Indocyanine green increases light-induced oxidative stress, senescence, and matrix metalloproteinases 1 and 3 in human RPE cells.

PURPOSE: Indocyanine green (ICG) is a commonly used vital dye for macular surgery. Recent reports implicate that its use might be associated with less favourable results regarding postoperative visual outcome and damage of retinal cells, and atrophic degeneration of the retinal pigment epithelium (RPE) has been described. This study investigates the effects of ICG on light-induced senescence of RPE cells. METHODS: Primary human RPE cells were either pre-incubated with ICG in concentrations of 0.005% and 0.05% or not and then exposed to white light. After 10 min of irradiation viability, induction of intracellular reactive oxygen species (ROS) and senescence-associated ß-galactosidase activity (SA ß-Gal) were determined. Expression and secretion of matrix metalloproteinases (MMPs) 1 and 3 and their mRNA were determined by RT-PCR and ELISA. RESULTS: Light exposure decreased RPE cell viability by 46%. Treatment with 0.005% and 0.05% ICG alone decreased RPE cell viability by 7% and 21%. In addition, expression of ROS, SA ß-Gal, and MMP-1 and 3 was significantly increased. When 0.005% and 0.05% ICG treatments were combined with light exposure, viability decreased by 69% and 82% compared to the untreated control. Effects on the expression of ROS, SA ß-Gal, and MMP-1 and 3 were, depending on the ICG dose, significantly increased when cells were pre-incubated with ICG and then illuminated. CONCLUSION: In this study, pretreatment with ICG significantly increased light-induced oxidative stress and senescence. This might indicate a potential, supplementary mechanism that could explain RPE alterations and reduced functional results after ICG-assisted internal limiting membrane peeling.

Intracameral moxifloxacin: in vitro safety on human ocular cells.

PURPOSE: The fourth-generation fluoroquinolone, moxifloxacin, covers most gram-positive and gram-negative isolates causing endophthalmitis. It is safe and effective for systemic and topical use, but only limited data are available on prophylactic intracameral administration to prevent endophthalmitis. This study uses a cell culture model to investigate the safety of moxifloxacin for intracameral application. METHODS: Endothelial toxicity of moxifloxacin was evaluated in cultured human corneas. Possible toxic effects of moxifloxacin (10-750 microg/mL) in corneal endothelial cells (CEC), primary human trabecular meshwork cells (TMC), and primary human retinal pigment epithelial (RPE) cells were evaluated after 24 hours and under conditions of oxidative and inflammatory stress by treatment with tumor necrosis factor alpha, lipopolysaccharides, or interleukin-6. Toxicity was evaluated by tetrazolium dye reduction assay, and cell viability was quantified by a microscopic live-dead assay. RESULTS: No corneal endothelial toxicity could be detected after 30 days of treatment with 500 microg/mL moxifloxacin. Concentrations up to 150 microg/mL had no influence on CEC, TMC, or RPE cell proliferation or on cell viability when administered for 24 hours. After preincubation with tumor necrosis factor alpha, lipopolysaccharides, or interleukin-6 for 24 hours and subsequent treatment with moxifloxacin at concentrations from 10 to 150 microg/mL for 24 hours, no significant decrease in proliferation or viability was observed. Hydrogen peroxide exposure did not increase cellular toxicity. CONCLUSIONS: This study showed no significant toxicity for moxifloxacin on CEC, TMC, RPE cells, or human corneal endothelium for concentrations up to 150 microg/mL. The minimum inhibitory concentration of moxifloxacin to inhibit 90% of pathogens commonly encountered in endophthalmitis is known to be in the range of 0.25-2.5 microg/mL. Therefore, prophylactic intracameral use of moxifloxacin at concentrations up to 150 microg/mL may be safely used to prevent endophthalmitis after intraocular surgery.

Cytoprotective effects of a blue light-filtering intraocular lens on human retinal pigment epithelium by reducing phototoxic effects on vascular endothelial growth factor-alpha, Bax, and Bcl-2 expression.

