelial cell transplants. METHODS: An immortalized heterogenous HCEC population and two immortalized, clonally grown HCEC lines (HCEC-B4G12 and HCEC-H9C1) were cultured on thermo-responsive substrates under serum-supplemented and serum-free culture conditions. Cell sheets were characterized by phase contrast microscopy and by immunofluorescent staining for ZO-1, Na(+),K(+)-ATPase, and vinculin. RESULTS: All tested HCEC populations were able to adhere, spread and proliferate on thermo-responsive substrates under serum-supplemented conditions. Under serum-free conditions, pre-coating of the polymer substrates with ECM proteins was necessary to facilitate attachment and spreading of the cells, except in the case of HCEC-B4G12 cells. The heterogenous HCEC population formed closed monolayers, properly localized ZO-1 to lateral cell borders, and had moderate vinculin levels under serum-free, and higher vinculin levels under serum-supplemented culture conditions. HCEC-B4G12 cells formed closed monolayers, showed proper localization of ZO-1 and Na(+),K(+)-ATPase to lateral cell borders, and had high vinculin levels irrespective of culture conditions. In contrast, HCEC-H9C1 cells had lowest vinculin levels under serum-supplemented, and higher vinculin levels under serum-free culture conditions. ZO-1 was detected throughout the cytoplasm under both culture conditions. These loosely adherent cells were only able to form a closed monolayer under serum-supplemented conditions. CONCLUSIONS: Serum-free production of HCEC sheets is possible. The extremely adherent clonal HCEC line B4G12 produced higher vinculin levels than the other two tested HCEC populations, and showed strong adherence to the thermo-responsive, polymeric culture substratum irrespective of culture conditions. This cell line closely resembles terminally differentiated HCEC in vivo, and was found to be particularly suitable for further studies on HCEC cell sheet engineering.

Volumetric and ionic regulation during the in vitro development of a corneal endothelial barrier.

Corneal endothelium is responsible for generating an ion flux between the corneal stroma and the anterior chamber of the eye that is necessary for the cornea to remain transparent. However, the ion transport regulatory mechanisms that develop during the formation of the endothelial barrier are not known. In this study, we determined the influence of cell confluence on cell volume and intracellular ionic content on the corneal endothelial cells of rabbits. Our results demonstrate that non-confluent endothelial cells display a hypertrophic volume increase, with higher intracellular contents of potassium and chlorine than those of confluent cells. In contrast, when cells reach confluence and the endothelial barrier forms, cell volume decreases and the intracellular contents of potassium and chlorine decrease. Our genetic analysis showed a higher expression of CFTR and CA2 genes in non-confluent cells, and of the gene KCNC3 in confluent cells. These results suggest that the normal ionic current that keeps the corneal stroma dehydrated and transparent is regulated by cell-cell contacts and endothelial cell confluence, and could explain why the loss of corneal endothelial cells is often associated with corneal edema and even blindness.

Dependence of cAMP meditated increases in Cl- and HCO(3)- permeability on CFTR in bovine corneal endothelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) is present on the apical membrane of corneal endothelial cells. Increasing intracellular [cAMP] with forskolin stimulates an NPPB and glibenclamide-inhibitable apical Cl(-) and HCO(3)(-) permeability [Sun, X.C., Bonanno, J.A., 2002. Expression, localization, and functional evaluation of CFTR in bovine corneal endothelial cells. Am. J. Physiol. Cell Physiol. 282, C673-C683]. To definitively determine that the increased permeability is dependent on CFTR, we used an siRNA knockdown approach. Apical Cl(-) and HCO(3)(-) permeability and steady-state HCO(3)(-) flux were measured in the presence or absence of forskolin using cultured bovine corneal endothelial cells that were transfected with CFTR siRNA or a scrambled sequence control. CFTR protein expression was reduced by approximately 80% in CFTR siRNA treated cultures. Forskolin (10 microM) increased apical chloride permeability by 7-fold, which was reduced to control level in siRNA treated cells. CFTR siRNA treatment had no effect on baseline apical chloride permeability. Apical HCO(3)(-) permeability was increased 2-fold by 10 microM forskolin, which was reduced to control level in siRNA treated cultures. Similarly, there was no effect on baseline apical HCO(3)(-) permeability by knocking down CFTR expression. The steady-state apical-basolateral pH gradient (DeltapH) at 4h in control cultures was increased approximately 2.5-fold by forskolin. In CFTR siRNA treated cells, the baseline DeltapH was similar to control, however forskolin did not have a significant effect. We conclude that forskolin induced increases in apical HCO(3)(-) permeability in bovine corneal endothelium requires CFTR. However, CFTR does not have a major role in determining baseline apical chloride or HCO(3)(-) permeability.