PURPOSE: To compare the possible protective effects of the ultraviolet (UV)-filtering and blue light-filtering SN60AT intraocular lens (IOL) and the untinted UV-filtering SA60AT IOL with regard to light-induced stress on human retinal pigment epithelium (RPE). SETTING: Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. METHODS: Primary human RPE cells were exposed to white light, and a tinted or untinted IOL was placed in the light beam. After 15 to 60 minutes of irradiation, cell viability was determined by a colorimetric test (tetrazolium dye-reduction assay) and a microscopic live/dead assay. The expression of vascular endothelial growth factor-alpha (VEGF-alpha), Bax, and Bcl-2 and their mRNA was determined by reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting. RESULTS: Without an IOL, white-light exposure decreased cell viability compared with the decrease with the nonirradiated control in a time-dependent manner. Light-induced cell death was significantly reduced by both the tinted IOL and untinted IOL. The combined UV and blue-light filtering attenuated light-induced cell damage significantly more than UV filtering alone. Results of RT-PCR and Western blotting showed a significant time-dependent decrease in Bcl-2 and increase in Bax and VEGF-alpha that were significantly less with the tinted IOL than with the untinted IOL. CONCLUSIONS: Both IOLs reduced light-induced RPE damage. The UV- and blue light-filtering IOL reduced damage more than the conventional IOL. This supports the hypothesis that blue light-filtering IOLs may prevent retinal damage in clinical use.

Capsular tension ring-based in vitro capsule opacification model.

PURPOSE: To evaluate a capsular tension ring (CTR)-supported anterior and posterior capsule opacification (PCO) model in cadaver eyes. The effect of CTR designs on lens capsule shape and lens epithelial cell (LEC) growth were investigated in vitro. SETTING: Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. METHODS: Following open-sky extracapsular cataract extraction, CTR models were implanted in 32 eyes of 16 human donors. The lens capsule expansion by the CTRs was evaluated. The capsular bags supported by the CTRs were excised and maintained at physiological conditions for up to 3 months. The area of LEC coverage over the posterior capsule surface was objectively determined twice a day using a graticule. RESULTS: After CTR implantation, all lens capsules could be safely excised and transferred into organ culture. The CTR designs resulted in different shapes of lens capsule expansion. Complete LEC confluence occurred after a mean of 8.25 days+/-2.87 (SD) with the AcriRing KR10 (AcriTec), 6.50+/-1.0 days with the Acrimed, 8.62+/-3.34 days with the InjectoRing (Corneal), 9.00+/-1.87 days with the Morcher 14C, 9.33+/-0.75 days with the Morcher 2A, and 6.25+/-0.5 days with the Ophthalmic Innovation CTR. CONCLUSION: The CTR-supported in vitro PCO model offers a physiological method to support the lens capsule and is a reproducible system for the study of LEC proliferation.

Intravitreal bevacizumab injections for treatment of central retinal vein occlusion: six-month results of a prospective trial.

PURPOSE: To evaluate the effect of intravitreal bevacizumab (Avastin; Genentech, Inc., South San Francisco, CA) injections on visual acuity and foveal retinal thickness in patients with central retinal vein occlusion (CRVO). METHODS: In this prospective, noncomparative, consecutive, interventional case series, 46 patients received repeated intravitreal injections (1.25 mg) of bevacizumab. Main outcome measures were visual acuity (Snellen and ETDRS charts) and optical coherence tomography measurements in a 6-month follow-up period. RESULTS: Mean visual acuity improved from 20/250 at baseline to 20/80 at the 6-month follow-up (P < 0.001). ETDRS chart findings revealed a mean letter gain +/-SD from baseline to 6 months of 13.9 +/- 14.4 letters. Mean central retinal thickness +/-SD decreased from 535 +/- 148 microm at baseline to 323 +/- 116 microm at the 6-month follow-up. Ischemic CRVO was associated with significantly lower visual acuity than nonischemic CRVO (P < 0.001). However, visual acuity gain was similar in both groups. Independent of duration of symptoms, CRVO was associated with a similar gain in visual acuity. CONCLUSION: Intravitreal injection of bevacizumab appears to be a new treatment option for patients with macular edema secondary to CRVO.

Pulsed electron avalanche knife: new technology for cataract surgery.

BACKGROUND: The pulsed electron avalanche knife (PEAK-fc) is a new pulsed electrosurgical device that allows for precise, "cold" and traction-free tissue dissection. AIM: To evaluate the surgical applicability, safety and potential complications of PEAK-fc in complicated cataract surgery. METHODS: The study included five children with congenital cataracts, two patients with advanced senile cataracts, six adults with mature cataracts, three of them with posterior iris synechia, three patients with post-traumatic cataracts with zonulolysis, one patient with intumescent traumatic cataract and three patients with massive anterior capsule opacification. Anterior and posterior capsulotomies, iris synechiolysis, dissection of anterior capsule opacification and fibrotic scar tissue were performed. PEAK-fc was set at voltages of 500-700 V, pulse duration of 0.1 m and repetition rate of 40-100 Hz. RESULTS: Anterior and posterior capsulotomies were successfully and safely performed in all eyes. The edges of capsulotomies appeared sharp, showing only limited collateral damage. PEAK-fc worked best by just gently touching the capsule, thereby avoiding tractional forces or pressure on the lens capsule. Posterior iris synechiae could be released and anterior capsule opacification was dissected without complications. CONCLUSIONS: PEAK-fc is a very helpful cutting device for complicated cases of cataract surgery, especially for mature and congenital cataracts, traumatic zonulolysis or anterior segment complications after intraocular inflammation.

Indications and clinical outcome of capsular tension ring (CTR) implantation: A review of 9528 cataract surgeries.

BACKGROUND: To report the indications and clinical outcomes of all capsular tension ring (CTR) implantations in a large series of consecutive cataract surgeries during a five year interval in a university eye hospital. METHODS: The study was designed as a restrospective analysis of a consecutive series of 9528 cataract surgeries. The records were checked for cases in which a CTR was implanted. The indications and clinical outcomes of CTR implantation were documented and an evaluation of posterior chamber intraocular lens (PCIOL) insertion, position, and centration. RESULTS: In this series, a CTR was implanted in 69 eyes of 67 patients. The indications were advanced or mature cataract in 40, post-traumatic cataract in 23, pseudoexfoliation syndrome in 4 and subluxated lens in 2 eyes. PCIOL implantation in the capsular bag was possible in 61 (90%) of these 69 eyes. In 5 (7%) additional eyes, PCIOL implantation in the ciliary sulcus was accomplished. In one eye (1%) no IOL implantation was performed because of high myopia. In only two of 69 eyes (2%), an anterior chamber intraocular lens had to be inserted despite prior CTR implantation. In 5 eyes (5%), a slight dislocation of the IOL was noted postoperatively, but none of these patients complained of visually relevant symptoms (eg, monocular diplopia). CONCLUSIONS: According to our experience CTRs are used very infrequently (0.7%), but remain useful in cataract surgeries with difficult preoperative or intraoperative conditions. If zonulolysis is less than two quadrants in extent, implantation of a PCIOL was possible in 98% of cases. Implantation of CTRs with special designs may have additional advantages (eg, inhibition of posterior capsule opacity) and warrant further investigation.

Keratinocyte transglutaminase in proliferative vitreoretinopathy.

PURPOSE: Proliferative vitreoretinopathy (PVR) is an excessive wound-healing process and the major complication in rhegmatogenous retinal detachment. The present study was designed to investigate the expression of keratinocyte transglutaminase (kTgase) in PVR membranes and retinal pigment epithelial (RPE) cells and to evaluate its expression in response to growth factors known to be increased in PVR disease. METHODS: Distribution of kTgase and its relation to fibronectin have been investigated immunohistochemically. PVR membranes and native and cultured RPE cells were analyzed by RT-PCR for the presence of kTgase mRNA. In vitro, RPE cells were treated with transforming growth factor (TGF)-beta2, basic fibroblast growth factor, interleukin-6, and interleukin-1beta. Expression of kTgase was studied by Northern and Western blot analysis. The effect of connective tissue growth factor (CTGF) silencing on the TGF-beta2-modulated expression of kTgase was investigated by transfection with CTGF small interfering (si)RNA before TGF-beta2 treatment. RESULTS: mRNA expression of kTgase was present in all PVR membranes. Immunohistochemical staining for kTgase showed an inhomogeneous pattern with localized accumulation and little colocalization with fibronectin. Although native RPE cells contained only a basal level of kTgase mRNA, the expression of kTgase was increased under culture conditions and was further enhanced by TGF-beta2 treatment. Silencing of CTGF expression did not attenuate the TGF-beta2-mediated induction of kTgase. CONCLUSIONS: The findings demonstrate that kTgase is present in PVR membranes. Its amount is related to the differentiation state of RPE cells and stimulation by TGF-beta2. TGF-beta2-mediated increase seems to be independent of CTGF.

Persistent serous retinal detachment after radial optic neurotomy.