Optimization of culture conditions for porcine corneal endothelial cells.

PURPOSE: To optimize the growth condition of porcine corneal endothelial cells (PCEC), we evaluated the effect of coculturing with a feeder layer (irradiated 3T3 fibroblasts) with the addition of various exogenous factors, such as epidermal growth factor (EGF), nerve growth factor (NGF), bovine pituitary extract (BPE), ascorbic acid, and chondroitin sulfate, on cell proliferation, size, and morphology. METHODS: PCEC cultures were seeded at an initial cell density of 400 cells/cm(2) in the presence or absence of 20,000 murine-irradiated 3T3 fibroblast/cm(2) in the classic media Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 20% fetal bovine serum (FBS). Mean cell size and bromodeoxyuridine incorporation was assessed at various passages. Growth-promoting factors were studies by seeding PCEC at 8,000 cells/cm(2) in DMEM with 20% FBS or Opti-MEM I supplemented with 4% FBS and one of the following additives: EGF (0.5, 5, 25 ng/ml), NGF (5, 20, 50 ng/ml), BPE (25, 50, 100, 200 microg/ml), ascorbic acid (10, 20, 40 microg/ml) and chondroitin sulfate (0.03, 0.08, 1.6%), alone or in combination. Cell number, size and morphology of PCEC were assessed on different cell populations. Each experiment was repeated at least twice in three sets. In some cases, cell cultures were maintained after confluence to observe post-confluence changes in cell morphology. RESULTS: Co-cultures of PCEC grown in DMEM 20% FBS with a 3T3 feeder layer improved the preservation of small polygonal cell shape. EGF, NGF, and chondroitin sulfate did not induce proliferation above basal level nor did these additives help maintain a small size. However, chondroitin sulfate did help preserve a good morphology. BPE and ascorbic acid had dose-dependent effects on proliferation. The combination of BPE, chondroitin sulfate, and ascorbic acid significantly increased cell numbers above those achieved with serum alone. No noticeable changes were observed when PCEC were cocultured with a 3T3 feeder layer in the final selected medium. CONCLUSIONS: Improvements have been made for the culture of PCEC. The final selected medium consistently allowed the growth of a contact-inhibited cell monolayer of small, polygonal-shaped cells.

A novel method of isolation, preservation, and expansion of human corneal endothelial cells.

PURPOSE: To explore new strategies for effective isolation, preservation, and expansion of human corneal endothelial cells (HCECs). METHODS: Human corneal Descemet's membrane and corneal endothelial cells were digested with collagenase A or Dispase II in supplemented hormonal epithelial medium (SHEM) for 1.5 to 16 hours. HCEC aggregates derived from collagenase A digestion were preserved in serum-free medium with low or high calcium for up to 3 weeks. Cryosections of HCEC aggregates were subjected to immunostaining with ZO-1, connexin 43, type IV collagen, laminin-5, and perlecan, and apoptosis was determined by TUNEL or cell-viability assay. For expansion, HCEC aggregates were seeded directly or after brief treatment with trypsin/EDTA in SHEM, with or without additional bovine pituitary extract (BPE), nerve growth factor (NGF), or basic fibroblast growth factor (bFGF). The resultant HCECs were immunostained with ZO-1, connexin 43, and Ki67. RESULTS: Digestion with collagenase A, but not Dispase, of the stripped Descemet's membrane generated HCEC aggregates, which preserved cell-cell junctions and basement membrane components. High cell viability of HCEC aggregates was preservable in a serum-free, high-calcium, but not low-calcium, medium for at least 3 weeks. Brief treatment of HCEC aggregates with trypsin/EDTA resulted in a higher proliferation rate than without, when cultured in SHEM, and the resultant confluent monolayer of hexagonal cells retained cell-cell junctions. However, additional BPE, NGF, or bFGF did not increase cell proliferation, whereas additional BPE or bFGF disrupted cell-cell junctions. CONCLUSIONS: Collagenase A digestion successfully harvested aggregates with viable HCECs that were preservable for at least 3 weeks in a serum-free, high-calcium medium and, with brief trypsin/EDTA treatment, expanded in the SHEM into a monolayer with hexagonal cells that exhibited characteristic cell junctions.

Induction of replication in human corneal endothelial cells by E2F2 transcription factor cDNA transfer.