We report the case of a patient presenting with serous retinal detachment following radial optic neurotomy for central retinal vein occlusion. Initially, the retinal detachment was successfully treated by a second vitrectomy and laser coagulation. After reabsorption of the gas tamponade, a recurrence of the retinal detachment was seen with no detectable retinal break. Although other mechanisms should be taken into account, this case indicates that it is possible to create a fistula between the subarachnoid and subretinal space. We conclude from this observation that special attention should be paid to the depth of the radial incision into the optic nerve head.

Potential role of tissue transglutaminase in glaucoma filtering surgery.

PURPOSE: Scarring of the filtering bleb site is the main cause of failure in glaucoma filtration surgery. In the present study, the role of tissue transglutaminase (tTgase) in the accumulation of extracellular matrix (ECM) proteins in these scars was investigated. Transglutaminases are enzymes capable of cross-linking ECM proteins to proteolysis-resistant complexes. METHODS: Expression of tTgase, its reaction product epsilon-(gamma-glutamyl)-lysine, and fibronectin and their colocalization were investigated immunohistochemically in failed blebs and in an in vitro trabeculectomy model. Failed blebs were analyzed by RT-PCR for the presence of tTgase mRNA. Human Tenon fibroblasts (HTFs) were treated with transforming growth factor-beta2 (TGF-beta2). The effect was studied with immunohistochemistry, Northern blot analysis, and Western blot analysis. tTgase activity was assayed by incorporation of biotinylated cadaverine into fibronectin. RESULTS: Expression of tTgase and epsilon-(gamma-glutamyl)-lysine was present in all failed blebs. Staining was most prominent at the rim of the Tenon cyst. In the in vitro trabeculectomy model, tTgase and epsilon-(gamma-glutamyl)-lysine were barely present at the incision side of the flap but were perspicuously increased by TGF-beta2 treatment. Enzyme and its reaction product were colocalized with fibronectin. Cultured HTFs contained a basal level of tTgase mRNA. After treatment with TGF-beta2, expression and activity of tTgase significantly increased. CONCLUSIONS: The findings demonstrated that tTgase is present and functionally active in failed blebs. Expression and activity of tTgase appeared to be stimulated by TGF-beta2, a growth factor known to be increased in primary open angle glaucoma. Intervention at this pathway might open a new approach to prevent scarring after glaucoma filtration surgery.

Pulsed electron avalanche knife for capsulotomy in congenital and mature cataract.

The pulsed electron avalanche knife (PEAK-fc, Carl Zeiss Meditec) is an electrosurgical cutting device that allows precise "cold" and traction-free tissue dissection. We describe its applicability and safety for anterior capsulotomy in a child with congenital cataract and an adult patient with mature cataract. The PEAK-fc was set at a voltage of 600 V and a pulse repetition rate of 80 Hz. Anterior capsulotomies were successfully and safely performed in both cases, with the edges of capsulotomies appearing sharp and showing only limited collateral damage. The PEAK-fc appears to be a helpful cutting device for complicated cases of cataract surgery, especially for mature and congenital cataracts.

Differential protein profiling of primary versus immortalized human RPE cells identifies expression patterns associated with cytoskeletal remodeling and cell survival.

Functional research of retinal pigment epithelium (RPE) most often relies on utilization of RPE-derived cell lines in vitro. However, no studies about similarities and differences of the respective cell lines exist so far. Thus, we here analyze the proteome of the most popular RPE cell lines: ARPE-19 and hTERT and compare their constitutive and de novo synthesized protein expression profiles to human early passage retinal pigment epithelial cells (epRPE) by 2-D electrophoresis and MALDI-TOF peptide mass fingerprinting. In all three cell lines the baseline protein expression pattern corresponded well to the de novo synthesized cellular proteome. However, comparison of the protein profile of epRPE cells with that of hTERT-RPE cells revealed a higher abundance of proteins related to cell migration, adhesion, and extracellular matrix formation, paralleled by a down-regulation of proteins attributed to cell polarization, and showed an altered expression of detoxification enzymes in hTERT-RPE. ARPE-19 cells, however, exhibited a higher abundance of components of the microtubule cytoskeleton and differences in expression of proteins related to proliferation and cell death. epRPE cells, hTERT-RPE, and ARPE-19 therefore may respond differently with respect to certain functional properties, a finding that should prove valuable for future in vitro studies.

Immunohistochemical findings after LASIK confirm in vitro LASIK model.

PURPOSE: To compare immunohistochemical findings in human donor corneas after successful laser in situ keratomileusis (LASIK) without clinical complications with a recently established human LASIK in vitro model. METHODS: Donor corneas with prior LASIK treatment were investigated. Cryostat sections were stained immunohistochemically for collagen types I, III, and VI and laminin and fibronectin. RESULTS: With light microscopy, the interface of the LASIK flap could hardly be detected. In all samples, fibronectin was consistently detected along the entire extent of the surgical wound. In contrast, collagen type III and laminin only stained the superficial portion of the LASIK incision site. Staining for collagen types I and VI showed no changes after LASIK. CONCLUSION: Histologic findings in donor corneas with prior LASIK treatment confirm histologic observations in a recently introduced human organ culture LASIK model. This strengthens the reliability of the latter LASIK model for further studies concerning wound healing after LASIK surgery.

Mapping of the retinal pigment epithelium in exudative age related macular degeneration.

PURPOSE: To evaluate a novel technique for three-dimensional mapping of the retinal pigment epithelium (RPE) layer in patients with subfoveal choroidal neovascularization (CNV) due to age-related macular degeneration. METHODS: Scanning with a recent generation retinal thickness analyzer (RTA) was performed in consecutive patients undergoing fluorescein angiography. From a 3 x 3 mm area centered on the fovea, three-dimensional area maps of the RPE level were calculated by external spreadsheet software. Included were 18 eyes with classic CNV, 18 eyes with occult CNV and 18 eyes from age-matched normal subjects. Repeatability was assessed by measuring 17 eyes with CNV 3 times. In ten additional patients, RTA imaging results were compared with cross-sections obtained by optical coherence tomography. RESULTS: By both methods, distinctive changes in RPE level maps were observed in classic and occult CNV. In classic CNV with the lesion extending over the RPE, only focal irregularities in the anteriorly displaced RPE surface were observed. In contrast, mapping of occult CNV showed a more irregular displacement of the RPE layer. The RPE map standard deviation indicating surface irregularity differed statistically significantly between the groups, with coefficients of variance of 5.9% for controls, 6.1% for classic and 8.8% for occult CNV (P < 0.001). Regarding repeatability, RPE level maps showed 1.2% coefficient of variance and an intra-class correlation coefficient of 0.87 for triplicate measurements in CNV patients. CONCLUSIONS: Topographic mapping of CNV lesions offers a fast, reproducible method for obtaining three-dimensional morphometric information on the RPE level and to quantify changes.

Galectin-1 influences migration of retinal pigment epithelial cells.

PURPOSE: To determine whether the beta-galactoside-binding matricellular protein Gal-1 is expressed in human specimens of proliferative vitreoretinopathy (PVR) and to evaluate its influence on RPE migration. METHODS: RT-PCR was used to detect Gal-1-specific transcripts in PVR membranes, and the expression pattern of Gal-1 was examined by immunohistochemistry. Expression of Gal-1 in native, low- and high-density cultured RPE cells was determined by Western blot analysis. Cultured human RPE cells were treated with bFGF, TGF-beta2, PDGF-BB, or HGF. The dose-response of Gal-1 mRNA expression was measured by by real-time quantitative RT-PCR and Northern blot analysis. Induction of Gal-1 protein was confirmed by Western blot analysis. To study the effect of Gal-1 on RPE migration in vitro, Gal-1 expression was silenced by RNA interference. beta-Lactose was used to saturate extracellular galectins. RPE cell migration was assessed by a modified Boyden chamber assay, with HGF as the chemoattractant. RESULTS: Gal-1 mRNA expression was present in human specimens of PVR membranes, and staining for Gal-1 was distributed throughout the extracellular matrix (ECM) of PVR membranes. Colocalization was found with laminin and fibronectin and cells of epithelial origin. Western blot analysis revealed greater baseline expression levels in low-density cultured RPE cells than in native and high-density cultured RPE cells. Treatment with HGF caused a dose-dependent increase in Gal-1 expression. Low expression levels of Gal-1 correlated with a reduction of RPE migration to 14% of control. beta-Lactose inhibited HGF-induced RPE cell migration to 23% of control. CONCLUSIONS: Gal-1 is present in the extracellular matrix of PVR membranes and may be derived from dedifferentiated RPE cells. The expression level of Gal-1 appears to be related to a migratory RPE phenotype and stimulation by HGF, both conditions implicated in the pathogenesis of early PVR. Furthermore, HGF-induced RPE migration may be dependent, at least in part, on Gal-1- and beta-galactoside-dependent mechanisms.