PURPOSE: Corneal endothelial cells in humans do not replicate to any meaningful extent. Diminishing density of the cell monolayer with age and in the disease states is a major cause of loss of corneal transparency. This study was conducted to test the hypothesis that overexpression of the transcription factor E2F2 results in replication in nonproliferating human corneal endothelial cells. METHODS: Whole human corneas were incubated for 2 hours in a solution of recombinant E1(-)/E3(-) adenovirus incorporating cDNA encoding E2F2 and green fluorescent protein (GFP) under control of a bidirectional promoter and subsequently maintained in ex vivo culture. Control specimens were incubated with an identical virus bearing the GFP sequence only, or virus-free medium. Efficiency of gene transfer and localization was examined by fluorescence microscopy. En face confocal microscopy of the corneal endothelial surface was used to image recombinant E2F2 expression. 5-bromodeoxyuridine (BrdU) incorporation was used to examine progression to the S phase. Changes in density of the corneal endothelium were quantified by specular microscopy and counting of trypan-blue-stained cells. Apoptosis was tested with a TUNEL assay. RESULTS: Recombinant proteins were expressed predominantly in the endothelium and in a high proportion of endothelial cells in the first week after exposure to virus, diminishing thereafter. Compared with the control, transduction with E2F2 resulted in progression from the G(1) to the S phase in a significant number of cells and in increased cell density. Apoptosis was not found to any significant extent. CONCLUSIONS: Overexpression of the transcription factor E2F2 in nonmitotic human corneal endothelial cells results in short-term expression, cell-cycle progression, and increased monolayer cell density.

Vitamins inhibit oxidant-induced apoptosis of corneal endothelial cells.

PURPOSE: To determine the effects of vitamins A, C, and E supplementation on lipid peroxidation and apoptosis in corneal endothelial cells. METHODS: Murine corneal endothelial cells were maintained in tissue culture medium supplemented with free iron ions, known to lead to increased lipid peroxidation. The concentration of antioxidative vitamins (ascorbic acid, tocopherol, and retinoic acid) in the cells and supernatant was determined using reversed-phase high-performance liquid chromatography. Apoptosis was assessed by quantification of caspase-3-like activity, using annexin-V/propidium iodide stains for flow cytometry. Lipid peroxidation was assessed using the malondialdehyde method. Supplementation of antioxidative vitamins was tested in the setting of apoptosis. RESULTS: Increasing levels of free iron led to a rapid loss of antioxidative vitamins in the supernatant and corneal endothelial cells. This was correlated with rising levels of malondialdehyde and increased apoptosis. Supplementation with ascorbic acid or alpha-tocopherol alone was not sufficient to prevent lipid peroxidation in the cells, whereas a combination of vitamins C and E was able to do so. In contrast, supplementation with vitamin A alone significantly reduced oxidative stress and apoptosis. CONCLUSIONS: We present an in vitro model to test the direct influence of vitamin supplementation on corneal endothelial cells with regard to lipid peroxidation and apoptosis. We show that supplementation with antioxidative vitamins of corneal endothelial cells significantly prevents the generation of free-radical injury, lipid peroxidation, and consequent apoptosis.

Increased endothelial survival of organ-cultured corneas stored in FGF-2-supplemented serum-free medium.

PURPOSE: To investigate the effect of FGF-2 on corneal endothelial cell survival in porcine and human corneas during corneal storage in a serum-free medium. METHODS: Porcine and paired human corneas were stored at 32 degrees C for 9 and 22 days, respectively. One cornea of each pair was stored in a serum-free culture medium, and the mate was preserved in the same medium supplemented with 10 ng/mL FGF-2. Quantitative analysis of corneal damage after storage was determined by the Janus green photometry technique. 5-Bromo-2-deoxyuridine (BrdU) labeling of the endothelium determined the effect of FGF-2 on endothelial proliferation during storage. Additional cell culture studies were performed to elucidate the role of FGF-2 on the incidence of endothelial apoptosis after serum deprivation. RESULTS: When FGF-2 was added to the serum-free medium, the damage rates of porcine endothelia were reduced from 15.1% +/- 8.7% (control) to 6.4% +/- 2.0% after 9 days and from 25.3% +/- 10.2% to 13.6% +/- 4.2% after 22 days of storage. In the human corneas stored during 22 days in FGF-2-supplemented medium, the amount of endothelial damage was 11.8% +/- 3.2%, which was significantly less damage than in the control fellow corneas stored in unsupplemented serum-free medium (19.3% +/- 6.3%; P < 0.01). DNA synthesis was not enhanced in corneas stored in serum-free medium, serum-free medium+FGF-2, or medium containing 10% FCS. Only a few (3.8%) TUNEL-positive endothelial cells were detected in cultures maintained in FGF-2-supplemented serum-free medium compared with a high number (48%) of apoptotic cells in control cultures. CONCLUSIONS: FGF-2 efficiently reduces human corneal endothelial damage that occurs during organ culture storage in a serum-free medium. This effect is truly protective, because no proliferative activity and a decreased rate of apoptosis were determined. FGF-2 emerges as an important component of a future serum-free corneal organ-culture medium established to replace fetal calf serum (FCS) as a potential source of recipient infection.