Pulsed electron avalanche knife (PEAK-fc) for dissection of retinal tissue.

OBJECTIVE: To evaluate the effectiveness and precision of tractionless retinal tissue dissection by the advanced version of the pulsed electron avalanche knife for fine cutting (PEAK-fc; Carl Zeiss Meditec, Jena, Germany). METHODS: Porcine retina (in vivo) and human retina (in vitro) were incised with the PEAK-fc using various pulse parameters. The globes were then processed for light microscopy. Evaluation of all specimens focused on depth of the retinal cuts and on the degree of collateral damage. RESULTS: Retinal cuts performed both in vivo on porcine eyes and on human donor eyes showed very sharp edges with only little collateral damage. With probes of 600 mum in length, the optimal pulse parameters for precise and reproducible cutting of the retina were an amplitude of 350 to 380 V, a repetition rate of 300 Hz, and 30 "minipulses" per pulse of 100-microsecond duration. With increasing voltage, cuts also affected the retinal pigment epithelium and the choroid, followed by intravitreal bleeding during in vivo application. CONCLUSION: We demonstrated that PEAK-fc is capable of precisely cutting retinal tissue in vivo and in vitro using optimal pulse parameters. Further in vivo studies will be necessary to determine the efficacy of this new tractionless cutting device in vitreoretinal surgery.

Pulsed electron avalanche knife in vitreoretinal surgery.

PURPOSE: To evaluate the advantages, disadvantages, safety, and surgical applicability of the pulsed electron avalanche knife (PEAK-fc), a new electrosurgical knife for "cold" and tractionless cutting, in vitreoretinal surgery. PEAK-fc is equipped with an integrated fiberoptic that makes bimanual procedures in intraocular surgery possible. METHODS: A prospective consecutive trial of 18 eyes in 18 patients who underwent vitreoretinal surgery for proliferative diabetic retinopathy, proliferative vitreoretinopathy, subretinal macular hemorrhage, or macular pucker was performed. The following specific maneuvers were performed with PEAK-fc: transection of epiretinal membranes, retinotomies, retinal vessel coagulation, and posterior membranectomy. RESULTS: Detached and attached retina could be dissected successfully in eight cases. Intraoperatively, incision edges were sharply demarcated, showing no visible collateral damage. Deeper layers than the neurosensory retina were not affected. With the bimanual approach, epiretinal avascular and vascular membranes could be removed in 10 cases. Hemorrhages occurring during transection of vascularized membranes could be stopped immediately using the coagulation mode of PEAK-fc. Posterior capsule fibrosis was successfully excised in one patient. No complications were observed. CONCLUSION: PEAK-fc offers precise and tractionless tissue cutting during ocular surgery. Using different waveform parameters, the same device performs cold cutting and/or "hot" coagulation, thus improving the precision, safety, and ergonomics of vitreoretinal surgery.

Absence of scar formation in human donor cornea with prior laser in situ keratomileusis.

PURPOSE: To investigate transglutaminases (enzymes capable of cross-linking extracellular matrix proteins to proteolysis-resistant complexes during scar tissue formation) in a human donor cornea after successful laser in situ keratomileusis (LASIK) without clinical complications and to compare with the results in a human donor cornea with corneal scarring after corneal injury. SETTING: Department of Ophthalmology, Ludwig-Maximilians University, Munich, Germany. METHODS: A donor cornea with prior uneventful LASIK treatment and 1 with corneal scarring after penetrating injury were investigated. Cryostat sections were stained immunohistochemically for tissue transglutaminase (tTG), keratocyte transglutaminase (kTG), and their reaction product epsilon-(gamma-glutamyl)-lysine. RESULTS: With light microscopy, the flap interface of the LASIK-treated eye could hardly be detected, while in the injured eye, infiltration of cells and a clear margin next to the scar formation were present. Immunohistochemistry demonstrated a distinct staining for tTG, kTG, and epsilon-(gamma-glutamyl)-lysine in the corneal scar. In contrast, neither transglutaminase nor epsilon-(gamma-glutamyl)-lysine staining could be observed at the flap margin or in the interface of the LASIK-treated donor eye. CONCLUSIONS: Irreversible protein cross-linking of transglutaminases via epsilon-(gamma-glutamyl)-lysine connections seem to be indicators for scarring in corneal wound healing. The absence of transglutaminases and their reaction product epsilon-(gamma-glutamyl)-lysine in a LASIK-treated cornea supports the idea of missing scar tissue formation after LASIK surgery.

An evaluation of novel vital dyes for intraocular surgery.

PURPOSE: To evaluate systematically the staining characteristics and safety of potential new dyes for intraocular surgery. METHODS: Six dyes were included in the investigation: light green SF (LGSF) yellowish, E68, bromophenol blue (BPB), Chicago blue (CB), rhodamine 6G, rhodulinblau-basic 3 (RDB-B3). All dyes were dissolved and diluted in a balanced saline saline solution. The light-absorbing properties of each dye were measured at a concentration of 0.05% between 200 and 1000 nm. Staining characteristics were examined by staining lens capsule tissue and epiretinal membranes (ERMs), removed intraoperatively, with dye concentrations of 1.0%, 0.5%, 0.2%, and 0.05%. Enucleated porcine eyes (postmortem time, 9 hours) were also stained. Dye-related toxicity was evaluated by a colorimetric test (MTT) measuring the inhibition of retinal pigment epithelium (RPE) cell proliferation (ARPE-19 and primary human RPE cells, passages 3-6). Cell viability was also quantified based on a two-color fluorescence cell-viability assay. Dyes were investigated in concentrations of 0.2% and 0.02%. RESULTS: All dyes investigated in this study stained human lens capsules, removed intraoperatively; ERMs, peeled during macular pucker surgery; and enucleated porcine eyes, depending on the concentration applied. The long-wavelength absorption maximum of the dyes was within the range of 527 to 655 nm at concentrations of 0.05%. Rhodamine G6 and RDB-B3 showed adverse effects on ARPE-19 cell proliferation at a concentration of 0.2% and were excluded from further investigation in primary RPE cells. The remaining four dyes showed no toxic effect on ARPE-19 and primary RPE cell proliferation at concentrations of 0.2% and 0.02%. Cell viability was affected by LGSF yellowish (0.2%) and CB (0.2% and 0.02%). Two dyes (E68 and BPB) showed no relevant toxicity in vitro. CONCLUSIONS: The systematic evaluation of dyes for intraocular use seems mandatory. In this study four dyes were identified with effective staining characteristics, with two of these dyes having no detectable toxic effect on RPE cells in vitro.

Functional outcome after trypan blue-assisted vitrectomy for macular pucker: a prospective, randomized, comparative trial.

PURPOSE: To evaluate functional outcome after the intraoperative application of 0.06% trypan blue during vitrectomy for macular pucker. DESIGN: Prospective, randomized, comparative study. METHODS: Forty-three eyes of 43 consecutive patients, 30 women and 13 men, were incorporated in the study. Patients were randomized into two groups: group 1 (n = 22) with trypan blue (0.06%) staining, and group 2 (n = 21) without trypan blue staining. Functional outcome (best-corrected visual acuity, Goldmann perimetry) was evaluated 1 day before surgery, at 6 weeks, and at intervals of 3, 6, and 12 or more months postoperatively. Only patients with an idiopathic macular pucker were included. In all patients, a standard three-port pars plana vitrectomy with peeling of an epiretinal membrane (ERM) and the internal limiting membrane was performed. No other modification of the surgical procedure, except the use of trypan blue was made between the two groups. RESULTS: Mean age was 68.9 years in group 1 (with trypan blue) and 69.4 in group 2 (without trypan blue). Median best-corrected visual acuity was 20/63 in both groups (range, 20/200-20/40; P >.5) before surgery. Mean follow-up time was 5.8 months in group 1 and 5.3 months in group 2. After surgery, median visual acuity had increased to 20/40 in both groups (range, 20/500 to 20/20 in group 1 and 20/100 to 20/20 in group 2; P <.001 for both groups). The difference between both groups was not statistically significant (P >.5). An improvement of visual acuity (gain of 2 or more lines) was seen in 16 patients of both groups. No postoperative visual field defects were noted. CONCLUSIONS: Trypan blue-assisted vitrectomy for macular pucker leads to good functional results with no dye-related adverse effects after short follow-up. Trypan blue might be especially applicable in cases in which the borders of the ERM are difficult to define. Hypothetical advantages, such as fewer recurrences of ERMs after trypan blue staining, will have to be evaluated during further follow-up of patients.