Comparison of the clinical performance of Healon 5 and Healon in phacoemulsification.

PURPOSE: Healon 5 is a high-molecular-mass fraction of sodium hyaluronate. Its density endows it with a number of viscoelastic characteristics. In this prospective, randomised clinical study we compared the performance of Healon 5 and Healon in phacoemulsification. SETTING: Institute of Ophthalmology, University of Modena and Reggio Emilia, Italy. METHODS: Two groups of patients underwent phacoemulsification and intraocular lens (IOL) implantation. In the first 27 patients Healon 5 was used as viscoelastic substance during surgery, and in the second 27 Healon was used. The surgeons subjective comments on the performance of these viscoelastic agents were recorded at the different steps of surgery: injection, capsulorhexis, phacoemulsification, IOL implantation, removal of viscoelastic agent and trasparency throughout the operation. The surgeon's overall impression of the viscoelastics during the whole operation was noted. Tonometry and endothelial cell count were performed in all patients before and after operation. RESULTS: There was no statistical difference between the two groups as regards visual acuity, ocular pressure and endothelial damage. Healon 5 showed excellent ability to maintain the anterior chamber during capsulorhexis, phacoemulsification and IOL implantation. Removal time with Healon 5 was not appreciably longer than Healon. CONCLUSIONS: Healon 5 emerges as a very interesting viscoelastic substance. Visibility is better if the anterior chamber is filled completely. Removal is easier if it is aspirated while moving the irrigation aspiration tip with circular movements over the top and around the border of the IOL.

Lidocaine versus ropivacaine for topical anesthesia in cataract surgery(1).

PURPOSE: To assess the anesthetic efficacy and safety of topical ropivacaine versus topical lidocaine in cataract surgery. SETTING: Institute of Ophthalmology, University of Modena and Reggio Emilia, Modena, Italy. METHODS: This prospective controlled randomized double-blind study comprised 64 patients scheduled for planned routine cataract extraction. Patients were randomized into 2 groups; 1 received topical ropivacaine 1% and the other, topical lidocaine 4%. The duration of surgery, intraoperative and early postoperative complications, and the need for supplemental intracameral anesthesia were recorded. Intraoperative and postoperative subjective pain was quantified by patients using a scale from 1 to 10. An endothelial cell count was performed preoperatively and 2 months after surgery. RESULTS: The mean endothelial cell density decreased from 2334 cells/mm(2) +/- 496 (SD) to 2016 +/- 674 cells/mm(2) in the ropivacaine group and from 2519 +/- 404 cells/mm(2) to 1847 +/- 607 cells/mm(2) in the lidocaine group. The difference in cell density between groups was not significant before (P =.154) or after surgery (P =.329); however, the difference in mean cell loss between groups was statistically significant (P =.031). The duration of surgery and intraoperative complications were the same in both groups. Four patients in the ropivacaine group and 5 in the lidocaine group required supplemental anesthesia (P >.05). The mean subjective analog pain score was slightly higher in the lidocaine group (P >.05). The day after surgery, 12 eyes in the ropivacaine group and 6 in the lidocaine group had transient corneal edema (P =.150). CONCLUSIONS: Topical ropivacaine performed at least as well as topical lidocaine in efficacy and safety in cataract surgery. It provided sufficient and long-lasting analgesia without the need for supplemental intracameral anesthesia in most cases.

Long-term endothelial cell loss after phacoemulsification: peribulbar anesthesia versus intracameral lidocaine 1%: prospective randomized clinical trial.

PURPOSE: To evaluate endothelial cell loss after phacoemulsification with posterior chamber intraocular lens implantation using peribulbar anesthesia or topical anesthesia combined with intracameral unpreserved lidocaine 1%. SETTING: Department of Ophthalmology, Charité, Humboldt-University of Berlin, Berlin, Germany. METHODS: Before and 20 months +/- 5.1 (SD) after surgery, specular microscopy was used to evaluate the number and morphology of endothelial cells in 78 eyes having peribulbar anesthesia or topical anesthesia combined with an intracameral injection of 0.15 cc unpreserved lidocaine 1%. RESULTS: The mean endothelial cell loss was 11.11% in the peribulbar group and 12.55% in the topical/lidocaine group. There was no statistically significant difference in the amount of endothelial cell loss or cell morphology between the 2 groups. CONCLUSION: The long-term postoperative endothelial cell course showed that topical anesthesia combined with an intracameral injection of 0.15 cc unpreserved lidocaine 1% is a safe alternative to peribulbar anesthesia.